U.S. patent application number 09/975719 was filed with the patent office on 2003-01-30 for virulence-associated nucleic acid sequences and uses thereof.
Invention is credited to Ausubel, Frederick M., Rahme, Laurence G..
Application Number | 20030022349 09/975719 |
Document ID | / |
Family ID | 22070007 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022349 |
Kind Code |
A1 |
Ausubel, Frederick M. ; et
al. |
January 30, 2003 |
Virulence-associated nucleic acid sequences and uses thereof
Abstract
Disclosed are bacterial virulence polypeptides and nucleic acid
sequences (e.g., DNA) encoding such polypeptides, and methods for
producing such polypeptides by recombinant techniques. Also
provided are methods for utilizing such polypeptides to screen for
antibacterial or bacteriostatic compounds.
Inventors: |
Ausubel, Frederick M.;
(Newton, MA) ; Rahme, Laurence G.; (Brookline,
MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
22070007 |
Appl. No.: |
09/975719 |
Filed: |
October 10, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09975719 |
Oct 10, 2001 |
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09199637 |
Nov 25, 1998 |
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6355411 |
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60066517 |
Nov 25, 1997 |
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Current U.S.
Class: |
435/219 ;
435/252.3; 435/320.1; 435/69.1; 536/23.7 |
Current CPC
Class: |
G01N 33/56911 20130101;
G01N 2333/21 20130101; A61K 49/0004 20130101; C12Q 1/18 20130101;
C07K 14/21 20130101; A61P 31/04 20180101; G01N 2500/10
20130101 |
Class at
Publication: |
435/219 ;
435/69.1; 435/252.3; 435/320.1; 536/23.7 |
International
Class: |
C12N 009/50; C12P
021/02; C12N 001/21; C12N 015/74; C07H 021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a sequence
substantially identical to SEQ ID NO:252.
2. The isolated nucleic acid molecule of claim 1, wherein said
nucleic acid molecule comprises the sequence shown in SEQ ID
NO:252
3. An isolated nucleic acid molecule comprising a sequence
substantially identical to SEQ ID NO:105.
4. The isolated nucleic acid molecule of claim 3, wherein said
nucleic acid molecule comprises the sequence shown in SEQ ID
NO:105.
5. An isolated nucleic acid molecule comprising a sequence
substantially identical to SEQ ID NO:106.
6. The isolated nucleic acid molecule of claim 5, wherein said
nucleic acid molecule comprises the sequence shown in SEQ ID
NO:106.
7. A substantially pure polypeptide comprising an amino acid
sequence that is substantially identical to the amino acid sequence
of SEQ ID NO:253.
8. The substantially pure polypeptide of claim 7, wherein said
amino acid sequence comprises the sequence shown in SEQ ID
NO:253.
9. A substantially pure polypeptide comprising an amino acid
sequence that is substantially identical to the amino acid sequence
of SEQ ID NO:107.
10. The substantially pure polypeptide of claim 9, wherein said
amino acid sequence comprises the sequence shown in SEQ ID
NO:107.
11. A substantially pure polypeptide comprising an amino acid
sequence that is substantially identical to the amino acid sequence
of SEQ ID NO:108.
12. The substantially pure polypeptide of claim 11, wherein said
amino acid sequence comprises the sequence shown in SEQ ID
NO:108.
13. A method for identifying a compound which is capable of
decreasing the expression of a pathogenic virulence factor, said
method comprising the steps of: (a) providing a pathogenic cell
expressing a nucleic acid molecule of claim 1; and (b) contacting
said pathogenic cell with a candidate compound, a decrease in
expression of said nucleic acid molecule following contact with
said candidate compound identifying a compound which decreases the
expression of a pathogenic virulence factor.
14. The method of claim 13, wherein said pathogenic cell infects a
mammal.
15. The method of claim 13, wherein said pathogenic cell infects a
plant.
16. A method for identifying a compound which is capable of
decreasing the expression of a pathogenic virulence factor, said
method comprising the steps of: (a) providing a pathogenic cell
expressing a nucleic acid molecule of claim 3; and (b) contacting
said pathogenic cell with a candidate compound, a decrease in
expression of said nucleic acid molecule following contact with
said candidate compound identifying a compound which decreases the
expression of a pathogenic virulence factor.
17. The method of claim 16, wherein said pathogenic cell infects a
mammal.
18. The method of claim 16, wherein said pathogenic cell infects a
plant.
19. A method for identifying a compound which is capable of
decreasing the expression of a pathogenic virulence factor, said
method comprising the steps of: (a) providing a pathogenic cell
expressing a nucleic acid molecule of claim 5; and (b) contacting
said pathogenic cell with a candidate compound, a decrease in
expression of said nucleic acid molecule following contact with
said candidate compound identifying a compound which decreases the
expression of a pathogenic virulence factor.
20. The method of claim 19, wherein said pathogenic cell infects a
mammal.
21. The method of claim 19, wherein said pathogenic cell infects a
plant.
22. A method for identifying a compound which binds a polypeptide,
said method comprising the steps of: (a) contacting a candidate
compound with a substantially pure polypeptide comprising an amino
acid sequence of claim 7 under conditions that allow binding; and
(b) detecting binding of the candidate compound to the
polypeptide.
23. A method for identifying a compound which binds a polypeptide,
said method comprising the steps of: (a) contacting a candidate
compound with a substantially pure polypeptide comprising an amino
acid sequence of claim 9 under conditions that allow binding; and
(b) detecting binding of the candidate compound to the
polypeptide.
24. A method for identifying a compound which binds a polypeptide,
said method comprising the steps of: (a) contacting a candidate
compound with a substantially pure polypeptide comprising an amino
acid sequence of claim 11 under conditions that allow binding; and
(b) detecting binding of the candidate compound to the
polypeptide.
25. A method of treating a pathogenic infection in mammal, said
method comprising the steps of: (a) identifying a mammal having a
pathogenic infection; and (b) administering to said mammal a
therapeutically effective amount of a composition which inhibits
the expression or activity of a polypeptide encoded by a nucleic
acid molecule of claim 1 in said pathogen.
26. A method of treating a pathogenic infection in mammal, said
method comprising the steps of: (a) identifying a mammal having a
pathogenic infection; and (b) administering to said mammal a
therapeutically effective amount of a composition which inhibits
the expression or activity of a polypeptide encoded by a nucleic
acid molecule of claim 3 in said pathogen.
27. The method of claim 26, wherein said pathogen is Pseudomonas
aeruginosa.
28. A method of treating a pathogenic infection in mammal, said
method comprising the steps of: (a) identifying a mammal having a
pathogenic infection; and (b) administering to said mammal a
therapeutically effective amount of a composition which inhibits
the expression or activity of a polypeptide encoded by a nucleic
acid molecule of claim 5 in said pathogen.
29. The method of claim 28, wherein said pathogen is Pseudomonas
aeruginosa.
30. A method of treating a pathogenic infection in a mammal, said
method comprising the steps of: (a) identifying a mammal having a
pathogenic infection; and (b) administering to said mammal a
therapeutically effective amount of a composition which binds and
inhibits a polypeptide encoded by an amino acid sequence of claim
5.
31. The method of claim 30, wherein said pathogen is Pseudomonas
aeruginosa.
32. A method of treating a pathogenic infection in a mammal, said
method comprising the steps of: (a) identifying a mammal having a
pathogenic infection; and (b) administering to said mammal a
therapeutically effective amount of a composition which binds and
inhibits a polypeptide encoded by an amino acid sequence of claim
7.
33. The method of claim 32, wherein said pathogen infection is
caused by Pseudomonas aeruginosa.
34. A method of treating a pathogenic infection in a mammal, said
method comprising the steps of: (a) identifying a mammal having a
pathogenic infection; and (b) administering to said mammal a
therapeutically effective amount of a composition which binds and
inhibits a polypeptide encoded by an amino acid sequence of claim
9.
35. The method of claim 34, wherein said pathogen infection is
caused by Pseudomonas aeruginosa.
36. A method of treating a pathogenic infection in mammal, said
method comprising the steps of: (a) identifying a mammal having a
pathogenic infection; and (b) administering to said mammal a
therapeutically effective amount of a composition which binds and
inhibits a substantially pure polypeptide comprising an amino acid
sequence of claim 11.
37. The method of claim 36, wherein said pathogen infection is
caused by Pseudomonas aeruginosa.
38. A method of identifying a compound which inhibits the virulence
of a Pseudomonas cell, said method comprising the steps of: (a)
providing a Pseudomonas cell; (b) contacting said cell with a
candidate compound; and (c) detecting the presence of a phenazine,
wherein a decrease in said phenazine relative to an untreated
control cell is an indication that the compound inhibits the
virulence of said Pseudomonas cell.
39. The method of claim 38, wherein said cell is Pseudomonas
aeruginosa.
40. The method of claim 38, wherein said phenazine is detected by
spectroscopy.
41. The method of claim 38, wherein said phenazine is a
pyocyanin.
42. The method of claim 41, wherein said pyocyanin is detected by
measuring the absorbance at 520 nm
43. The method of claim 38, wherein said cell is present in a cell
culture.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. utility application
09/199,637, filed Nov. 25, 1998, which is still pending and which
claims benefit of U.S. provisional application serial number
60/066,517, filed Nov. 25, 1997.
BACKGROUND OF THE INVENTION
[0002] This invention relates to nucleic acid molecules, genes, and
polypeptides that are related to microbial pathogenicity.
[0003] Pathogens employ a number of genetic strategies to cause
infection and, occasionally, disease in their hosts. The expression
of microbial pathogenicity is dependent upon complex genetic
regulatory circuits. Knowledge of the themes in microbial
pathogenicity is necessary for understanding pathogen virulence
mechanisms and for the development of new "anti-virulence or
anti-pathogenic" agents, which are needed to combat infection and
disease.
[0004] In one particular example, the opportunistic human pathogen,
Pseudomonas aeruginosa, is a ubiquitous gram-negative bacterium
isolated from soil, water, and plants (Palleroni, J. N. In:
Bergey's Manual of Systematic Bacteriology, ed., J. G. Holt,
Williams & Wilkins, Baltimore, Md., pp. 141-172, 1984). A
variety of P. aeruginosa virulence factors have been described and
the majority of these, such as exotoxin A, elastase, and
phospholipase C, were first detected biochemically on the basis of
their cytotoxic activity (Fink, R. B., Pseudomonas aeruginosa the
Opportunist: Pathogenesis and Disease, Boca Raton, CRC Press Inc.,
1993). Subsequently, the genes corresponding to these factors or
genes that regulate the expression of these factors were
identified. In general, most pathogenicity-related genes in
mammalian bacterial pathogens were first detected using a
bio-assay. In contrast to mammalian pathogens, simple systematic
genetic strategies have been routinely employed to identify
pathogenicity-related genes in plant pathogens. Following random
transposon-mediated mutagenesis, thousands of mutant clones of the
phytopathogen are inoculated separately into individual plants to
determine if they contain a mutation that affects the pathogenic
interaction with the host (Boucher et al., J. Bacteriol.
168:5626-5623, 1987; Comai and Kosuge, J. Bacteriol. 149:40-46,
1982; Lindgren et al., J. Bacteriol. 168:512-522, 1986; Rahme et
al., J. Bacteriol. 173:575-586, 1991; Willis et al., Mol.
Plant-Microbe Interact. 3:149-156, 1990). Comparable experiments
using whole-animal mammalian pathogenicity models are not feasible
because of the vast numbers of animals that must be subjected to
pathogenic attack.
Summary of the Invention
[0005] We have identified and characterized a number of nucleic
acid molecules, polypeptides, and small molecules (e.g.,
phenazines) that are involved in conferring pathogenicity and
virulence to a pathogen. This discovery therefore provides a basis
for drug-screening assays aimed at evaluating and identifying
"anti-virulence" agents which are capable of blocking pathogenicity
and virulence of a pathogen, e.g., by selectively switching
pathogen gene expression on or off, or which inactivate or inhibit
the activity of a polypeptide which is involved in the
pathogenicity of a microbe. Drugs that target these molecules are
useful as such anti-virulence agents.
[0006] In one aspect, the invention features an isolated nucleic
acid molecule including a sequence substantially identical to any
one of
1 BI48 (SEQ ID NO:1), ORF2 (SEQ ID NO:2), ORF3 (SEQ ID NO:4),
ORF6O2c (SEQ ID NO:6), ORF214 (SEQ ID NO:8) ORF1242c (SEQ ID
NO:10), ORF594 (SEQ ID NO:12), ORF1O4O (SEQ ID NO:14), ORF1640c
(SEQ ID NO:16), ORF2228c (SEQ ID NO:18), ORF2068c (SEQ ID NO:20),
ORF1997 (SEQ ID NO:22), ORF2558c (SEQ ID NO:24), ORF2929c (SEQ ID
NO:26), ORF3965c (SEQ ID NO:28), ORF3218 (SEQ ID NO:30), ORF3568
(SEQ ID NO:32), ORF4506c (SEQ ID NO:34), ORF3973 (SEQ ID NO:36),
ORF4271 (SEQ ID NO:38), ORF4698 (SEQ ID NO:40), ORF5028 (SEQ ID
NO:42), ORF5080 (SEQ ID NO:44), ORF6479c (SEQ ID NO:46), ORF5496
(SEQ ID NO:48), ORF5840 (SEQ ID NO:50), ORF5899 (SEQ ID NO:52),
ORF6325 (SEQ ID NO:54), ORF7567c (SEQ ID NO:56), ORF7180 (SEQ ID
NO:58), ORF7501 (SEQ ID NO:60), ORF7584 (SEQ ID NO:62), ORF8208c
(SEQ ID NO:64), ORF8109 (SEQ ID NO:66), ORF9005c (SEQ ID NO:68),
ORF8222 (SEQ ID NO:70), ORF8755c (SEQ ID NO:72), ORF9431c (SEQ ID
NO:74), ORF9158 (SEQ ID NO:76), ORF10125c (SEQ ID NO:78), ORF9770
(SEQ ID NO:80), ORF9991 (SEQ ID NO:82), ORF10765c (SEQ ID NO:84),
ORF10475 (SEQ ID NO:86), ORF11095c (SEQ ID NO:88), ORF11264 (SEQ ID
NO:90), ORF11738 (SEQ ID NO:92), ORF12348c (SEQ ID NO:94),
ORF12314c (SEQ ID NO:96), ORF13156c (SEQ ID NO:98), ORF12795 (SEQ
ID NO:100), ORF13755c (SEQ ID NO:210), ORF13795c (SEQ ID NO:212),
ORF14727c (SEQ ID NO:214), ORF13779 (SEQ ID NO:216), ORF14293c (SEQ
ID NO:218), ORF14155 (SEQ ID NO:220), ORF14360 (SEQ ID NO:222),
ORF15342c (SEQ ID NO:224), ORF15260c (SEQ ID NO:226), ORF14991 (SEQ
ID NO:228), ORF15590c (SEQ ID NO:230), ORF15675c (SEQ ID NO:232),
ORF16405 (SEQ ID NO:234), ORF16925 (SEQ ID NO:236), ORF17793c (SEQ
ID NO:238), ORF18548c (SEQ ID NO:240), ORF17875 (SEQ ID NO:242),
ORF18479 (SEQ ID NO:244), ORF19027c (SEQ ID NO:246), ORF19305 (SEQ
ID NO:248), ORF19519 (SEQ ID NO:250), ORF19544 (SEQ ID NO:252),
ORF20008 (SEQ ID NO:254), ORF20623c (SEQ ID NO:256), ORF21210c (SEQ
ID NO:258), ORF21493c (SEQ ID NO:260), ORF21333 (SEQ ID NO:262),
ORF22074c (SEQ ID NO:264), ORF21421 (SEQ ID NO:266), ORF22608c (SEQ
ID NO:268), ORF22626 (SEQ ID NO:270), ORF23228 (SEQ ID NO:272),
ORF23367 (SEQ ID NO:274), ORF25103c (SEQ ID NO:276), ORF23556 (SEQ
ID NO:278), ORF26191c (SEQ ID NO:280), ORF23751 (SEQ ID NO:282),
ORF24222 (SEQ ID NO:284), ORF24368 (SEQ ID NO:286), ORF24888c (SEQ
ID NO:288), ORF25398c (SEQ ID NO:290), ORF25892c (SEQ ID NO:292),
ORF25110 (SEQ ID NO:294), ORF25510 (SEQ ID NO:296), ORF26762c (SEQ
ID NO:298), ORF26257 (SEQ ID NO:300), ORF26844c (SEQ ID NO:302),
ORF26486 (SEQ ID NO:304), ORF26857c (SEQ ID NO:306), ORF27314c (SEQ
ID NO:308), ORF27730c (SEQ ID NO:310), ORF26983 (SEQ ID NO:312),
ORF28068c (SEQ ID NO:314), ORF27522 (SEQ ID NO:316), ORF28033c (SEQ
ID NO:318), ORF29701c (SEQ ID NO:320), ORF28118 (SEQ ID NO:322),
ORF28129 (SEQ ID NO:324), ORF29709c (SEQ ID NO:326), ORF29189 (SEQ
ID NO:328), ORF29382 (SEQ ID NO:330), ORF30590c (SEQ ID NO:332),
ORF29729 (SEQ ID NO:334), ORF30221 (SEQ ID NO:336), ORF30736c (SEQ
ID NO:338), ORF30539 (SEQ ID NO:340), ORF31247c (SEQ ID NO:342),
ORF31539c (SEQ ID NO:346), ORF31222 (SEQ ID NO:348), ORF31266 (SEQ
ID NO:350), ORF31661c (SEQ ID NO:352), ORF32061c (SEQ ID NO:354),
ORF32072c (SEQ ID NO:356), ORF31784 (SEQ ID NO:358), ORF32568c (SEQ
ID NO:360), ORF33157c (SEQ ID NO:362), ORF32539 (SEQ ID NO:364),
ORF33705c (SEQ ID NO:366), ORF32832 (SEQ ID NO:368), ORF33547c (SEQ
ID NO:370), ORF33205 (SEQ ID NO:372), ORF33512 (SEQ ID NO:374),
ORF33771 (SEQ ID NO:376), ORF34385c (SEQ ID NO:378), ORF33988 (SEQ
ID NO:380), ORF34274 (SEQ ID NO:382), ORF34726c (SEQ ID NO:384),
ORF34916 (SEQ ID NO:386), ORF35464c (SEQ ID NO:388), ORF35289 (SEQ
ID NO:390), ORF35410 (SEQ ID NO:392), ORF35907c (SEQ ID NO:394),
ORF35534 (SEQ ID NO:396), ORF35930 (SEQ ID NO:398), ORF36246 (SEQ
ID NO:400), ORF26640c (SEQ ID NO:402), ORF36739 (SEQ ID NO:404),
ORF37932c (SEQ ID NO:406), ORF38640c (SEQ ID NO:408), ORF39309c
(SEQ ID NO:410), ORF38768 (SEQ ID NO:412), ORF40047c (SEQ ID
NO:414), ORF40560c (SEQ ID NO:416), ORF40238 (SEQ ID NO:418),
ORF40329 (SEQ ID NO:420), ORF40709c (SEQ ID NO:422), ORF40507 (SEQ
ID NO:424), ORF41275c (SEQ ID NO:426), ORF42234c (SEQ ID NO:428),
ORF41764c (SEQ ID NO:430), ORF41284 (SEQ ID NO:432), ORF41598 (SEQ
ID NO:434), ORF42172c (SEQ ID NO:436), ORF42233c (SEQ ID NO:438),
33A9 (SEQ ID NO:102, 189, 190, 191, 192, 193, 194, 195, 196, 197,
and 198), 34B12 (SEQ ID NO:104), 34B12-ORF1 (SEQ ID NO:105),
34B12-ORF2 (SEQ ID NO:106), 36A4 (SEQ ID NO:109), 36A4 contig(SEQ
ID NO:111), 23A2 (SEQ ID NO:112), 3E8 phn(-) (SEQ ID NO:114), 3E8
contigPA01 (SEQ ID NO:115), 34H4 (SEQ ID NO:118), 33C7 (SEQ ID
NO:119), 25a12.3 (SEQ ID NO:120), 8C12 (SEQ ID NO:121), 2A8 (SEQ ID
NO:122), 41A5 (SEQ ID NO:123), 50E12 (SEQ ID NO:124), 35A9 (SEQ ID
NO:125), pho23 (SEQ ID NO:126), 16G12 (SEQ ID NO:127), 25F1 (SEQ ID
NO:128), PA14 degP (SEQ ID NO:131), 1126 contig (SEQ ID NO:135),
contig 1344 (SEQ ID NO:136), ORFA (SEQ ID NO:440), ORFB (SEQ ID
NO:441), ORFC (SEQ ID NO:442), phzR (SEQ ID NO:164, and 1G2 (SEQ ID
NO:137).
[0007] Preferably, the isolated nucleic acid molecule includes any
of the above-described sequences or a fragment thereof; and is
derived from a pathogen (e.g., from a bacterial pathogen such as
Pseudomonas aeruginosa). Additionally, the invention includes a
vector and a cell, each of which includes at least one of the
isolated nucleic acid molecules of the invention; and a method of
producing a recombinant polypeptide involving providing a cell
transformed with a nucleic acid molecule of the invention
positioned for expression in the cell, culturing the transformed
cell under conditions for expressing the nucleic acid molecule, and
isolating a recombinant polypeptide. The invention further features
recombinant polypeptides produced by such expression of an isolated
nucleic acid molecule of the invention, and substantially pure
antibodies that specifically recognize and bind such a recombinant
polypeptides.
[0008] In an another aspect, the invention features a substantially
pure polypeptide including an amino acid sequence that is
substantially identical to the amino acid sequence of any one
of
2 ORF2 (SEQ ID NO:3), ORF3 (SEQ ID NO:5), ORF602c (SEQ ID NO:7),
ORF214 (SEQ ID NO:9), ORF1242c (SEQ ID NO:11), ORF594 (SEQ ID
NO:13), ORF1040 (SEQ ID NO:15), ORF1640c (SEQ ID NO:17), ORF2228c
(SEQ ID NO:19), ORF2068c (SEQ ID NO:21), ORF1997 (SEQ ID NO:23),
ORF2558c (SEQ ID NO:25), ORF2929c (SEQ ID NO:27), ORF3965c (SEQ ID
NO:29), ORF3218 (SEQ ID NO:31), ORF3568 (SEQ ID NO:33), ORF4506c
(SEQ ID NO:35), ORF3973 (SEQ ID NO:37), ORF4271 (SEQ ID NO:39),
ORF4698 (SEQ ID NO:41), ORF5028 (SEQ ID NO:43), ORF5080 (SEQ ID
NO:45), ORF6479c (SEQ ID NO:47), ORF5496 (SEQ ID NO:49), ORF5840
(SEQ ID NO:51), ORF5899 (SEQ ID NO:53), ORF6325 (SEQ ID NO:55),
ORF7567c (SEQ ID NO:57), ORF7180 (SEQ ID NO:59), ORF7501 (SEQ ID
NO:61), ORF7584 (SEQ ID NO:63), ORF8208c (SEQ ID NO:65), ORF8109
(SEQ ID NO:67), ORF9005c (SEQ ID NO:69), ORF8222 (SEQ ID NO:71),
ORF8755c (SEQ ID NO:73), ORF9431c (SEQ ID NO:75), ORF9158 (SEQ ID
NO:77), ORF10125c (SEQ ID NO:79), ORF9770 (SEQ ID NO:81), ORF9991
(SEQ ID NO:83), ORF10765c (SEQ ID NO:85), ORF10475 (SEQ ID NO:87),
ORF11095c (SEQ ID NO:89), ORF11264 (SEQ ID NO:91), ORF11738 (SEQ ID
NO:93), ORF12348c (SEQ ID NO:95), ORF12314c (SEQ ID NO:97),
ORF13156c (SEQ ID NO:99), ORF12795 (SEQ ID NO:101), ORF13755c (SEQ
ID NO:211), ORF13795c (SEQ ID NO:213), ORF14727c (SEQ ID NO:215),
ORF13779 (SEQ ID NO:217), ORF14293c (SEQ ID NO:219), ORF14155 (SEQ
ID NO:221), ORF14360 (SEQ ID NO:223), ORF15342c (SEQ ID NO:225),
ORF15260c (SEQ ID NO:227), ORF14991 (SEQ ID NO:229), ORF15590c (SEQ
ID NO:231), ORF15675c (SEQ ID NO:233), ORF16405 (SEQ ID NO:235),
ORF16925 (SEQ ID NO:237), ORF17793c (SEQ ID NO:239), ORF18548c (SEQ
ID NO:241), ORF17875 (SEQ ID NO:243), ORF18479 (SEQ ID NO:245),
ORF19027c (SEQ ID NO:247), ORF19305 (SEQ ID NO:249), ORF19519 (SEQ
ID NO:251), ORF19544 (SEQ ID NO:253), ID NO:253), ORF20008 (SEQ ID
NO:255), ORF20623c (SEQ ID NO:257), ORF21210c (SEQ ID NO:259),
ORF21493c (SEQ ID NO:261), ORF21333 (SEQ ID NO:263), ORF22074c (SEQ
ID NO:265), ORF21421 (SEQ ID NO:267), ORF22608c (SEQ ID NO:269),
ORF22626 (SEQ ID NO:271), ORF23228 (SEQ ID NO:273), ORF23367 (SEQ
ID NO:275), ORF25103c (SEQ ID NO:277), ORF23556 (SEQ ID NO:279),
ORF26191c (SEQ ID NO:281), ORF23751 (SEQ ID NO:283), ORF24222 (SEQ
ID NO:285), ORF24368 (SEQ ID NO:287), ORF24888c (SEQ ID NO:289),
ORF25398c (SEQ ID NO:291), ORF25892c (SEQ ID NO:293), ORF25110 (SEQ
ID NO:295), ORF25510 (SEQ ID NO:297), ORF26762c (SEQ ID NO:299),
ORF26257 (SEQ ID NO:301), ORF26844c (SEQ ID NO:303), ORF26486 (SEQ
ID NO:305), ORF26857c (SEQ ID NO:307), ORF27314c (SEQ ID NO:309),
ORF27730c (SEQ ID NO:311), ORF26983 (SEQ ID NO:313), ORF28068c (SEQ
ID NO:315), ORF27522 (SEQ ID NO:317), ORF28033c (SEQ ID NO:319),
ORF29701c (SEQ ID NO:321), ORF28118 (SEQ ID NO:323), ORF28129 (SEQ
ID NO:325), ORF29709c (SEQ ID NO:327), ORF29189 (SEQ ID NO:329),
ORF29382 (SEQ ID NO:331), ORF30590c (SEQ ID NO:333), ORF29729 (SEQ
ID NO:335), ORF30221 (SEQ ID NO:337), ORF30736c (SEQ ID NO:339),
ORF30539 (SEQ ID NO:341), ORF31247c (SEQ ID NO:343), ORF30963c (SEQ
ID NO:345), ORF31539c (SEQ ID NO:347), ORF31222 (SEQ ID NO:349),
ORF31266 (SEQ ID NO:351), ORF31661c (SEQ ID NO:353), ORF32061c (SEQ
ID NO:355), ORF32072c (SEQ ID NO:357), ORF31784 (SEQ ID NO:359),
ORF32568c (SEQ ID NO:361), ORF33157c (SEQ ID NO:363), ORF32530 (SEQ
ID NO:365), ORF33705c (SEQ ID NO:367), ORF32832 (SEQ ID NO:369),
ORF33547c (SEQ ID NO:371), ORF33205 (SEQ ID NO:373), ORF33512 (SEQ
ID NO:375), ORF33771 (SEQ ID NO:377), ORF34385c (SEQ ID NO:379),
ORF33988 (SEQ ID NO:381), ORF34274 (SEQ ID NO:383), ORF34726c (SEQ
ID NO:385), ORF34916 (SEQ ID NO:387), ORF35464c (SEQ ID NO:389),
ORF35289 (SEQ ID NO:391), ORF35410 (SEQ ID NO:393), ORF35907c (SEQ
ID NO:395), ORF35534 (SEQ ID NO:397), ORF35930 (SEQ ID NO:399),
ORF36246 (SEQ ID NO:401), ORF26640c (SEQ ID NO:403), ORF36769 (SEQ
ID NO:405), ORF37932c (SEQ ID NO:407), ORF38640c (SEQ ID NO:409),
ORF39309c (SEQ ID NO:411), ORF38768 (SEQ ID NO:413), ORF40047c (SEQ
ID NO:415), ORF40560c (SEQ ID NO:417), ORF40238 (SEQ ID NO:419),
ORF40329 (SEQ ID NO:421), ORF40709c (SEQ ID NO:423), ORF40507 (SEQ
ID NO:425), ORF41275c (SEQ ID NO:427), ORF42234c (SEQ ID NO:429),
ORF41764c (SEQ ID NO:431), ORF41284 (SEQ ID NO:433), ORF41598 (SEQ
ID NO:435), ORF42172c (SEQ ID NO:437), ORF42233c (SEQ ID NO:439),
33A9 (SEQ ID NOS:103, 199,200,201,202,203,204,205,206,207,and 208),
34B12-ORF1 (SEQ ID NO:107), 34B12-ORF2 (SEQ ID NO:108), 36A4 (SEQ
ID NO:110), 3E8phzA (SEQ ID NO:116), 3E8phzB (SEQ ID NO:117), PhzR
(SEQ ID NO:165), ORFA (SEQ ID NO:443), ORFB (SEQ ID NO:444), ORFC
(SEQ ID NO:445), and PA14 degP (SEQ ID NO:132).
[0009] Preferably, the substantially pure polypeptide includes any
of the above-described sequences of a fragment thereof; and is
derived from a pathogen (e.g., from a bacterial pathogen such as
Pseudomonas aeruginosa).
[0010] In yet another related aspect, the invention features a
method for identifying a compound which is capable of decreasing
the expression of a pathogenic virulence factor (e.g., at the
transcriptional or post-transcriptional levels), involving (a)
providing a pathogenic cell expressing any one of the isolated
nucleic acid molecules of the invention; and (b) contacting the
pathogenic cell with a candidate compound, a decrease in expression
of the nucleic acid molecule following contact with the candidate
compound identifying a compound which decreases the expression of a
pathogenic virulence factor. In preferred embodiments, the
pathogenic cell infects a mammal (e.g., a human) or a plant.
[0011] In yet another related aspect, the invention features a
method for identifying a compound which binds a polypeptide,
involving (a) contacting a candidate compound with a substantially
pure polypeptide including any one of the amino acid sequences of
the invention under conditions that allow binding; and (b)
detecting binding of the candidate compound to the polypeptide.
[0012] In addition, the invention features a method of treating a
pathogenic infection in a mammal, involving (a) identifying a
mammal having a pathogenic infection; and (b) administering to the
mammal a therapeutically effective amount of a composition which
inhibits the expression or activity of a polypeptide encoded by any
one of the nucleic acid molecules of the invention. In preferred
embodiments, the pathogen is Pseudomonas aeruginosa.
[0013] In yet another aspect, the invention features a method of
treating a pathogenic infection in a mammal, involving (a)
identifying a mammal having a pathogenic infection; and (b)
administering to the mammal a therapeutically effective amount of a
composition which binds and inhibits a polypeptide encoded by any
one of the amino acid sequences of the invention. In preferred
embodiments, the pathogenic infection is caused by Pseudomonas
aeruginosa.
[0014] Moreover, the invention features a method of identifying a
compound which inhibits the virulence of a Pseudomonas cell,
involving (a) providing a Pseudomonas cell; (b) contacting the cell
with a candidate compound; and (c) detecting the presence of a
phenazine, wherein a decrease in the phenazine relative to an
untreated control culture is an indication that the compound
inhibits the virulence of the Pseudomonas cell. In preferred
embodiments, the cell is Pseudomonas aeruginosa; the cell is
present in a cell culture; and the phenazine is detected by
spectroscopy (e.g., pyocyanin is detected at an absorbance of 520
nm). Pyocyanin is generally detected according to any standard
method, e.g., those described herein.
[0015] By "isolated nucleic acid molecule" is meant a nucleic acid
(e.g., a DNA) that is free of the genes which, in the
naturally-occurring genome of the organism from which the nucleic
acid molecule of the invention is derived, flank the gene. The term
therefore includes, for example, a recombinant DNA that is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote; or that exists as a separate molecule (for example, a
cDNA or a genomic or cDNA fragment produced by PCR or restriction
endonuclease digestion) independent of other sequences. In
addition, the term includes an RNA molecule which is transcribed
from a DNA molecule, as well as a recombinant DNA which is part of
a hybrid gene encoding additional polypeptide sequence.
[0016] By "polypeptide" is meant any chain of amino acids,
regardless of length or post-translational modification (for
example, glycosylation or phosphorylation).
[0017] By a "substantially pure polypeptide" is meant a polypeptide
of the invention that has been separated from components which
naturally accompany it. Typically, the polypeptide is substantially
pure when it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the preparation is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, a polypeptide of the invention. A substantially pure
polypeptide of the invention may be obtained, for example, by
extraction from a natural source (for example, a pathogen); by
expression of a recombinant nucleic acid encoding such a
polypeptide; or by chemically synthesizing the protein. Purity can
be measured by any appropriate method, for example, column
chromatography, polyacrylamide gel electrophoresis, or by HPLC
analysis.
[0018] By "substantially identical" is meant a polypeptide or
nucleic acid molecule exhibiting at least 25% identity to a
reference amino acid sequence (for example, any one of the amino
acid sequences described herein) or nucleic acid sequence (for
example, any one of the nucleic acid sequences described herein).
Preferably, such a sequence is at least 30%, 40%, 50%, 60%, more
preferably 80%, and most preferably 90% or even 95% identical at
the amino acid level or nucleic acid to the sequence used for
comparison.
[0019] Sequence identity is typically measured using sequence
analysis software (for example, Sequence Analysis Software Package
of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. In an exemplary approach to determining
the degree of identity, a BLAST program may be used, with a
probability score between e.sup.-3 and e.sup.-100indicating a
closely related sequence.
[0020] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding (as used herein) a polypeptide
of the invention.
[0021] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, for example, a recombinant polypeptide of the
invention, or an RNA molecule).
[0022] By "purified antibody" is meant antibody which is at least
60%, by weight, free from proteins and naturally-occurring organic
molecules with which it is naturally associated. Preferably, the
preparation is at least 75%, more preferably 90%, and most
preferably at least 99%, by weight, antibody. A purified antibody
of the invention may be obtained, for example, by affinity
chromatography using a recombinantly-produced polypeptide of the
invention and standard techniques.
[0023] By "specifically binds" is meant a compound or antibody
which recognizes and binds a polypeptide of the invention but which
does not substantially recognize and bind other molecules in a
sample, for example, a biological sample, which naturally includes
a polypeptide of the invention.
[0024] By "derived from" is meant isolated from or having the
sequence of a naturally-occurring sequence (e.g., a cDNA, genomic
DNA, synthetic, or combination thereof).
[0025] By "inhibiting a pathogen" is meant the ability of a
candidate compound to decrease, suppress, attenuate, diminish, or
arrest the development or progression of a pathogen-mediated
disease or an infection in a eukaryotic host organism. Preferably,
such inhibition decreases pathogenicity by at least 5%, more
preferably by at least 25%, and most preferably by at least 50%, as
compared to symptoms in the absence of the candidate compound in
any appropriate pathogenicity assay (for example, those assays
described herein). In one particular example, inhibition may be
measured by monitoring pathogenic symptoms in a host organism
exposed to a candidate compound or extract, a decrease in the level
of symptoms relative to the level of pathogenic symptoms in a host
organism not exposed to the compound indicating compound-mediated
inhibition of the pathogen.
[0026] By "pathogenic virulence factor" is meant a cellular
component (e.g., a protein such as a transcription factor, as well
as the gene which encodes such a protein) without which the
pathogen is incapable of causing disease or infection in a
eukaryotic host organism.
[0027] The invention provides a number of targets that are useful
for the development of drugs that specifically block the
pathogenicity of a microbe. In addition, the methods of the
invention provide a facile means to identify compounds that are
safe for use in eukaryotic host organisms (i.e., compounds which do
not adversely affect the normal development and physiology of the
organism), and efficacious against pathogenic microbes (i.e., by
suppressing the virulence of a pathogen). In addition, the methods
of the invention provide a route for analyzing virtually any number
of compounds for an anti-virulence effect with high-volume
throughput, high sensitivity, and low complexity. The methods are
also relatively inexpensive to perform and enable the analysis of
small quantities of active substances found in either purified or
crude extract form.
[0028] Other features and advantages of the invention will be
apparent from the detailed description, and from the claims.
DETAILED DESCRIPTION
[0029] The drawings will first be described.
DRAWINGS
[0030] FIG. 1 is a schematic diagram showing the physical map of
cosmid BI48 (SEQ ID NO: 1) and the orientation of the identified
open reading frames (ORFs).
[0031] FIG. 2 shows the nucleotide sequence of cosmid B148 (SEQ ID
NO:1).
[0032] FIG. 3 shows the nucleotide sequences for
3 ORF2 (SEQ ID NO:2), ORF3 (SEQ ID NO:4), ORF602c (SEQ ID NO:6),
ORF214 (SEQ ID NO:8), ORF1242c (SEQ ID NO:10), ORF594 (SEQ ID
NO:12), ORF1040 (SEQ ID NO:14), ORF1640c (SEQ ID NO:16), ORF2228c
(SEQ ID NO:18), ORF2068c (SEQ ID NO:20), ORF1997 (SEQ ID NO:22),
ORF2558c (SEQ ID NO:24), ORF2929c (SEQ ID NO:26), ORF3965c (SEQ ID
NO:28), ORF3218 (SEQ ID NO:30), ORF3568 (SEQ ID NO:32), ORF4506c
(SEQ ID NO:34), ORF3973 (SEQ ID NO:36), ORF4271 (SEQ ID NO:38),
ORF4698 (SEQ ID NO:40), ORF5028 (SEQ ID NO:42), ORF5080 (SEQ ID
NO:44), ORF6479c (SEQ ID NO:46), ORF5496 (SEQ ID NO:48), ORF5840
(SEQ ID NO:50), ORF5899 (SEQ ID NO:52), ORF6325 (SEQ ID NO:54),
ORF7567c (SEQ ID NO:56), ORF7180 (SEQ ID NO:58), ORF7501 (SEQ ID
NO:60), ORF7584 (SEQ ID NO:62), ORF8208c (SEQ ID NO:64), ORF8109
(SEQ ID NO:66), ORF9005c (SEQ ID NO:68), ORF8222 (SEQ ID NO:70),
ORF8755c (SEQ ID NO:72), ORF9431c (SEQ ID NO:74), ORF9158 (SEQ ID
NO:76), ORF10125c (SEQ ID NO:78), ORF9770 (SEQ ID NO:80), ORF9991
(SEQ ID NO:82), ORF10765c (SEQ ID NO:84), ORF10475 (SEQ ID NO:86),
ORF11095c (SEQ ID NO:88), ORF11264 (SEQ ID NO:90), ORF11738 (SEQ ID
NO:92), ORF12348c (SEQ ID NO:94), ORF12314c (SEQ ID NO:96),
ORF13156c (SEQ ID NO:98), ORF12795 (SEQ ID NO:100), ORF13755c (SEQ
ID NO:210), ORF13795c (SEQ ID NO:212), ORF14727c (SEQ ID NO:214),
ORF13779 (SEQ ID NO:216), ORF14293c (SEQ ID NO:218), ORF14155 (SEQ
ID NO:220), ORF14360 (SEQ ID NO:222), ORF15342c (SEQ ID NO:224),
ORF15260c (SEQ ID NO:226), ORF14991 (SEQ ID NO:228), ORF15590c (SEQ
ID NO:230), ORF15675c (SEQ ID NO:232), ORF16405 (SEQ ID NO:234),
ORF16925 (SEQ ID NO:236), ORF17793c (SEQ ID NO:238), ORF18548c (SEQ
ID NO:240), 0RF17875 (SEQ ID NO:242), ORF18479 (SEQ ID NO:224),
ORF19027c (SEQ ID NO:246), ORF19305 (SEQ ID NO:248), ORF19519 (SEQ
ID NO:250), ORF19544 (SEQ ID NO:252), ORF20008 (SEQ ID NO:254),
ORF20623c (SEQ ID NO:256), ORF21210c (SEQ ID NO:258), ORF21493c
(SEQ ID NO:260), ORF21333 (SEQ ID NO:262), ORF22074c (SEQ ID
NO:264), ORF21421 (SEQ ID NO:266), ORF22608c (SEQ ID NO:268),
ORF22626 (SEQ ID NO:270), ORF23228 (SEQ ID NO:272), ORF23367 (SEQ
ID NO:274), ORF25103c (SEQ ID NO:276), ORF23556 (SEQ ID NO:278),
ORF26191c (SEQ ID NO:280), ORF23751 (SEQ ID NO:282), ORF24222 (SEQ
ID NO:284), ORF24368 (SEQ ID NO:286), ORF24888c (SEQ ID NO:288),
ORF25398c (SEQ ID NO:290), ORF25892c (SEQ ID NO:292), ORF25110 (SEQ
ID NO:294), ORF25510 (SEQ ID NO:296), ORF26762c (SEQ ID NO:298),
ORF26257 (SEQ ID NO:300), ORF26844c (SEQ ID NO:302), ORF26486 (SEQ
ID NO:304), ORF26857c (SEQ ID NO:306), ORF27314c (SEQ ID NO:308),
ORF27730c (SEQ ID NO:310), ORF26983 (SEQ ID NO:312), ORF28068c (SEQ
ID NO:314), ORF27522 (SEQ ID NO:316), ORF28033c (SEQ ID NO:318),
ORF29701c (SEQ ID NO:320), ORF28118 (SEQ ID NO:322), ORF28129 (SEQ
ID NO:324), ORF29709c (SEQ ID NO:326), ORF29189 (SEQ ID NO:328),
ORF29382 (SEQ ID NO:330), ORF30590c (SEQ ID NO:332), ORF29729 (SEQ
ID NO:334), ORF30221 (SEQ ID NO:336), ORF30736c (SEQ ID NO:338),
ORF30539 (SEQ ID NO:340), ORF31247c (SEQ ID NO:342), ORF31539c (SEQ
ID NO:346), ORF31222 (SEQ ID NO:348), ORF31266 (SEQ ID NO:350),
ORF31661c (SEQ ID NO:352), ORF32061c (SEQ ID NO:354), ORF32072c
(SEQ ID NO:356), ORF31784 (SEQ ID NO:358), ORF32568c (SEQ ID
NO:360), ORF33157c (SEQ ID NO:362), ORF32539 (SEQ ID NO:364),
ORF33705c (SEQ ID NO:366), ORF32832 (SEQ ID NO:368), ORF33547c (SEQ
ID NO:370), ORF33205 (SEQ ID NO:372), ORF33512 (SEQ ID NO:374),
ORF33771 (SEQ ID NO:376), ORF34385c (SEQ ID NO:378), ORF33988 (SEQ
ID NO:380), ORF34274 (SEQ ID NO:382), ORF34726c (SEQ ID NO:384),
ORF34916 (SEQ ID NO:386), ORF35464c (SEQ ID NO:388), ORF35289 (SEQ
ID NO:390), ORF35410 (SEQ ID NO:392), ORF35907c (SEQ ID NO:394),
ORF35534 (SEQ ID NO:396), ORF35930 (SEQ ID NO:398), ORF36246 (SEQ
ID NO:400), ORF26640c (SEQ ID NO:402), ORF36739 (SEQ ID NO:404),
ORF37932c (SEQ ID NO:406), ORF38640c (SEQ ID NO:408), ORF39309c
(SEQ ID NO:410), ORF38768 (SEQ ID NO:412), ORF40047c (SEQ ID
NO:414), ORF40560c (SEQ ID NO:416), ORF40238 (SEQ ID NO:418),
ORF40329 (SEQ ID NO:420), ORF40709c (SEQ ID NO:422), ORF40507 (SEQ
ID NO:424), ORF41275c (SEQ ID NO:426), ORF42234c (SEQ ID NO:428),
ORF41764c (SEQ ID NO:430), ORF41284 (SEQ ID NO:432), ORF41598 (SEQ
ID NO:434), ORF42172c (SEQ ID NO:436), and ORF42233c (SEQ ID
NO:438).
[0033] FIG. 4 shows the deduced amino acid sequences for
4 ORF2 (SEQ ID NO:3), ORF3 (SEQ ID NO:5), ORF602c (SEQ ID NO:7),
ORF214 (SEQ ID NO:9), ORF1242c (SEQ ID NO:11), ORF594 (SEQ ID
NO:13), ORF1040 (SEQ ID NO:15), ORF1640c (SEQ ID NO:17), ORF2228c
(SEQ ID NO:19), ORF2068c (SEQ ID NO:21), ORF1997 (SEQ ID NO:23),
ORF2558c (SEQ ID NO:25), ORF2929c (SEQ ID NO:27), ORF3965c (SEQ ID
NO:29), ORF3218 (SEQ ID NO:31), ORF3568 (SEQ ID NO:33), ORF4506c
(SEQ ID NO:35), ORF3973 (SEQ ID NO:37), ORF4271 (SEQ ID NO:39),
ORF4698 (SEQ ID NO:41), ORF5028 (SEQ ID NO:43), ORF5080 (SEQ ID
NO:45), ORF6479c (SEQ ID NO:47), ORF5496 (SEQ ID NO:49), ORF5840
(SEQ ID NO:51), ORF5899 (SEQ ID NO:53), ORF6325 (SEQ ID NO:55),
ORF7567c (SEQ ID NO:57), ORF7180 (SEQ ID NO:59), ORF7501 (SEQ ID
NO:61), ORF7584 (SEQ ID NO:63), ORF8208c (SEQ ID NO:65), ORF8109
(SEQ ID NO:67), ORF9005c (SEQ ID NO:69), ORF8222 (SEQ ID NO:71),
ORF8755c (SEQ ID NO:73), ORF9431c (SEQ ID NO:75), ORF9158 (SEQ ID
NO:77), ORF10125c (SEQ ID NO:79), ORF9770 (SEQ ID NO:81), ORF9991
(SEQ ID NO:83), ORF10765c (SEQ ID NO:85), ORF10475 (SEQ ID NO:87),
ORF11095c (SEQ ID NO:89), ORF11264 (SEQ ID NO:91), ORF11738 (SEQ ID
NO:93), ORF12348c (SEQ ID NO:95), ORF12314c (SEQ ID NO:97),
ORF13156c (SEQ ID NO:99), ORF12795 (SEQ ID NO:101), ORF13755c (SEQ
ID NO:211), ORF13795c (SEQ ID NO:213), ORF14727c (SEQ ID NO:215),
ORF13779 (SEQ ID NO:217), ORF14293c (SEQ ID NO:219), ORF14155 (SEQ
ID NO:221), ORF14360 (SEQ ID NO:223), ORF15342c (SEQ ID NO:225),
ORF15260c (SEQ ID NO:227), ORF14991 (SEQ ID NO:229), ORF15590c (SEQ
ID NO:231), ORF15675c (SEQ ID NO:233), ORF16405 (SEQ ID NO:235),
ORF16925 (SEQ ID NO:237), ORF17793c (SEQ ID NO:239), ORF18548c (SEQ
ID NO:241), ORF17875 (SEQ ID NO:243), ORF18479 (SEQ ID NO:245),
ORF19027c (SEQ ID NO:247), ORF19305 (SEQ ID NO:249), ORF19519 (SEQ
ID NO:251), ORF19544 (SEQ ID NO:253), ORF20008 (SEQ ID NO:255),
ORF20623c (SEQ ID NO:257), ORF21210c (SEQ ID NO:259), ORF21493c
(SEQ ID NO:261), ORF21333 (SEQ ID NO:263), ORF22074c (SEQ ID
NO:265), ORF21421 (SEQ ID NO:267), ORF22608c (SEQ ID NO:269),
ORF22626 (SEQ ID NO:271), ORF23228 (SEQ ID NO:273), ORF23367 (SEQ
ID NO:275), ORF25103c (SEQ ID NO:277), ORF23556 (SEQ ID NO:279),
ORF26191c (SEQ ID NO:281), ORF23751 (SEQ ID NO:283), ORF24222 (SEQ
ID NO:285), ORF24368 (SEQ ID NO:287), ORF24888c (SEQ ID NO:289),
ORF25398c (SEQ ID NO:291), ORF25892c (SEQ ID NO:293), ORF25110 (SEQ
ID NO:295), ORF25510 (SEQ ID NO:297), ORF26762c (SEQ ID NO:299),
ORF26257 (SEQ ID NO:301), ORF26844c (SEQ ID NO:303), ORF26486 (SEQ
ID NO:305), ORF26857c (SEQ ID NO:307), ORF27314c (SEQ ID NO:309),
ORF27730c (SEQ ID NO:311), ORF26983 (SEQ ID NO:313), ORF28068c (SEQ
ID NO:315), ORF27522 (SEQ ID NO:317), ORF28033c (SEQ ID NO:319),
ORF29701c (SEQ ID NO:321), ORF28118 (SEQ ID NO:323), ORF28129 (SEQ
ID NO:325), ORF29709c (SEQ ID NO:327), ORF29189 (SEQ ID NO:329),
ORF29382 (SEQ ID NO:331), ORF30590c (SEQ ID NO:333), ORF29729 (SEQ
ID NO:335), ORF30221 (SEQ ID NO:337), ORF30736c (SEQ ID NO:339),
ORF30539 (SEQ ID NO:341), ORF31247c (SEQ ID NO:343), ORF30963c (SEQ
ID NO:345), ORF31539c (SEQ ID NO:347), ORF31222 (SEQ ID NO:349),
ORF31266 (SEQ ID NO:351), ORF31661c (SEQ ID NO:353), ORF32061c (SEQ
ID NO:355), ORF32072c (SEQ ID NO:357), ORF31784 (SEQ ID NO:359),
ORF32568c (SEQ ID NO:361), ORF33157c (SEQ ID NO:363), ORF32530 (SEQ
ID NO:365), ORF33705c (SEQ ID NO:367), ORF32832 (SEQ ID NO:369),
ORF33547c (SEQ ID NO:371), ORF33205 (SEQ ID NO:373), ORF33512 (SEQ
ID NO:375), ORF33771 (SEQ ID NO:377), ORF34385c (SEQ ID NO:379),
ORF33988 (SEQ ID NO:381), ORF34274 (SEQ ID NO:383), ORF34726c (SEQ
ID NO:385), ORF34916 (SEQ ID NO:387), ORP35464c (SEQ ID NO:389),
ORF35289 (SEQ ID NO:391), ORF35410 (SEQ ID NO:393), ORF35907c (SEQ
ID NO:395), ORF35534 (SEQ ID NO:397), ORF35930 (SEQ ID NO:399),
ORF36246 (SEQ ID NO:401), ORF26640c (SEQ ID NO:403), ORF36769 (SEQ
ID NO:405), ORF37932c (SEQ ID NO:407), ORF38640c (SEQ ID NO:409),
ORF39309c (SEQ ID NO:411), ORF38768 (SEQ ID NO:413), ORF40047c (SEQ
ID NO:415), ORF40560c (SEQ ID NO:417), ORF40238 (SEQ ID NO:419),
ORF40329 (SEQ ID NO:421), ORF40709c (SEQ ID NO:423), ORP40507 (SEQ
ID NO:425), ORF41275c (SEQ ID NO:427), ORF42234c (SEQ ID NO:429),
ORF41764c (SEQ ID NO:431), ORF41284 (SEQ ID NO:433), ORF41598 (SEQ
ID NO:435), ORF42172c (SEQ ID NO:437), and ORF42233c (SEQ ID
NO:439).
[0034] FIG. 5 shows the nucleotide sequence (SEQ ID NO:102)
encoding a protein encoded by the 33A9 sequence.
[0035] FIG. 6A shows the deduced amino acid sequence (SEQ ID
NO:103) a protein encoded by the 33A9 sequence.
[0036] FIG. 6B shows the nucleotide sequences of several ORFs1-10
(SEQ ID NOS:189, 190, 191, 192, 193, 194, 195, 196, 197, and 198)
identified in the 33A9 sequence and their respective amino acid
sequences (ORFs1-10; SEQ ID NOS: 199, 200, 201, 202, 203, 204, 205,
206, 207, and 208).
[0037] FIG. 7 shows the physical map of the 34B12 EcoR1 fragment
map identifying the positions of three ORFs: ORF1 (L-S), ORF2, and
ORF 1S. The nucleotide sequence corresponding to the pho34B12
insertion (SEQ ID NO:104) containing ORF1 (L-S) (SEQ ID NOS:105 and
107), ORF2 (SEQ ID NOS:106 and 108), and ORF1-S(SEQ ID NOS:208 and
209) is also shown.
[0038] FIG. 8 shows the deduced amino acid sequence of ORF1(L-S)
(SEQ ID NO:107) which is depicted in FIG. 7.
[0039] FIG. 9 shows the deduced amino acid sequence of ORF2 (SEQ ID
NO:108) which is depicted in FIG. 7.
[0040] FIG. 10 shows the nucleotide sequence (SEQ ID NO:109)
corresponding to the 36A4 insertion.
[0041] FIG. 11 shows the deduced amino acid sequence of the peptide
(SEQ ID NO: 110) encoded by the 36A4 sequence. The predicted
peptide encoded by the 36A4 sequence has homology to the hrpM gene
of Pseudomonas syringae (Loubens, et al. Mol. Microbiol. 10:
329-340, 1993).
[0042] FIG. 12 shows the nucleotide sequence (SEQ ID NO:111) of
contig 2507 identified using 36A4 nucleotide sequence.
[0043] FIG. 13 shows the nucleotide sequence (SEQ ID NO:112)
corresponding to the 23A2 insertion.
[0044] FIG. 14A shows the deduced amino acid sequence of the
peptide (SEQ ID NO: 113) encoded by the 23A2 sequence. The peptide
predicted by the 23A2 sequence is homologous to a known protein in
Pseudomonas aeruginosa (strain CD10): the mexA gene. This gene is
part of an operon that also contains two other genes: mexB and oprM
(Poole et al., Mol Microbiol. 10: 529-544, 1993); GenBank
submission: LI 1616.
[0045] FIG. 14B shows the nucleotide sequence (SEQ ID NO:148) and
predicted partial amino acid sequences of PA14 mexA and mexB (SEQ
ID NOS: 149 and 150, respectively).
[0046] FIG. 15 shows the nucleotide sequence (SEQ ID NO:114) of the
PAO1 phenazine operon that was identified using the 3E8 sequence
tag.
[0047] FIG. 16A shows the nucleotide sequence (SEQ ID NO:115) of
the 3E8 sequence tag.
[0048] FIG. 16B shows the nucleotide sequences flanking the 3E8
sequence tag (SEQ ID NO:160).
[0049] FIG. 17 shows the deduced 3E8 PHZA amino acid sequence (SEQ
ID NO:116).
[0050] FIG. 18A shows the deduced 3E8 PHZB amino acid sequence (SEQ
ID NO:117).
[0051] FIG. 18B shows the deduced 3E8 PHZA partial amino acid
sequence (SEQ ID NO:161).
[0052] FIG. 18C shows the deduced 3E8 PHZB partial amino acid
sequence (SEQ ID NO:162).
[0053] FIG. 18D shows the deduced 3E8 PHZC partial amino acid
sequence (SEQ ID NO:163).
[0054] FIG. 18E shows the nucleotide sequence (SEQ ID NO:164) and
predicted partial amino acid sequence (SEQ ID NO:165) of PA14
phzR.
[0055] FIG. 19 shows the nucleotide sequence (SEQ ID NO:118) of the
34H4 sequence tag.
[0056] FIG. 20 shows the nucleotide sequence (SEQ ID NO:119) of the
33C7 sequence tag.
[0057] FIG. 21 shows the nucleotide sequence (SEQ ID NO:120) of the
25a12.3 sequence tag.
[0058] FIG. 22 shows the nucleotide sequence (SEQ ID NO:121) of the
8C12 sequence tag.
[0059] FIG. 23 shows the nucleotide sequence (SEQ ID NO:122) of the
2A8 sequence tag.
[0060] FIG. 24A shows the nucleotide sequences (SEQ ID NOS:123,
124, 125, 126, 127, and 128) of the 41A5, 50E12, 35A9, pho23,
16G12, and 25F1 TnphoA sequence tags, respectively.
[0061] FIG. 24B shows the nucleotide sequence (SEQ ID NO:166) and
predicted amino acid sequence (SEQ ID NO:167) of PA14 pho 15.
[0062] FIG. 24C shows the nucleotide sequence (SEQ ID NO:168) of
PA14 50E12 encoding YgdP.sub.Pa (SEQ ID NO:169) and PtsP.sub.Pa
(SEQ ID NO:170).
[0063] FIG. 24D shows the nucleotide sequence (SEQ ID NO:171) of
PA14 35A9 encoding mtrR.sub.Pa (SEQ ID NO:172).
[0064] FIG. 24E shows the nucleotide sequence (SEQ ID NO:173) of
PA14 25F1 encoding ORFT (SEQ ID NO:174), ORFU (SEQ ID NO:175), and
DjlA.sub.Pa (SEQ ID NO:176).
[0065] FIG. 25 shows the nucleotide sequence (SEQ ID NO:129) of the
phnA and phnB genes of Pseudomonas aeruginosa of PAO1 and PA14,
respectively.
[0066] FIG. 26 shows the deduced amino acid sequence (SEQ ID
NO:130) of PHNA.
[0067] FIG. 27 shows the nucleotide sequence (SEQ ID NO:131) of the
PA14 degP gene.
[0068] FIG. 28 shows the deduced amino acid sequence (SEQ ID
NO:132) of the PA14 degP gene.
[0069] FIG. 29 shows the nucleotide sequence (SEQ ID NO:133) of the
algD gene of Pseudomonas aeruginosa strain 8830.
[0070] FIG. 30 shows the deduced amino acid sequence (SEQ ID
NO:134) of the algD gene of Pseudomonas aeruginosa strain 8830.
[0071] FIG. 31 shows the nucleotide sequence (SEQ ID NO:135) of the
1126 contig identified using 25A12.
[0072] FIG. 32 shows the physical map of the 1344 (SEQ ID NO:136)
contig identified using 33C7 which illustrates three identified
ORFs: ORFA (SEQ ID NO:440), ORFB (SEQ ID NO:441), and ORFC (SEQ ID
NO:442). The amino acid sequences of ORFA (SEQ ID NO:443), ORFB
(SEQ ID NO:444), and ORFC (SEQ ID NO:445) encoded by their
respective ORF is also shown.
[0073] FIG. 33 shows the nucleotide sequence (SEQ ID NO:137) of the
1G2 sequence tag.
[0074] FIGS. 34A-D are graphs showing the complementation of the
worm pathogenicity phenotype of 4 TnphoA mutants using the C.
elegans slow-killing assay.
[0075] FIG. 34A is a graph showing that the nonpathogenic phenotype
of mutant 12A1 (open diamonds) could be fully complemented to the
wild-type PA14 levels (filled squares) by the lasR gene from PAO1
under the control of the constitutive lacZ promoter in trans in
strain 12A1 (pKDT17) (open circles). The reconstructed lasR mutant,
PA14 lasR-G (open squares) is as nonpathogenic as 12A1 (open
diamonds). Results from an experiment using one-day-old adults is
shown.
[0076] FIG. 34B is a graph showing the complementation of the
delayed-killing phenotype of pho 15. Strains pho 15(pEcdsbA) (open
diamonds) and pho 15(pPAdsbA), carry the dsbA gene from E. coli and
P. aeruginosa, respectively, in trans under the control of the
constitutive lacZ promoter.
[0077] FIG. 34C is a graph showing that the delayed killing
phenotype of 25F1 was only partially restored by strains
25F1(pORF338) and 25F1(p3-ORFs) carrying plasmids containing orf338
and orf338-orf224-djlA.sub.Pa, respectively.
[0078] FIG. 34D is a graph showing the complementation of 50E12 by
the orf159-ptsP.sub.Pa operon. Strain 50e12(pUCP18), like mutant
12A1, does not kill worms even after 63 hours. Both strains
50E12(pMT205-lac) and 50E12(pMT206-nat), expressing the putative
orf159-ptsP.sub.Pa operon were able to kill C. elegans. In
50E12(pMT205-lac), transcription of orf159-ptsP.sub.Pa is under the
control of the constitutive lacZ promoter, whereas in
50E12(pMT206-nat), the operon is controlled by its native promoter.
Each data point represents means.+-.SD of 3-4 replicates. Unless
indicated otherwise, synchronized L4 worms were used in the
experiments. At least two independent experiments were performed
for each complementation analysis.
[0079] FIG. 35A is a schematic illustration showing the
anthranilate synthase complex that is encoded by the phnA and phnB
genes which catalyzes the conversion of chorismate to anthranilate.
Antranilate serves as a precursor for pyocyanin production in P.
aeruginosa, strain PAO1 (Essar et al., J. Bacteriol. 172: 884-900,
1990). The double arrows indicate the involvement of multiple,
undefined steps, leading from the conversion of anthranilate to
pyocyanin.
[0080] FIG. 35B is a schematic illustration showing the generation
of the .DELTA.phnAphnB mutant by an in-frame deletion of 1602 bp
within the phnA and phnB genes.
[0081] FIG. 35C is a graph showing the effect of the
.DELTA.phnAphnB mutant on fast killing in C. elegans. Fast- killing
assays were conducted using the wild type PA14 strain, the TnphoA
mutant 3E8 or the .DELTA.phnAphnB strain. Worm mortality was
monitored 3 hours after initial exposure to the bacteria and the
defect in fast killing seen with .DELTA.phnAphnB strain was
comparable to that of another phenazine mutant, 3E8.
Virulence Factor Identification and Characterization
[0082] As described herein, plants were used as an in vivo
pathogenesis model for the identification of virulence factors of
the human opportunistic pathogen Pseudomonas aeruginosa. Nine out
of nine TnphoA mutant derivatives of P. aeruginosa strain
UCBPP-PA14 that were identified in a plant leaf assay for less
pathogenic mutants also exhibited significantly reduced
pathogenicity in a mouse burn assay, suggesting that P. aeruginosa
utilized many common strategies to infect both hosts. Seven of
these nine mutants contained TnphoA insertions in previously
unknown genes. These results demonstrated that an alternative
non-vertebrate host of a human bacterial pathogen could be used in
an in vivo high throughput screen to identify novel bacterial
virulence factors involved in mammalian pathogenesis. These
experimental examples are intended to illustrate, not limit, the
scope of the claimed invention.
[0083] These experiments were carried out using the following
techniques.
[0084] Strains, Growth Conditions and Plasmids. P. aeruginosa
strain UCBPP-PA14 is a human clinical isolate that was used in
these experiments for the identification of novel virulence-related
genes (Ausubel et al., Methods of Screening Compounds Useful for
Prevention of Infection or Pathogenicity, U.S. Ser. Nos.
08/411,560, 08/852,927, and 08/962,750, filed on Mar. 25, 1995, May
7, 1997, and Nov. 3, 1997, respectively; Rahme et al., Science
268:1899-1902, 1995), and P. aeruginosa strains PAK (Ishimoto and
Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and PAO1
(Holloway et al., Microbiol. Rev. 43:73-102, 1979) have been
studied extensively in many laboratories. Luria Bertani broth and
agar were used for the growth of P. aeruginosa and Escherichia coli
strains at 37.degree. C. Minimal medium (M9) was also used for the
growth of P. aeruginosa.
[0085] Transposon Mutagenesis. Transposon-mediated mutagenesis of
UCBPP-PA14 was performed using TnphoA carried on the suicide
plasmid pRT731 in E. coli strain SM10 .lambda.pir (Taylor et al.,
J. Bacteriol. 171:1870-1878, 1989). Donor and recipient cells grown
in this medium were plated together on Luria Bertani agar plates
and incubated at 37.degree. C. for eight to ten hours and
subsequently plated on Luria Bertani plates containing rifampicin
(100 .mu.g/ml) (to select against the E. coli donor cells) and
kanamycin (200 .mu.g/ml) (to select for TnphoA containing P.
aeruginosa cells). Colonies which grew on the rifampicin and
kanamycin media were replicated to Luria Bertani containing
ampicillin (300 .mu.g/ml); ampicillin resistant colonies indicated
pRT731 integration into the UCBPP-PA14 genome and were
discarded.
[0086] Alkaline Phosphatase Activity. Two thousand five hundred
(2,500) prototrophic UCBPP-PA14 TnphoA mutants were screened on
peptone glucose agar plates (Ostroff et al., J. Bacteriol.
172:5915-5923, 1990) containing 40 .mu.g/ml
5-bromo-4-chloro-3-indoly phosphate (XP). Peptone medium was
selected because it suppressed the production of the endogenous
blue-green pigment pyocyanin and the fluorescent yellow pigment
pyoverdin, permitting visualization of the blue color that resulted
from dephosphorylation of XP by periplasmic alkaline phosphatase
generated by PhoA.sup.+ mutants.
[0087] Growth Conditions and Mutant Isolation Strategy. P.
aeruginosa strains that were grown to saturation in L-broth at
37.degree. C. were washed in 10 mM MgSO.sub.4, resuspended at an
optical density of 0.2 (OD.sub.600=0.2) in 10 mM MgSO.sub.4 and
diluted 1:100 and 1:1000 (corresponding to a bacterial density of
approximately 10.sup.6 and 10.sup.5 cfu/ml, respectively).
Approximately 10 ml of the diluted cells were inoculated with a
Pipetman into stems of approximately twelve-week old lettuce plants
(variety Romain or Great lake) grown in MetroMix potting soil in a
greenhouse (26.degree. C.). The stems were washed with 0.1% bleach
and placed on 15 cm diameter petri dishes containing one Whatman
filter (Whatman #1) that was impregnated with 10 mM MgSO.sub.4. The
midrib of each lettuce leaf was inoculated with three different
TnphoA-generated P. aeruginosa mutants to be tested and the wild
type UCBPP-PA14 strain as a control. The plates were kept in a
growth chamber during the course of the experiment at 28-30.degree.
C. and 90-100% relative humidity. Symptoms were monitored daily for
five days.
[0088] In the Arabidopsis leaf infiltration model, P. aeruginosa
strains grown and washed as above were diluted 1:100 in 10 mM
MgSO.sub.4 (corresponding to a bacterial density of 10.sup.3
/cm.sup.2 leaf disk area) and were injected into leaves of six-week
old Arabidopsis plants as described for infiltration of Pseudomonas
syringae (Ausubel et al., Methods of Screening Compounds Useful for
Prevention of Infection or Pathogenicity, U.S. Ser. Nos.
08/411,560, 08/852,927, and 08/962,750, filed on Mar. 25, 1995, May
7, 1997, and Nov. 3, 1997, respectively; Rahme et al., Science
268:1899-1902, 1995; Dong et al., Plant Cell 3:61-72, 1991).
Incubation conditions and monitoring of symptoms were the same as
in the lettuce experiments. Leaf intercellular fluid containing
bacteria was harvested, and bacterial counts were determined as
described (Rahme et al., Science 268:1899-1902, 1995; Dong et al.,
Plant Cell 3:61-72, 1991). Four different samples were taken using
two leaf discs per sample. Control plants inoculated with 10 mM
MgSO.sub.4 showed no symptom developement.
[0089] Mice Mortality Studies. A 5% total surface area burn was
fashioned on the outstreached abdominal skin of six-week-old male
AKRIJ mice (Jackson Laboratories) weighing between 25 and 30 gm as
previously described (Ausubel et al., Methods of Screening
Compounds Useful for Prevention of Infection or Pathogenicity, U.S.
Ser. Nos. 08/411,560, 08/852,927, and 08/962,750, filed on Mar. 25,
1995, May 7, 1997, and Nov. 3, 1997, respectively; Rahme et al.,
Science 268:1899-1902, 1995; Stevens, J. Burn Care Rehabil.
15:232-235, 1994). Immediately following the burn, mice were
injected with 5.times.10.sup.3 or 5.times.10.sup.5 P. aeruginosa
cells, and the number of animals that died of sepsis was monitored
each day for ten days. Animal study protocols were reviewed and
approved by the subcommittee on Animal Studies of the Massachusetts
General Hospital. Statistical significance for mortality data was
determined using a .chi..sup.2 test with Yates' correction or
Fisher's exact test. Differences between groups were considered
statistically significant at P.ltoreq.0.05.
[0090] DNA Manipulation, Molecular Cloning, and Sequence Analysis
of TnphoA Mutants. P. aeruginosa chromosomal DNA was isolated by
phenol extraction (Strom and Lory, J. Bacteriol. 165:367-372,
1986), and DNA blotting and hybridization studies were performed as
described in Ausubel et al. (Current Protocols in Molecular
Biology, Wiley, New York, 1996).
[0091] The oligonucleotides 5'-AATATCGCCCTGAGCAGC- 3' (LGR1) (SEQ
ID NO:138) and 5'-AATACACTCACTATGCGCTG- 3' (LGR2) (SEQ ID NO:139)
corresponded to sequences on opposite strands at the 5'- end of
TnphoA. The oligonucleotides 5'-CCATCTCATCAGAGGGTA-3' (LGR3) (SEQ
ID NO:140) and 5'-CGTTACCATGTTAGGAGGTC-3' (LGR4) (SEQ ID NO:141)
corresponded to sequences on opposite strands at the of the 3'- end
of TnphoA. LGR1+LGR2 or LGR3 +LGR4 were used to amplify by inverse
PCR (IPCR) DNA sequences adjacent to the sites of TnphoA insertion
as described (Ochman et al., 1993, A Guide to Methods and
Applications, eds., Innis, M. A., States, D. J., 1990). Amplified
DNA fragments ranging in size from 350 to 650 base pairs were
cloned into pBlueScript SK+/- by filling in the ends of the IPCR
products prior to subcloning into the EcoRV site of pBlueScript
SK+/-. To determine the sequence of IPCR-amplified products,
double-stranded DNA sequencing was performed using the Sequenase
2.0 kit (U. S. Biochemical, Inc.). Sequences obtained were compared
to the non-redundant peptide sequence databases at the National
Center for Biotechnology Information (NCBI) using the BLASTX
program (Gish and States, Nat. Genet. 3:266-272, 1993).
[0092] Isolation and DNA Manipulation of the Wild Type Clone
Containing the Gene Corresponding to the pho34B12 Mutation from the
UCBPP-PA14 Genomic Library. The IPCR product that was generated
from UCBPP-PA14 TnphoA mutant pho34B12 mutant was labeled using a
random primed DNA labeling kit (Boehringer Mannheim, Indianapolis,
Ind.) and used to probe a genomic library of UCBPP-PA14 chromosomal
DNA in pJSR1 (Rahme et al., Science 268:1899-1902, 1995) for a
clone containing the gene corresponding to the pho34B12 mutation. A
3.7 kb EcoRI fragment, identified in cosmid clone pLGR34B12 which
corresponded to the pho34B12 mutation, was subcloned into EcoRI
site of pRR54 (Roberts et al., J. Bacteriol. 172:6204-6216, 1990)
after filling-in the ends of both vector and fragment to construct
pLGRE34B12. The same fragment (made blunt ended) was subcloned into
the SmaI site of pCVD (Donnenberg and Kaper, Infect. Immun.
59:4310-4317, 1991) to construct pLGR34. pLGR34 was used to replace
the mutated pho34B12 gene with a wild-type copy as described
(Donnenberg and Kaper, Infect. Immun. 59:4310-4317, 1991). The 3.7
kb EcoRI fragment was also subcloned into the EcoRI site of
pBlueScript SK+/- to construct pBSR34B12 and used for DNA sequence
analysis.
[0093] A 1,659 base pair sequence corresponding to the pho34B12
insertion that contains two overlapping open reading frames (ORF1
and ORF2) on opposing strands was submitted to GenBank and was
assigned Accession number AF031571. ORF1 is 1,148 bp (nucleotides
361 to 1509) and ORF2 is 1,022 bp (nucleotides 1458 to 436). The
overlap of the two ORFs is from nucleotide 436 to 1458. ORF1
contains a second putative translational start site at nucleotide
751 corresponding to a coding region of 758 bp. The oligonucleotide
primers 5'-CGCATCGTCGAAACGCTGGCGGCC-3' (SEQ ID NO:142) and
5'-GCCGATGGCGAGATCATGGCGATG-3' (SEQ ID NO:143) were used to amplify
a 1100 bp fragment from pBSR34B12 containing ORF1. Because of the
two putative initiation sites present in ORF1, the oligonucleotide
primers 5'-TGCGCAACGATACGCCGTTGCCGACGATC-3' (SEQ ID NO:144) and
5'-GATTCCACCTTCGCAGCGCAGCCC-3' (Reg3) (SEQ ID NO:145) were also
used to amplify a 1659 bp from pBSR34B12 containing ORF1. The
oligonucleotide primers 5'-GATTCCACCTTCGCAGCGCAGCCC-3' (SEQ ID
NO:146) and 5'-GCCGATGGCGAGATCATGGCGATG-3' (SEQ ID NO:147) were
used to amplify a 1302 bp fragment from pBSR34B12 containing ORF2.
All primer combinations were designed to contain the putative
upstream regulatory elements of each ORF. The PCR products obtained
(1100, 1659, and 1302 bp) were cloned into pCR2.1 (Invitrogen Inc.)
to construct pLE15, pLE1, and pLE2, respectively. All three PCR
products were subcloned into pRR54 to construct pRRLE15, pRRLE1,
and pRRLE2, respectively.
[0094] Enzymatic Activities of TnphoA Mutants. P. aeruginosa
strains grown for eighteen hours in LB medium were used for assays
of enzymatic activities. Proteolytic and elastolytic activities
were determined as described previously (Toder et al., Mol.
Microbiol. 5:2003-2010, 1991). Quantitation of pyocyanin was
determined as described (Essar et al., J. Bact. 172:884-900, 1990).
Hemolytic activity was detected following incubation on plates
containing Trypticase soy agar (BBL) supplemented with 5% Sheep red
blood cells (Ostroff and Vasil, J. Bacteriol. 169:4957-4601,
1987).
[0095] Generation of a Non-Polar GacA Mutation. A non-polar gacA
mutation in UC BPP-PA14 was constructed by cloning a 3.5 kb PstI
fragment containing the gacA gene from cosmid pLGR43 (Rahme et al.,
Science 268:1899-1902, 1995) into the unique BamHI restriction site
in the suicide vector pEGBR (Akerley et al., Cell 80:611-620, 1995)
using BamHI linkers. A 950 bp EcoRI-HincII Klenow end-filled
fragment containing the kanamycin resistance gene cassette from
pUC18K (Menard et al., J. Bacteriol. 175:5899-5906, 1993) was then
cloned into the unique BamH1 restriction site (made blunt ended) in
gacA, such that transcription was maintained and translation of the
downstream portion of gacA was reinitiated at the 3' end of the
kanamycin cassette. The resultant construct, SW 7-4, containing the
kanamycin gene cassette within the gacA gene and in the orientation
of its transcription, was used to marker-exchange by homologous
recombination the disrupted gacA gene into the wild-type UCBPP-PA14
genome.
[0096] Isolation and Characterization of P. aeruginosa Virulence
Factors. Using the procedures described above, the P. aeruginosa
UCBPP-PA14 genome was mutagenized with transposon TnphoA, and 2,500
prototrophic mutants were screened for impaired pathogenicity in
the lettuce stem assay. This lettuce assay allowed for the testing
of several mutants on a single lettuce stem. Interestingly, we
found that lettuce was not only susceptible to infection by
UCBPP-PA14 but also was susceptible to the well characterized P.
aeruginosa strains PAK (Ishimoto and Lory, Proc. Natl. Acad. Sci
USA 86:1954-1957, 1989) and PAO1 (Holloway et al., Microbiol. Rev.
43:73, 1979). Both of these latter strains proliferated in lettuce
leaves and elicited disease symptoms similar to those elicited by
UCBPP-PA14, characterized by water soaking followed by soft rot
four to five days post-infection. In later stages of infection, all
three P. aeruginosa strains invaded the entire midrib of a lettuce
leaf resulting in complete maceration and collapse of the
tissue.
[0097] As summarized in Table 1, we identified nine
TnphoA-generated mutants of UCBPP-PA14 among the 2,500 prototrophs
screened that elicited either null, weak, or moderate rotting
symptoms on lettuce stems compared to the wild-type strain.
5 TABLE 1 Growth in Symptoms % Mouse Arabidopsis Elicited in
Mortality.sup.c Gene Strain leaves.sup.a Arabidopsis.sup.b 5
.times. 10.sup.3 5 .times. 10.sup.5 Identity PA14 5.5 .times.
10.sup.7 severe 53 100 33C7 8.3 .times. 10.sup.4 none 0 0
unknown.sup.d 1D7 7.5 .times. 10.sup.5 weak 0 50 gacA 25A12 1.7
.times. 10.sup.6 weak 11 87 unknown 33A9 5.1 .times. 10.sup.6
moderate 0 0 unknown 25F1 1.5 .times. 10.sup.4 moderate 0 20
unknown 34H4 3.8 .times. 10.sup.6 moderate 0 33 unknown pho34B12
4.0 .times. 10.sup.6 moderate 0 56 unknown pho15 3.9 .times.
10.sup.4 moderate 0 62 dsbA 16G12 2.3 .times. 10.sup.5 moderate 20
100 unknown .sup.aFour different samples were taken using two leaf
discs/sample. Control plants inoculated with 10 mM MgSO.sub.4
showed no symptoms during the course of the experiments. Three
independent experiments gave similar results. .sup.bSymptoms
observed four to five days after infection. None, no symptoms;
chlorosis, chlorosis circumscribing the inoculation site; weak,
localized water-soaking and chlorosis of tissue circumscribing the
inoculation site; moderate, moderate water-soaking and chlorosis
with most of the tissue softened around the inoculation site;
severe, #severe soft-rotting of the entire leaf characterized by a
water-soaked reaction zone and chlorosis around the inoculation
site at two to three days post-infection. .sup.cAll animal
experiments were conducted at least twice using 8-10
animals/experiment. Independent experiments showed similar
percentage mortality rates. Mice were injected with .about.5
.times. 10.sup.3 or 5 .times. 10.sup.5 cells. .sup.dBLASTX analysis
yielded no encoded proteins with significant homology.
[0098] Severe maceration of the leaf was not observed with any of
the mutants. DNA blot analysis showed that each of the nine mutants
contained a single TnphoA insertion, using as a probe a 1542 base
pair BglI-BamHI fragment containing the kanamycin resistance
conferring gene of TnphoA (Taylor et al., J. Bact. 171:1870-1878,
1989). Two of the nine UCBPP-PA14 TnphoA mutants, pho34B1, and pho
15, expressed alkaline phosphatase activity suggesting that the
genes containing these TnphoA insertions encoded membrane-spanning
or secreted proteins (Taylor et al., J. Bact. 171:1870-1878, 1989;
Manoil and Beckwith, Proc. Natl. Acad. Sci USA 82:5117, 1985).
[0099] The nine TnphoA mutants were further tested by measuring
their growth rate over the course of four days in Arabidopsis
leaves as a quantitative measure of pathogenicity (Rahme et al.,
Science 268:1899-1902, 1995; Dong et al., Plant Cell 3:61-72,
1991). Although none of the mutants showed any significant
differences in their growth rates as compared to the wild-type
strain in both rich and minimal media, the growth rate over time of
all nine mutants in Arabidopsis leaves was lower than the wild-type
strain. Table 1 lists the maximal levels of growth reached by each
mutant at the fourth day post-infection. In the case of all nine
mutants, less severe symptom development reflected reduced
bacterial counts in leaves. All of the mutants except 33C7 elicited
either weak or moderate rot and water soaking symptoms with varying
amounts of chlorosis (yellowing) (Table 1). Interestingly, however,
as summarized in Table 1, the levels of proliferation of the
individual mutants did not directly correlate with the severity of
symptoms that they elicited. For example, even though mutant 25A12
(FIG. 21) grew to similar levels as mutants 33A9 (FIGS. 5 and
6A-B), pho34B12 (FIGS. 7, 8, and 9), and 34H4 (FIG. 19), and only
ten-fold less than wild-type UCBPP-PA14, mutant 25A12 elicited very
weak symptoms. Similarly, mutants 33C7 (FIG. 20), pho 15 (FIG.
24B), and 25F1 (FIG. 24A) all reached similar maximal levels of
growth (approximately 10.sup.3-fold less than the growth of the
wild type); however, only mutant 33C7 failed to cause any disease
symptoms (Table 1). The differences observed in the degree of
symptoms and proliferation levels among the ten mutants suggested
that these mutants likely carried insertions in genes that are
involved in various stages of the plant infectious process.
[0100] The pathogenicity of each of the nine TnphoA-generated
mutants that were less pathogenic in the plant leaf assay was
measured in a full-thickness skin thermal bum mouse model (Rahme et
al., Science 268:1899-1902, 1995; Stevens et al., J. of Burn Care
and Rehabil. 15:232-235, 1994). As shown in Table 1, all nine
mutants were significantly different from the wild-type with a
P.ltoreq.0.05 at both doses except for 25A12 and 16G12 (FIG. 24A),
which were not significantly different from wild-type at the higher
dose of 5.times.10.sup.5 cells. In addition to the data shown in
Table 1, mutant 33A9 also caused no mortality even at a higher dose
of 5.times.10.sup.6.
[0101] We used DNA blot analysis and DNA sequence analysis to
determine whether TnphoA in the nine less pathogenic mutants had
inserted in known genes. DNA blot analysis revealed that mutant 1D7
contained a TnphoA insertion in the gacA gene (Laville et al.,
Proc. Natl. Acad. Sci. USA 89:1562-1566, 1992; Gaffney et al., Mol.
Plant-Microbe Interact. 7:455-463, 1994) which we had shown
previously to be an important pathogenicity factor for P.
aeruginosa in both plants and animals (Ausubel et al., Methods of
Screening Compounds Useful for Prevention of Infection or
Pathogenicity, U.S. Ser. Nos. 08/411,560, 08/852,927, and
08/962,750, filed on March 25, 1995, May 7, 1997, and Nov. 3, 1997,
respectively; Rahme et al., Science 268:1899-1902, 1995). For the
other eight mutants we used the inverse polymerase chain reaction
(IPCR) to generate amplified products corresponding to DNA
sequences adjacent to the sites of the TnphoA insertions (Ochman et
al., A Guide to Methods and Applications, eds., Innis, M. A.,
States, D. J., 1990). The IPCR products were cloned and then
subjected to DNA sequence analysis. Mutant pho15 contained TnphoA
inserted into a P. aeruginosa gene (from strain PA01) previously
deposited in GenBank (Accession # U84726) that shows a high degree
similarity to the Azotobacter vinelandii dsbA gene, which encodes a
periplasmic disulfide bond forming enzyme (Bardwell et al., Cell
67:581-589, 1991). Homologues of dsbA in the bacterial
phytopathogen Erwinia chrysanthemi and in the human pathogens
Shigella flexneri and Vibrio cholera are required for pathogenesis
(Shevchik et al., Mol. Microbiol 16:745-753, 1995; Peek and Taylor,
Proc. Natl. Acad. Sci. USA 89:6210-6214, 1992; Watarai et al.,
Proc. Natl. Acad. Sci. USA 92:4927-4931, 1995). Computer analysis
using the program BLASTX showed that when the DNA sequences
corresponding to the remaining seven TnphoA insertions were
translated in all possible reading frames, no significant
similarities to any known genes were found (Table 1).
[0102] We performed a variety of biochemical tests to categorize
the nine less pathogenic UCBPP-PA14 mutants on the basis of whether
they carried defects in previously described primary virulence
factors and/or metabolic pathways. All mutants were assayed for
protease, elastase, and phospholipase activities and for their
ability to secrete the secondary metabolite pyocyanin (Toder et
al., Mol. Microbiol. 5:2003-2010, 1991; Essar et al., J. Bact.
172:884-900, 1990; Ostroff and Vasil, J. Bacteriol. 169:45974601,
1987). Pyocyanin is a redox-active phenazine compound excreted by
most clinical strains of P. aeruginosa that kills mammalian and
bacterial cells through the generation of reactive oxygen
intermediates and which has been implicated as a P. aeruginosa
virulence factor (Hassett et al. Infect. Immun. 60:328-336, 1992;
Kanthakumar et al., Infect. Immun. 61:2848-2853, 1993; Miller et
al. Infect. Immun. 64:182, 1996). Mutants 33C7, 33A9, 34H4, 25F1,
and 16G12 showed no defects in any of the biochemical assays used.
Mutant pho34B12 showed decreased hemolytic activity on blood agar
plates, reduced elastase activity (.about.50%), and no detectable
pyocyanin production. Mutant pho15 showed only traces of elastase
activity and a decrease in proteolytic activity (60-70%) compared
to the wild-type. Mutant 25A12 showed a 50% decreased elastolytic
activity. Finally, mutant 1D7 which contained an insertion in gacA,
showed reduced levels of pyocyanin (.about.50%) as compared to the
wild-type. In addition to mutant 1D7 a second independent
gacA::TnphoA mutant was identified from our plant screen, mutant
33D11. This latter mutant also exhibited a similar reduction in
pyocyanin production and reduced virulence in both plants and
mice.
[0103] On the basis of the DNA sequence analysis and biochemical
testing of the mutants, the genes targeted by the TnphoA insertions
in mutants 1D7 and pho34B12 were chosen for further analysis. As
discussed above, 1D7 contained an insertion in gacA which we had
shown previously to encode a virulence factor in P. aeruginosa
(Rahme et al., Science 268:1899-1902, 1995). Recently a gacA-like
gene has also been shown to be an important virulence factor for
Salmonella typhimurium (Johnston et al., Mol. Microbiol. 22:715,
1996). However, the two gacA::TnphoA insertions (1D7 and 33D11),
the gacA insertion mutant that we constructed previously (Ausubel
et al., Methods of Screening Compounds Useful for Prevention of
Infection or Pathogenicity, U.S. Ser. Nos. 08/411,560, 08/852,927,
and 08/962,750, filed on Mar. 25, 1995, May 7, 1997, and Nov. 3,
1997, respectively; Rahme et al., Science 268:1899-1902, 1995), and
an independently constructed P. aeruginosa gacA mutation that
affects the production of several known virulence factors (Hassett
et al., Infect. Immun. 60:328-336, 1992) all exert a polar effect
on at least one gene, a homologue of the E. coli uvrC gene
immediately downstream of gacA (Rahme et al., Science
268:1899-1902, 1995; Laville et al., Proc. Natl. Acad. Sci USA
89:1562-1566, 1992; Reimmann et al., Mol. Microbiol. 24:309-319,
1997). To provide definitive evidence that the loss of
pathogenicity phenotypes of the gacA mutants described herein was
due to the disruption of the gacA open reading frame per se rather
than due to a polar effect on a gene downstream of gacA, we
constructed a non-polar gacA mutation in UCBPP-PA14 using a DNA
cassette encoding a gene that confers kanamycin resistance.
Importantly, the non-polar gacA mutant exhibited the same
diminished level of pathogenicity in the mouse assay (50%
mortality) and in the Arabidopsis assay (growth to 3.times.10.sup.5
cfu/cm.sup.2 after four days) as the gacA::TnphoA mutant (1D7), but
did not exhibit the extreme UV sensitivity of the polar gacA
mutants. Like 1D7, the non-polar gacA mutant also excreted lower
levels of pyocyanin (50%) compared to the wild-type.
[0104] Mutant pho34B12 was chosen for further analysis for the
following reasons. First, the insertion in pho34B12 was situated
directly downstream of the P. aeruginosa pyocyanin biosynthetic
genes phnA and phnB (Essar et al. J. Bact. 172:884-900, 1990), in a
previously uncharacterized region of the P. aeruginosa genome.
Second, the pho34B12 insertion caused a pleiotropic phenotype that
included reduced elastase and hemolytic activities, suggesting that
the gene in which the pho34B12 TnphoA insertion was situated might
encode a regulator of diverse pathogenicity factors.
[0105] To rule out the possibility that a secondary mutation in
pho34B12 was responsible for the loss of pathogenicity phenotype
rather than the TnphoA insertion, we replaced the pho34B12::TnphoA
mutation by homologous recombination with the corresponding wild
type gene. This resulted in restoration of the pathogenicity defect
in both plants and animals as well as restoration of hemolytic and
elastolytic activity and pyocyanin production to wild-type levels
(Table 2, below).
6TABLE 2 Growth in Symptoms % mouse Arabidopsis Elicited in
mortality Strain Leaves Arabidopsis 5 .times. 10.sup.5 % pyocyanin
PA14 5.5 .times. 10.sup.7 severe 100 100 pho34B12 4.0 .times.
10.sup.6 moderate 56 .ltoreq.1 pho34B12 3.9 .times. 10.sup.5 severe
100 120 reconstructed to wild-type pho34B12 6.1 .times. 10.sup.5
moderate 0 600 +pLGRE34B12 pho34B12 7.0 .times. 10.sup.5 moderate
13 40 +pRRLE2 pho34B12 5.0 .times. 10.sup.5 moderate 13 1,400
+pRRLE1 pho34B12 1.0 .times. 10.sup.5 moderate 22 1,360 +pRRLE15
.sup.aSee Table 1 for an explanation of table entries.
[0106] These results in Table 2 show that the TnphoA insertion in
pho34B12 was the cause of the pleiotropic phenotype of this strain,
including the loss of pathogenicity phenotype. The fact that no
putative ORFs were present in the next 500 bp downstream of the
stop codon following the pho34B12::TnphoA insertion (see below)
made it unlikely that TnphoA exerted a polar effect on a downstream
gene which was responsible for the phenotype of mutant pho34B12.
Genetic complementation analysis of pho34B12 with a plasmid
(pLGRE34B12) containing a 3.7 kb insert which included pho34B12 and
part of the phnAB region resulted in restoration of the elastase
and hemolytic activities to wild-type levels and more than a
ten-fold overproduction of pyocyanin (Table 2). However, the
impaired pathogenicity phenotype of pho34B12 in both Arabidopsis
and mice was not complemented by pLGRE34B12 (Table 2), most likely
due to the presence of multiple copies of the wild-type gene
corresponding to pho34B12.
[0107] Further DNA sequence analysis showed that the region
containing the pho34B12 mutation encoded two almost completely
overlapping open reading frames (ORFs) (ORF1 and ORF2) that were
transcribed in opposite directions. Moreover, ORF1 had two
potential methionine start codons (designated OFR1-S and ORF1-L).
The predicted proteins encoded by ORF1-S and ORF1-L, which were
transcribed in the same direction as the phnA, phnB, and phoA
genes, contained a consensus motif that corresponded to a lipid
attachment site found in a variety of prokaryotic membrane
lipoproteins (Hayashi and Wu, J. Bioenerg. Biomembr. 22:451-471,
1990). These membrane lipoproteins are synthesized with a precursor
signal peptide, providing an explanation for the Pho+phenotype of
the pho34B12 insertion (Hayashi and Wu, J. Bioenerg. Biomembr.
22:451-471, 1990). The predicted protein encoded by ORF2 contained
an N-terminal `helix-tum-helix` DNA-binding motif similar to the
`helix-turn-helix` motif found in the LysR family of
transcriptional regulators (Henikoff et al., Proc. Natl. Acad. Sci.
USA 85:6602-6606, 1988; Viale et al., J. Bacteriol., 173:5224-5229,
1991). This class of proteins includes regulators involved in both
mammalian and plant pathogenesis (Finlay and Falkow, Microbiol. and
Mol. Biol. Rev. 61:136-169, 1997). The existence of two functional
almost completely overlapping ORFs is unusual in bacterial
genomes.
[0108] To determine which of the ORFs encoded in the pho34B 12
region were functional, additional complementation analysis was
carried out using plasmids that contained PCR products
corresponding to ORF1-S, ORF1-L, and ORF2 (FIG. 7). The production
of both pyocyanin and elastolytic activity was restored to 20-40%
of wild type levels by the plasmid synthesizing the protein encoded
by ORF2 (pRRLE2). Similarly, the hemolytic ability of this
complemented strain was partially restored. Complementation of
pho34B12 with plasmids pRRLE1 and PRRLE15, corresponding to ORF1-S
and ORF1-L, respectively, also restored the hemolytic, pyocyanin,
and elastolytic activities. Interestingly, however, the presence of
plasmids pRRLE1 and pRRLE15 resulted in a 10-fold higher production
of pyocyanin and a 2-fold higher level of elastase activity.
Neither pRRLE1, pRRLE15, nor pRRLE2 complemented the loss of
pathogenicity phenotypes of mutant pho34B12 in either plants or
animals (Table 2). Further characterization of this region
including site directed mutagenesis will further elucidate which of
the three ORFs is (are) required for pathogenicity in plants and
animals.
[0109] The data presented above demonstrated that previously
unknown P. aeruginosa virulence factors (genes) that play a
significant role in mammalian pathogenesis can be readily
identified by screening random P. aeruginosa mutants for ones that
display attenuated pathogenic symptoms in plants. This is
consistent with our previous study in which we demonstrated that at
least three P. aeruginosa genes encode virulence factors involved
in both plant and animal pathogenesis (Ausubel et al., Methods of
Screening Compounds Useful for Prevention of Infection or
Pathogenicity, U.S. Ser. Nos. 08/411,560, 08/852,927, and
08/962,750, filed on Mar. 25, 1995, May 7, 1997, and Nov. 3, 1997,
respectively; Rahme et al., Science 268:1899-1902, 1995). On the
other hand, we did not expect to find that nine out of nine mutants
that we isolated that were less virulent in plants would also be
less virulent in mice. The simplest interpretation of this result
is that P. aeruginosa pathogenesis in plants and animals utilizes a
substantially overlapping set of genes which may be considered to
be basic virulence genes. Another possible interpretation is that
some of the identified genes may encode regulatory proteins (i.e.,
pho34B12), that control different effector molecules, a subset of
which may be specific for either plants or animals. We also did not
expect that the majority of mutants that would be identified in
this study (7 out of 9) would correspond to previously unknown
genes. Using the Poisson distribution, a genome size for P.
aeruginosa of 5.9 Mb and an average gene size of 1.1 kb, we
calculated that the 2,500 mutants tested represents 25% of the
total number that needs to be tested to give approximately 95%
probability of testing each gene in the assay. Therefore, since our
screen for P. aeruginosa virulence mutants is not nearly saturated,
it is likely that many additional P. aeruginosa genes with
important roles in pathogenicity await discovery.
[0110] Importantly, at least two of the previously known virulence
factors (genes) identified in our model as being important in plant
pathogenesis, are not only important virulence factors for P.
aeruginosa in a mouse bum model, but have also been described as
important virulence factors in other gram-negative pathogens. These
latter pathogenicity factors (genes) include dsbA, and gacA
(Shevchik et al. Mol. Microbiol. 16:745-753, 1995; Peek and Taylor,
Proc. Natl. Acad. Sci. USA 89:6210-6214, 1992; Watarai et al.,
Proc. Natl. Acad. Sci. USA 92:4927-4931, 1995; Johnston, et al.,
Mol. Microbiol. 22:715, 1996). This makes it likely that many of
the previously unknown factors identified in P. aeruginosa will be
generally relevant for gram-negative pathogenesis.
[0111] Another important conclusion from this study is that the
high throughput in vivo screening method that we have developed can
lead to the identification of pathogenicity factors that do not
correlate with obvious biochemical defects. Mutants 33C7, 33A9,
34H4, 25F1, and 16G12 exhibited no detectable defects in several
known P. aeruginosa pathogenicity factors and, importantly, mutants
33C7 and 33A9 were among the most debilitated in the mouse model.
Moreover, even though mutants pho34B12 and 25A12 did exhibit
diminished production of known virulence factors, the genes
corresponding to these mutants have not been identified previously,
most likely because the biochemical defects in these mutants cannot
be readily identified efficiently in a simple high throughput
screen. This attests to the sensitivity of our screen for loss of
pathogenicity phenotypes.
[0112] In the last few years, other high throughput screens for
identifying bacterial pathogenicity factors have been described.
The IVET (in vivo expression technology) identifies promoters that
are specifically activated during pathogenesis (Wang et al., Proc.
Natl. Acad. Sci. USA. 93:10434-10439, 1996; Mahan et al., Science
259:686-688, 1993), STM (signature-tagged transposon method)
identifies genes that are required for survival in a host (Hensel,
Science 268:400-403, 1995) and DFI (differential fluorescence
induction) utilizes green fluorescent protein and fluorescence
activated cell sorting to identify genes that are activated under
specific conditions or in specific host cell types (Valdivia and
Falkow, Mol. Microbiol. 22:367-378, 1996). These approaches are
complimentary with the one that we have described in this
application and each approach has advantages and disadvantages. One
advantage of our screening procedure in a non-vertebrate host is
that it directly measures pathogenicity whereas the IVET and DFI
methods measure pathogenicity-associated gene expression. Unlike
the STM procedure, which identifies genes whose function cannot be
complemented in trans by the mixed population of bacterial mutants
used for the inoculum, the present screen in a non-vertebrate
involves testing each mutant clone separately.
Other Virulence Targets
[0113] The 33A9 nucleic acid sequence (FIGS. 5 and 6A-B) was also
identified in a cosmnid clone designated B148 (FIG. 1). This cosmid
was sequenced in its entirety and its nucleic acid sequence is
shown in FIG. 2. Using standard database analysis, the nucleotide
sequences and deduced amino acid sequences of several additional
open reading frames were identified (FIGS. 3 and 4). A summary of
this analysis is presented in Table 3. Like the sequences described
above, any one of the sequences found in FIGS. 3 and 4 can be used
to screen for compounds (e.g., using the methods described herein)
that reduce the virulence of a pathogen.
[0114] The sequence obtained from the pBI48 cosmid of strain PA14
revealed that 33A9 was located approximately 5 kb upstream of a
pili gene cluster (FIG. 1, Table 3). This cluster contains the
pilS/pilR genes, known to be involved in the regulation of pili
formation. Moreover, the analysis of the sequence upstream of 33A9
did not show any homology with previously identified sequences
suggesting the possibility that the entire region surrounding 33A9
could define a pathogenicity island. FIGS. 3 (orf 19544), FIG. 4
(orf 19544), 5, 6A, and 6B show the 33A9 nucleotide sequence, as
well as the identified ORFs.
[0115] In addition, analysis of the sequence obtained from the
pBI48 cosmid clone indicated the presence of a sequence located
approximately 2 kb downstream of 33A9, which showed strong homology
with tRNA sequences (ORF 22626, FIG. 1). Because the analysis of
the region located upstream of the tRNA sequence did not show any
homology with sequences present in the database, and because tRNA
sequences represent "hot spots" for DNA insertions, we hypothesized
that the tRNA sequence represented the right boundary for the
insertion of a pathogenicity island present in PA14. As seen in
FIG. 1 the size of the region that could represent the piece of
foreign DNA that was inserted is approximately 25 kb. The
identification of the boundary that is located upstream of the
presumptive pathogenicity island will assist to establish the exact
size of the inserted piece of DNA. Moreover, the analysis of the
33A9 region also indicated the presence of more than one sequence
with homology at the protein level to integrases and transposases
(ORF21421, ORF8109 respectively). Finally, our data showed that the
33A9 locus was present in several highly pathogenic P. aeruginosa
clinical isolates, and absent in PAO1, a less pathogenic strain of
P. aeruginosa.
[0116] The analysis of the sequencing data obtained from the pBI48
cosmid also indicated the presence of two sequences flanking the
33A9 gene which contained recognition motifs involved in cell
attachment. Sequence analysis of ORF11738 (2436 bp) and ORF23228
(2565 bp), upstream and downstream of 33A9 respectively (FIG. 1),
indicated the presence of RGD motifs in these two open reading
frames. RGD tripeptide sequences are a characteristic eukaryotic
recognition motif that binds to host cell surface integrins and
have been found to be involved in bacterial adherence. By mimicking
host molecules, bacterial adhesins that contain these RGD motifs
can effect responses in the host that are required to promote
cell-cell adhesion.
[0117] The expression of these two RGD-containing ORFs was
evaluated in both 33A9 and the wild type strain PA14. Transcript
levels were determined by hybridization with a radiolabeled DNA
probe that corresponded to an internal region of ORF11738 and
ORF23228. The data obtained for the two ORFs in the mutant 33A9
showed reduced transcript levels compared to the wild type PA14,
indicating that the genes encoded by ORF11738 and ORF23228 are both
regulated by 33A9. These data indicated that 33A9 plays a role as a
multigene regulator responsible for the regulation of the
expression of genes involved in bacterial attachment to host cell
surfaces.
7TABLE 3 ORF Start Stop Length Blast n BlastP Motif Terminator
Shine-Delgarno 244c 244 35 210 602c 602 42 561 730 214 214 792 579
594 594 3734 3141 Conjugal transfer prtn ATP/GTP BINDING 730 1205C
1205 987 219 1640C 1640 1206 435 1615C 1615 1439 177 rev
transcriptase 2929c 2929 2288 642 adhesin precursor 3994c 3994 3818
177 outer memb. protein 4506C 4506 3862 645 lipoprotein 4442 4901c
4901 4668 234 atp-dep. rna helicase 4726 10475 10475 10828 354 unk.
mycobacterium 11738 11738 14173 2436 mycobact. unk. ATP/GTP BINDING
14155 14155 16101 1947 DNA helicase ATP/GTP BINDING 15915 21421
21421 22761 1341 several P. a. genes integrase 22982 21464 22505
22505 22657 153 t-RNAs, oprL, prenylation 23228 23228 26197 2970
atp dep. protease zin protease 26191c 26191 23612 2580 clp
proteases, ClpB ATP/GTP BINDING 26844c 26844 26332 513 ClpB 26486
26486 27160 675 Memb. glycoprotein 26857c 26857 26516 342 viral
nucl. antigen 28068c 28068 27055 1014 PilS yabO (hypothetical)
28118 28118 29188 1071 PilS lipoprotien 29382 29382 31172 1791 PilS
31186 31247c 31247 30591 657 FABprotien 31222 31222 32523 1302
AlgB, PilR sigma54interaction domain 31518 32568c 32568 32065 504
tonB (Fe receptor) 32567 33705c 33705 32569 1137 PilR, D-AA 32567;
32609 33678 dehydrogenase 34274 34274 34915 642 pilin Nterm mrthyl
(pilin) 34916 34916 35449 534 prepilin leader 36246 36246 36875 630
Pil genes} pilx, pily 1 41284 41284 42234 951 sugar transport
41175; 41170 42236c 42236 41185 1052 LYTB In addition, using the
plant and nematode screening assays (slow- or fast-killing assays)
described in Ausubel et al.
[0118] (Methods of Screening Compounds Useful for Prevention of
Infection or Pathogenicity, U.S. Ser. Nos. 08/411,560, 08/852,927,
and 08/962,750, filed on Mar. 25, 1995, May 7, 1997, and Nov. 3,
1997, respectively), several other mutant Pseudomonas aeruginosa
strains were identified as having decreased virulence. The slow-
and fast-killing assays utilized for these studies are described
below.
[0119] Slow-killing assay. For the slow-killing assay, 10 .mu.l of
an overnight bacterial culture was spread on an NG plate (modified
from NGM agar described in Sulston and Hodgkin (In: The Nematode
Caenorhabditis elegans, W. B. Wood, ed., Cold Spring Harbor, N.Y.:
Cold Spring Harbor Laboratory, 188, pp. 587-606): (0.35% instead of
0.25% peptone was used) and incubated at 37.degree. C. for 24
hours. After 8-24 hours at room temperature (23-25.degree. C.) each
plate (3.5 cm diameter) was seeded with 40-50 hermaphrodite L4 C.
elegans strain Bristol; for statistical purposes, 3-4 replicates
per trial were carried out. Plates were incubated at 25.degree. C.,
and the number of dead worms were scored every 4-6 hours. A worm
was considered dead when it no longer moved when touched with an
eyelash and failed to display any pharyneal pumping action. For
each batch of mutants assayed, PA14 and E. coli OP50 were used as
positive and negative controls. Any worms that died as a result of
being immobilized to the wall of the plate were excluded from the
analysis. In order to determine LT.sub.50s, data were plotted on a
graph (percentage of worms killed vs. time after exposure to test
strains (hour)). A curve of the form: percentage
killed=A+(1-A)/(1+exp(B-G.times.- log(hours after exposure))) was
fitted to the data using the SYSTAT 5.2.1 computer program, where A
represented the fraction of worms dying in a OP.sub.50 control
experiment, and B and G are parameters which were varied to fit the
curve. Once B and G have been determined, LT.sub.50 is calculated
by the formula LT.sub.50=exp(B/G).times.(1-2.times.A) (1/G).
[0120] In developing the screen, we took advantage of two
observations. First, the longer it took for the worms to be killed,
the more progeny were produced. Second, early larval stages are
apparently more resistant to killing by P. aeruginosa. This
provided us with a convenient and very sensitive assay for the
identification of TnphoA mutants that are only slightly impaired in
their pathogenic potential. These attenuated mutants would be less
efficient at killing worms, and the production of progeny by
survivors effectively "amplifies" even a weak defect into a readily
observable phenotype. Thus, on plates containing attenuated
PA14::TnphoA mutants, from the initial seeded hermaphrodites,
hundreds of worms were obtained. On plates seeded with a
nonpathogenic mutant, thousands of worms were seen by day five and
the bacterial lawn was completely consumed, whereas none or very
few live worms were found on the plates seeded with the wild-type
strain. Putative nonpathogenic or attenuated mutants identified in
the preliminary screen were retested, and subjected to a virulence
assay to determine the C. elegans-killing kinetics.
[0121] Fast-killing assay. The fast-killing assay, like the
slow-killing assay, is useful for identifying disease-causing
microbial virulence factors. In addition, the assay is useful for
identifying therapeutics that are capable of either inhibiting
pathogenicity or increasing an organism's resistance capabilities
to a pathogen. In preferred embodiments, the fast-killing assay is
carried out using a nematode strain having increased permeability
to a compound, e.g., a toxin such as colchicine. Examples of
nematodes having such increased permeability include, without
limitation, animals having a mutation in a P-glycoprotein, e.g.,
PGP-1, PGP-3, or MRP-1. Such mutant nematodes are useful in the
fast-killing assay because of their increased sensitivity to toxins
that is due to increased membrane permeability. This characteristic
results in an assay with an increased differential between full
susceptibility and full resistance to toxic compounds. The
fast-killing assay may also be carried out by increasing the
osmolarity of the culture medium as described below.
[0122] The fast-killing assay conditions utilized herein are as
follows, 5 .mu.l of a PA14 culture grown overnight in Kings B was
spread on plates (3.5 cm diameter) containing peptone-glucose
medium (PG), (1% Bacto-Peptone, 1% NaCl, 1% glucose, 1.7%
Bacto-Agar). Since the efficacy of fast-killing was found to depend
on osmolarity, PG medium was modified by the addition of 0.15 M
sorbitol. After spreading the bacterial culture, plates were
incubated at 37 .degree. C. for 24 hours and then placed at room
temperature for 8-12 hours. Fifteen to twenty worms were placed on
the assay plate, which was then incubated at 25.degree. C. Each
independent assay consisted of 3-4 replicates. Worm mortality was
scored over time, and a worm was considered dead when it failed to
respond to touch as is described above. The E. coli strain DH5a was
used as a control for the fast-killing assays.
[0123] An analysis of these strains, together with those identified
above, indicated that they fell into several different classes
including the following: some mutants were less pathogenic on both
plants and nematodes, whereas others were reduced in either plants
or nematodes, but not both. Bacterial mutants less pathogenic in
plants were defined as those which, at four days post-infiltration
(DPI), had a mean maximum titer (from 5 leaf samples) of two
standard deviations lower relative to wild-type within the same set
of experiments. The wild-type control was necessary because the
maximal level reached by wild-type at four DPI could vary as much
as an order of magnitude between experiments due to the effects of
minor variations in growth conditions on the plant defense
responses. Similarly, a mutant was characterized as reduced in
pathogenicity in worms if the mean time required to kill 50% of the
worms feeding on it (LT.sub.50 from 3 replicates) was two standard
deviations less than LT.sub.50 of wild-type PA14 in the same
experiment.
[0124] In general, those mutant strains having reduced
pathogenicity in plants included 16G12, 25A12, 33A9, and 33C7;
those having reduced pathogenicity in nematodes included the 35A9,
44B1, 1G2, 8C12, and 2A8, and those having reduced pathogenicity in
plants and nematodes included 25F1, 41A5, 5OE12, pho15, 12A1,
pho23, 34B12, 34H4. 3E8, 23A2, and 36A4. Tables 4 and 5 (below)
summarize the pathogenicity phenotypes of these mutant strains.
Sequence analysis was carried out for each of these strains having
decreased virulence due to insertional mutagenesis. The DNA
sequence analyses, summarized in Tables 4 and 5, showed that both
novel and known genes were identified in our screening assays.
Sequences from 5OE12 and 41C1 each show strong similarity to
previously described open reading frames (ORFs) of unknown function
in E. coli. Mutant 35A9 identified a mtrR homologue of N.
gonorrhoeae (SwissProt P39897). Mutant 25F1 identified an operon
encoding 3 proteins having identity to orfT of C. tepidium, MPK,
and DjlA.sub.Ec. Sequences from 48D9, 35H7, and 12A1 corresponded
to the lemA, gacA, and lasR genes, respectively. The sequences
disrupted in mutants 44A5 and 44B1 do not have significant
similarity to any sequence deposited in GenBank. (The 44B1-sequence
tag is only 148 bp because and there were no sequences
corresponding to the 44B1 insertion in the PAO1 database were
identified). Accordingly, these sequences identify additional
virulence factors. The nucleotide and amino acid sequences obtained
from these experiments are shown in FIGS. 10, 11, 12, 13, 14A, 14B,
15, 16, 16A, 16B, 17, 18A, 18B, 18C, 18D, and 18E and FIGS. 22, 23,
24A, 24B, 24C, 24D, 24E, 25, 26, 27, and 28.
[0125] We also carried out a battery of standard biochemical tests
on TnphoA mutants 41A5, 50E12, 41 C1, 35A9, 48D9, 12A1, 44B1, and
35H7 to assess if any co lesions in known P. aeruginosa virulence
factors important for mammalian pathogenicity. These tests
included: a standard plate assay for sensitivity to H.sub.2O.sub.2,
as well as standard quantitative analysis of extracellular
protease, elastase, phospholipase C, and pyocyanin. Except for the
following, the majority of the PA14 TnphoA mutants were
indistinguishable biochemically from the parent PA14 strain. Mutant
12A1 exhibited decreased elastolytic and proteolytic activities but
overproduced pyocyanin. Mutant 50E12 produced 3-fold higher levels
of pyocyanin than PA14. Mutant 41A5 had only about 70% of wild-type
levels of proteolytic activity.
[0126] A detailed description of the DNA sequence analysis and
biochemical analysis of each of these mutants identified using the
slow-killing assay (described above) is now presented in the
following sections.
[0127] Mutant 12A1. The Tn phoA insertion in 12A1 was inserted into
codon 154 of the previously described lasR gene of P. aeruginosa
PA01. The phenotype of 12A1, like other known lasR mutants, is
pleiotropic, and includes decreased elastase and protease
production. In addition 12A1 produced 2-3 times more pyocyanin than
the parent PA14 strain at stationary phase. Furthermore, a lasR
mutant expressing GFP (PA141asR::GFP19-1) failed to establish
itself in the worm gut as very little fluorescence was detected in
C. elegans intestines after 48 hours of feeding.
[0128] FIG. 34A shows that the defective nematode slow-killing
phenotype of 12A1 was completely restored when the P. aeruginosa
PAO1 lasR gene was expressed in trans under the control of the
constitutive lacZpromoter in strain 12A1 (pKDT17). The production
of elastase was also found to be restored to wild-type levels in
12A1 (pKDT17), but not the overproduction of pyocyanin. Because the
pyocyanin-overproduction phenotype was not expected, we constructed
a new lasR mutant, lasR::Gm, by marker exchanging a lasR gene
interrupted by a gentamicin cassette into the PA14 genome. The
lasR::Gm mutant was as nonpathogenic as 12A1 (FIG. 34A), but
produced normal levels of pyocyanin, suggesting that 12A1 may
harbor a second mutation that resulted in the upregulation of
pyocyanin production. The result also indicated that the
upregulation of pyocyanin production during the stationary phase is
not related to the attenuated pathogenicity phenotype.
[0129] Mutant pho 15. Disruption of the dsbA gene in pho15 was
found to be responsible for the nonpathogenic phenotypes. FIG. 24B
shows the nucleotide sequence (SEQ ID NO:166) and predicted amino
acid sequence (SEQ ID NO:167) of PA14 pho15. The pathogenicity
defective phenotype of pho15 in C. elegans was also found to be
fully restored by constitutive expression of the E. coli dsbAEC
gene or the PA14 dsbA.sub.Pa gene in trans in the pho15 background
(FIG. 34B). For these experiments, the E. coli dsbA.sub.EC gene was
cloned into pUCP18 as follows. The PCR-amplified E. coli dsbA was
cloned into the KpnI and XbaI sites of pBAD18 to form pCH3. This
placed the E. coli dsbA under the E. coli arabinose promoter. A 700
bp KpnI/SphI fragment containing the E. coli dsbA was cloned into
the KpnI/SphI sites of pUCP18, to make pEcdsbA, placing the E. coli
dsbA under the constitutive E. coli lacZ promoter. pEcdsbA was
subsequently used to transform PA14 and pho 15 to construct strains
PA14(pEcdsbA) and pho 15(pEcdsbA), respectively.
[0130] PA14dsbA.sub.Pa was constructed as follows. Based on the
dsbA sequences of PA01 (GenBank Accession number U84726), primers
TMW8 (5'-GCACTGATCGCTGCGTAGCACGGC-3'; SEQ ID NO:177) and TMW9
(5'-TGACGTAGCCGGAACGCAGGCTGC-3'; SEQ ID NO:178) were used to
amplify a 1126 bp fragment containing the dsbA gene plus 176 bp
upstream of the translational start of the dsbA gene from genomic
DNA of PA14. This fragment was cloned, using the TA cloning kit
(Invitrogen), into the pCR2.1 vector to generate pCRdsbA. The
SacI/XbaI fragment-containing dsbA was cloned into SacI/XbaI
digested pUCP18 to construct pPAdsbA, placing the transcription of
dsbA under the constitutive lacZ promoter. Strain pho15(PAdsbA) was
constructed by transforming pho15 with pPAdsbA.sub.Pa.
[0131] Mutant 25F1. In 25F1, TnphoA was found to be inserted within
codon 100 of a putative gene (orf338) that encodes a 338 amino acid
protein, the first gene of a putative 3-gene operon. The predicted
downstream genes (orf224 and orf252) encode 224 and 252 amino acid
proteins, respectively. GAP analysis showed that orf338 is 28.5%
identical (37.7% similar) to orfT of C. tepidum (GenBank Accession
number U58313). BLASTP of ORF224 identified mannose- 1-phosphate
guanylyltransferase (MPG; EC 2.7.7.13) from eukaryotes,
archeabacteria, cyanobacteria, and mycobacteria, but not
proteobacteria, close relatives of P. aeruginosa. It is not clear
if ORF224 is a functional MPG since all known MPGs consist of
359-388 amino acid residues, whereas OFR224 consists of only 224
amino acid residues. ORF252 is homologous to E. coli DjlA.sub.Ec.
DjlA.sub.Ec is thought to play a role in the correct assembly,
activity and/or maintenance of a number of membrane proteins,
including the two-component histidine kinase signal-transduction
systems.
[0132] To test if orf338 is the gene responsible for reduced
pathogenicity in worms, we compared the killing kinetics of a
strain carrying orf338 alone, 25F1 (pORF338), to wild type PA14 and
25F1 carrying vector alone. The 25Fl(pORF338) was constructed as
follows.
[0133] A 1.8 kb PCR-fragment containing 482 bp upstream promoter
sequence, the entire orf338 and a truncated orf224 was amplified
(Expand.TM. High Fidelity System, Boehringer Mannheim) from PA14
genomic DNA using primers F2327 (5'-CGAGGAATCCAGTCGAGGTG-3'; SEQ ID
NO:179) and R4180 (5'-GCAAGATGCAGCCGAGAGTAG-3'; SEQ ID NO:180). The
product was cloned into vector pCR2.1 (TA Cloning, Invitrogen) to
construct plasmid pMT403C-R. The SacI/XbaI fragment from pMT403C-R,
which contained the PCR product, was cloned into the SacI/XbaI of
pUCP18 to construct pORF338, placing orf338 under the control of
its native promoter. 25F1 were transformed with pORF338 to make
strain 25F1(pORF338).
[0134] In addition, a strain which contained the entire operon
(orf338, orf224, and djlA.sub.Pa) was constructed as follows. A PCR
strategy was used to amplify a 3.6 kb genomic fragment containing
orf338, orf224, and djlA.sub.Pa and their upstream transcriptional
sequences using primers RIF3115
(5'-GTCAGAATTCTCAGCTTGACGTTGTTGCCC-3'; SEQ ID NO:181) and RIR6757
(5'-GTCAGAATTCGACTTCTATTACCGCGACGCC-3'; SEQ ID NO:182). EcoRI sites
(underlined) are present in the primers, but absent in the genomic
sequence. Both strands of the PCR product were sequenced to
determine the sequence of orf338, orf224, and djlA.sub.Pa in strain
PA14. The PCR EcoRI digestion product was cloned into the EcoRI
site of pUCP18, and the orientation of insertion determined by
restriction digest. Plasmid p3-ORFs, where orf338, orf224, and
djlA.sub.Pa are under the control by its native promoter was then
used to transform 25F1 to make strain 25F1 (p3-ORFs).
[0135] As is shown in FIG. 34C, strain 25F1 (pORF338) failed to
complement fully the slow-killing phenotype. Strain 25F1 (p3-ORFs),
which contained the entire operon (orf338, orf224, and
djlA.sub.Pa), also showed only partial complementation of the
mutant phenotype. This result indicated that the TnphoA is
responsible for the pathogenicity phenotype; partial
complementation may be a consequence of gene dosage. The higher
mortality achieved by strain 25F1 (p3-ORFs) compared to strain 25F1
(pORF338) further suggested that the downstream genes, ORF224
and/or DjlA.sub.Pa may also play a role in PA14 virulence.
[0136] FIG. 24E shows the nucleotide sequence (SEQ ID NO:173) of
PA14 25F1 encoding ORFT (SEQ ID NO:174), ORFU (SEQ ID NO:175), and
DjlA.sub.Pa (SEQ ID NO:176).
[0137] Mutant 50E12. The TnphoA insertion in 50E12 was inserted
within codon 39 of a predicted 759 amino acid protein that is 43%
identical (54% similar) to the PtsP.sub.Ec protein of E. coli.
Based on sequence analysis, ptsP.sub.Ec is predicted to encode
Enzyme INtr, a 738 amino acid protein which contains an N-terminal
Nif-A domain and a C-terminal Enzyme I domain; the latter functions
in the phosphoenolpyruvate-dependen- t phosphotransferase system.
It is thought the Nif-A domain serves a signal transduction
function, either directly sensing small molecule signals or
receiving signals from a NifL-like protein. Either mechanism may
modulate the catalytic activity of the Enzyme I domain; which in
turn is suggested to phosphorylate NPr (nitrogen-related HPr) and
thereby regulate transcription of RpoN-dependent operons.
Immediately upstream of the PA14 ptSP.sub.Pa homologue is open
reading frame (orf159) predicted to encode a 159 amino acid protein
that appears to be co-transcribed with ptsPPa. FIG. 24C shows the
nucleotide sequence (SEQ ID NO:168) of PA14 50E12 encoding
YgdP.sub.Pa (SEQ ID NO:169) and Pts.sub.Pa (SEQ ID NO:170). ORF159
is 62.3-64.8% identical to YgdP proteins of unknown function found
in H. influenzae (GenBank Accession number Q57045) and E. coli
(GenBank Accession number Q46930). These proteins are closely
related to invasion protein A in Helicobacter pylori and Bartonella
bacilliformis. B. bacilliformis invasion protein A (SwissProt
Accession number P35640) is encoded by ailA, which when present
together with an adjacent but independently transcribed gene, ailB,
confers on E. coli the ability to invade human erythrocytes .
[0138] For the complementation of 50E12, two strains were tested:
50E12(pMT206-lac) and 50E12(pMT206-nat). Strain 50E12(pMT206-lac)
carried plasmid pMT206-lac, where the transcription of orf159 and
ptsPPa is under the control of the constitutive lacZ promoter. For
strain 50E12(pMT206-nat), the transcription of orf159 and
ptSP.sub.Pa is controlled only by their native promoter. Each of
these strains were constructed as follows.
[0139] A 4.3 kb PCR fragment, containing the EcoRI site at both
ends was amplified from genomic DNA of P. aeruginosa PA14 using
these primers: RIF1698 (5'-GTCAGAATTCGATGTTCCAGTCCCAGATCCC-3'; SEQ
ID NO:183) and RIR6002 (5'-GTCAGAATTCCAGTAGACCACCGCCGAGAG-3': SEQ
ID NO:184). This fragment was cloned into the EcoRI site of pUCP 18
to make pMT206-lac and pMT206-nat; their identity confirmed by
restriction digest. In pMT206-lac, the transcription of orf159 and
ptsPPa is under the control of both the constitutive lacZ promoter
and their native promoter. Only their native promoter controls the
transcription of orf159 and ptsPPa in pMT206-nat.
[0140] As is shown in FIG. 34D, both strains partially complemented
the mutant phenotype, with the time required by these complemented
strains to kill 100% of the worms being longer than the wild-type
strain. Partial complementation was observed in the burned-mouse
assay: Mortality of mice after infection by 5.times.10.sup.5
bacteria from strain 50E12(pMT206-nat) was 39%, compared to 100%
and 0% mortality when infected by the wild-type strain and 50E12,
respectively. These results indicated that the putative
orf159-ptsP.sub.Pa operon is involved in P. aeruginosa pathogenesis
in nematode and mice.
[0141] Mutant 35A9. The TnphoA insertion in 35A9 is located in a
putative 210 amino acid protein (encoded by orf210) that is most
closely related (31.5% identity) to the N. gonorrhoeae MtrRNg
protein, which belongs to the TetR family of helix-turn-helix
containing bacterial transcription regulation proteins. ORF210 is
adjacent to, and divergently transcribed from, three genes that are
homologous to components of the energy dependent efflux (EDE)
system in P. aeruginosa. Analyses of sequences from PA01 showed
that together, these four genes defined a novel energy dependent
efflux (EDE) system in P. aeruginosa. The other EDE systems in P.
aeruginosa described previously are the mexR, mexA-mexB-oprK
system, the nfxB, mexC-mexD-oprJ system and the nfxC,
mexE-mexF-oprN system. FIG. 24D shows the nucleotide sequence (SEQ
ID NO:171) of PA14 35A9 encoding mtrR.sub.Pa (SEQ ID NO:172).
[0142] Mutants 37H7 and 1D7. Analysis of the IPCR product from
mutant 37H7 showed that there is a TnphoA insertion within codon
188 of the 214 amino acid GacA protein. DNA blot analysis showed
that 1D7 also contained an insertion in the gacA gene.
[0143] Mutant 48D9. TnphoA is inserted between codon 491 and 492 of
the 925 amino acid LemA-homologue, a sensor kinase belonging to a
family of bacterial two-component regulators . The cognate response
regulator of LemA in P. syringae is GacA and GacA+LemA have been
shown to affect the expression of a variety number of virulence
factors .
[0144] Mutant 41C1. TnphoA is inserted in the AefA-homologue of the
putative E. coli integral membrane protein (SwissProt P77338) in
mutant 41C1. It is a member of the 30-40 kD UPF0003 protein family
(PROSITE PDOC00959). In addition to E. coli, it is also present
Synechocystis strain PCC 6803 and Methanococcus jannaschii.
[0145] In addition, strains pho34B12, 3E8, 8C12, 1G2, 35A9, and
23A2, were also found to have a phenazine-minus mutant phenotype.
Moreover, pho34B12, 3E8, 8C12, and 1G2 mutants were found to be
reduced in pigment production. An additional mutant, 6A6, was also
identified having reduced pigment. The characteristic color of P.
aeruginosa strains has been attributed to a group of tricyclic
secondary metabolites collectively known as phenazines, the most
extensively characterized of which is the blue-green pigment,
pyocyanin (1-hydroxy-5-methyl phenazine). In order to test whether
the reduction of pigmentation in the bacterial mutants was at least
in part due to the reduction in pyocyanin, levels of this pigment
were quantified in wild type PA14 as well as in all the mutants
obtained using the fast-killing assay. The results of this analysis
showed that the pho34B12, 3E8, 8C12, 1G2, and 6A6 mutants that had
a reduced pigment phenotype were also reduced in pyocyanin
production, with levels ranging from 10 to 50% of the wild type
strain. The other mutants, 13C9, 23A2, and 36A4 had levels of
pyocyanin comparable with the wild type strain.
[0146] In addition, the sequence interrupted by the TnphoA mutation
in 3E8 was found to predict a protein with homology to the phzB
gene from Pseudomonas fluorescens, that is part of an operon
involved in the production of the secondary metabolite, phenazine
(GenBank Accession number: L48616). The phzB gene also has a
homolgue in Psuedomonas aureofaciens, referred to as phzY. (GenBank
Accession number AF007801). Using the sequence tag, a cosmid
(1G2503), containing this region in the Pseudomonas aeruginosa
database was identified, that contains both the phzA and phzB
genes, as well as other genes that are thought to play a role in
phenazine biosynthesis, the pcnC and D genes (GenBank Accession
number AF005404). Four of these strains, 34B12, 3E8, 23A12, and
35A9, were examined for pathogenicity in the mouse-burn assay.
Surprisingly, these experiments showed that the phenazine defective
strains have reduced pathogenesis, indicating that the genes
interrupted by the TnphoA insertions are mammalian virulence
factors. The nucleotide and deduced amino acid sequences, including
sequence tags, for these strains are shown in FIGS. 7-9, 13, 14A,
14B, 15, 16A, 16B, 17, 18A, 18B, 18C, 18D, 18E, 22, 24A, 24B, 24C,
24D, 24E, addition, FIGS. 25 and 26 show the nucleotide sequence of
the phnA and phnB genes of Pseudomonas aeruginosa and the deduced
amino acid sequence of PHNA, respectively.
[0147] A detailed description of the DNA sequence and biochemical
analyses of each of the mutants identified using the fast-killing
assay (described above) is now presented in the following
sections.
[0148] Mutants 36A4, 23A2, and 13C9. The DNA sequence tags obtained
from all three of the mutants that produced wild type levels of
pyocyanin, had homologies to known genes in Pseudomonads. Mutant
36A4 contained TnphoA inserted into a gene homologous to hrpM,
previously identified as a locus controlling pathogenicity in the
plant pathogen Pseudomonas syringae (Mills and Mukhopadhyay, In:
Pseudomonas: biotransformations, pathogenesis, and evolving
technology, S. Silver, A. M. Chakrabarty, B. Iglewiski, and S.
Kaplan, eds., American Society for Microbiology, 1990, pp. 47-57,
Mukhopadhyay et al., J. Bacteriol. 170:5479-5488, 1988); GenBank
Accession number 140793). This locus also has homology to the E.
coli mdoH gene, which encodes an enzyme involved in the
biosynthesis of periplasmic glucans (Loubens et al., Mol.
Microbiol. 10:329-340, 1993; GenBank Accession number X64197). The
TnphoA insertion in mutant 23A2 was inserted into a gene previously
identified in P. aeruginosa strain PAO1 as mexA (Poole et al., Mol.
Microbiol. 10:529-544, 1993; GenBank Accession number L11616). The
product of mexA, predicted to be a cytoplasmic-membrane-associated
lipoprotein, likely functions together with the products of the
other two genes contained in the same operon, mexB and oprM, as a
non-ATPase efflux pump with broad substrate specificity (Li et al.,
Antimicrob. Agents. Chemother. 39:1948-1953, 1995). Sequence
analysis of the DNA flanking the third mutant that was wild type
for pigment production, 13C9, showed that it corresponded to
another previously known gene in P. aeruginosa strain PAO1, orp
(GenBank Accession number U54794). Orp, or osmoprotectant-dependent
regulator of phospholipase C, was identified as a factor
controlling the expression of the pathogenicity factor PlcH, one of
the two isoforms of phosholipase C produced by P. aeruginosa (Sage
et al.,Mol. Microbiol. 23: 43-56, 1997).
[0149] Mutants 1G2 and 8C12. Molecular analysis of two of the
non-pigmented mutants 1G2 and 8C12 showed that they contained
insertions into novel genes, although DNA flanking the 1G2
insertion contained a motif characteristic of histidine sensor
kinases. This gene was not present in the PAO1 genome database.
Although the 8C12 sequence tag identified a homologous gene in the
PAO1 database, no significant motifs were found within this
gene.
[0150] Mutants 3E8 and 6A6. Two mutants, 3E8 and 6A6, contained
TnphoA insertions into the same gene, which was homologous to the
previously identified phzB gene in P. fluorescens strain 2-79
(GenBank Accession number AF007801) and phzY in P. aureofaciens,
strain 30-84 (GenBank Accession number L48616). These two mutants
contained the TnphoA insertion in exactly the same position,
however, they were independent isolates since they were obtained
from two different mutant libraries. Although phzB and phzY
contained no identifiable sequence motifs, they were present in
operons known to regulate production of phenazine-1-carboxylate
(PCA) in both P. fluorescens and P. aureofaciens (Mavrodi et al.,
J. Bacteriol. 180:2541-2548, 1998).
[0151] Mutant pho34A12. DNA flanking the TnphoA insertion in the
final non-pigmented mutant pho34B12, was previously cloned and
shown to be a novel locus as described infra. Interestingly, this
insertion is immediately downstream of the phenazine biosynthetic
genes, phnA and phnB, as identified in P. aeruginosa strain PAO1
(Essar et al., J. Bacteriol. 172:884-900, 1990).
[0152] Phenazines are Required for Fast Killing of C. elegans
[0153] The isolation of both pigmented and non-pigmented mutants in
the fast-killing screen indicated that the fast-killing process
involved more than one factor. However, the molecular analysis of
the 3E8 and 6A6 mutants (containing insertions in an operon known
to regulate phenazine production) strongly suggested that
phenazines represented one class of toxin that mediate fast
killing. In order to directly test this hypothesis, an additional
mutation, .DELTA.phnA phnB, was generated and studied as
follows.
[0154] The phenazine biosynthetic genes phnA and phnB (Essar et
al., J. Bacteriol. 172:884-900, 1990) genes lie upstream of the
previously characterized pho34B12 TnphoA insertion in PA14; GenBank
Accession number AF031571). A 3.7 kb EcoRI fragment corresponding
to the wild type sequence of this region (from the plasmid pLGR34)
was subcloned into pBluescript SK/+to yield Bs34B12. This plasmid
contained 944 bp of phnA (full length of 1591 bp), the entire phnB
(600 bp) gene and 1.7 kb of downstream sequences. The missing 605
bp of phnA and 405 bp upstream were amplified using PCR from
genomic PA14 DNA with the oligonucleotide primers PHNA3
(5'-GGTCTAGACGAACTGAGCGAGGAG-3'; SEQ ID NO:185) and PHNA2
(5'-GCCTGCAGGCGTTCTACCTG-3'; SEQ ID NO:186). The primers were based
on the sequence of the previously cloned phnA and phnB genes from
P. aeruginosa strain PAOI (Essar et al., J. Bacteriol. 172:884-900,
1990, GenBank Accession number M33811). The 1010 bp amplified
sequence was subcloned into the PstI sites of pBs34B12 to give the
construct, pBs34B12phnA. An in-frame deletion within phnA, phnB was
generated by replacing 2.6 kb of the wild type sequence of the
genes with a 1 kb fragment (FIG. 35) amplified by PCR using the
primers PHNDEL1 (5'-GGCTGCAGTGATTGACTGAGCGTCTGCTGGAGAACG-3'; SEQ ID
NO:187) and PHNDEL2 (5'-GGGAAGCTTCGTCTAGAATCACGTGAACATGTTCC-3': SEQ
ID NO:188) to yield the plasmid pBs34b12phndel. A 1.8 kb XbaI
fragment containing the phnAphnB in-frame deletion was subcloned
into the positive-sucrose-selection suicide vector pCVD442
(Donnenberg and Kaper, Infect. Immun. 59:4310-4317, 1991). The
resulting construct, pCVD34B12phndel, was used to introduce the
disrupted phnA, phnB genes into the wild-type PA14 genome by
homologous recombination resulting in the mutant PA14
.DELTA.phnAphnB. DNA restriction and DNA blot analyses using DNA
from the parental PA14 and derivative PA14 .DELTA.phnAphnB strains
were undertaken in order to verify that the mutant contained the
desired deletion.
[0155] Although little is known about the nature of the enzymes
that catalyze the formation of phenazines in P. aeruginosa and
related Pseudomonads, the conversion of chorismate to anthranilate
is thought to be a key step in the pathway (FIG. 35A). In P.
aeruginosa strain PAO1, this step is most-likely catalyzed by the
anthranilate synthase encoded by the phnA and phnB genes, since
mutations in these genes result in decreased production of the
phenazine pyocyanin (Essar et al., J. Bacteriol. 172:884-900,
1990). The phnA and phnB genes were cloned from PA14 and a
.DELTA.phnAphnB mutant containing a 1602 bp deletion in these genes
was generated (FIG. 35B). Importantly, this mutation was designed
to be non-polar and therefore did not affect the two ORFs shown to
be directly downstream of phnA and phnB (infra). Measurement of
pyocyanin in the .DELTA.phnAphnB mutant showed that it generated
only 10% of wild type levels, confirming that phnA and phnB are
involved in pyocyanin production in strain PA14 just as in PAO1.
Assays conducted using .DELTA.phnAphnB revealed that this strain
was severely reduced in fast killing. As seen in FIG. 35C, less
than 5% of the worms were dead three hours after exposure to
.DELTA.phnAphnB in contrast to almost 100% that were exposed to the
wild type strain. The .DELTA.phnAphnB strain behaved in a manner
similar to the other phenazine mutant, 3E8, which served as the
control for an attenuated mutant in this experiment. These results
demonstrated that phenazines are required for the fast killing of
C. elegans.
[0156] To discover whether the bacterial factors that mediated fast
killing are relevant to pathogenesis in other hosts, the
fast-killing mutants were tested for virulence in the Arabidopsis
leaf infiltration model as well as the mouse full thickness skin
bum model (infra). Five fast-killing mutants were tested for growth
over the course of four days in Arabidopsis leaves as a
quantitative measure of their pathogenicity and also in the mouse
full thickness skin bum model. As shown in Tables 4 and 5, the
maximal level of growth in Arabidopsis leaves on the fourth day
postinfection was significantly lower for 2 of the phenazine
mutants, 3E8 and 8C12. In the mouse model these two mutants caused
significantly less mortality than the wild type strain with a
P<0.05 when an inoculum of 5.times.10.sup.5 cells was used. The
third phenazine mutant 1G2, was not significantly different from
the wild type strain in either the plant or the mouse models.
[0157] Both the hrpM mutant, 36A4, and the mexA mutant, 23A2, were
severely debilitated in growth in Arabidopsis leaves, indicating a
strong pathogenicity defect in this model. In the mouse model,
mutant 36A4, had a dramatic effect causing no mortality at the dose
tested. In contrast, the mexA mutant, 23A2 was only marginally
affected. These results demonstrated that the fast killing screen
is extremely effective at identifying genes required for
pathogenesis in both plants and mice, and further, provide the
first in vivo demonstration that phenazines are required for
pathogenesis in these two hosts.
[0158] We also note that we have identified a regulator, phzR, of
the phz operon. FIG. 18E shows the nucleotide sequence (SEQ ID
NO:164) and predicted partial amino acid sequence (SEQ ID NO:165)
of PA14 phzR.
Phenazines and Pathogenesis
[0159] PA14 mutants reduced in fast killing also affected pigment
synthesis. Our molecular analysis revealed that the association
between pigment production and pathogenesis was not simply due to
the coordinate regulation of pigmentation and toxin production by
regulatory factors. Instead we found that mutations in phenazine
biosynthetic genes were reduced in virulence, strongly implicating
phenazines as toxins in the fast-killing process. Phenazines,
tri-cyclic pigmented compounds that give Pseudomonads their
characteristic colors (Turner and Messenger, Adv. Microb. Physiol.
27:211-273, 1986), are secondary metabolites thought to increase
the survival of organisms under competitive conditions (Maplestone
et al., Gene 115:151-157, 1992). Although the repertoire of
phenazines produced by PA14 is unknown, P. aeruginosa strain PAO1
produces at least six different phenazines, including the well
characterized blue-green pigment pyocyanin. Phenazines including
pyocyanin, have been demonstrated to have antibiotic action against
several species of bacteria, fungi, and protozoa, a quality
attributed to their redox active. In their highly-reactive reduced
state, phenazines have been described to undergo redox cycling in
the the presence of various reducing agents or molecular oxygen
resulting in the formation of superoxide and hydrogen peroxide
(Hassan and Fridovich, J. Bacteriol. 141:1556-163, 1980). In vitro,
these moderately cytotoxic oxygen radicals can be converted by an
iron catalyst to the highly cytotoxic hydroxyl radical (Britigan et
al., J. Clin. Invest. 90:2187-2196, 1992). Formation of reactive
oxygen species by phenazines is also thought to contribute to their
cytotoxic effects observed on eukaryotic cells in vitro. These
effects include the inhibition of mammalian cell respiration, the
disruption of ciliary beating, and immunomodulatory effects such as
stimulation of the inflammatory response, inhibition of lymphocyte
proliferation and alteration of the T lymphocyte response to
antigens.
[0160] The biosynthetic pathways leading to the production of
phenazines in P. aeruginosa have been poorly defined making it
difficult to identify the steps in the pathway blocked by the PA14
mutants defective in phenazine production. However, the transposon
insertion in two mutants, 3E8 and 6A6, disrupted a gene with
homology to phzB, which was previously characterized as being
involved in phenazine production in the related Pseudomonads, P.
fluorescens, and P. aureofaciens. In P. fluorescens, phzB was shown
to be part of a seven gene operon (phzA-G) involved in the
production of phenazine-1-carboxylic acid. Comparison of this
operon in P. flourescens and P. aureofaciens showed that the two
were highly homologous, suggesting that pathways leading to
phenazine production are conserved in fluorescent Pseudomonads
(Mavrodi et al., J. Bacteriol. 180:2541-2548, 1998). Although the
DNA flanking the phza and phzB genes has only been partially
sequenced in P. aeruginosa strain PA14, our analysis suggests that
the region shares a conserved structure with the P. fluorescens
phzA-F operon. The predicted translated products of the phza and
phzb genes from PA14 and P. fluorescens share 68 and 74% identity,
respectively. In addition, a region containing phzA-F-like genes is
present in P. aeruginosa strain PAO1, and the predicted translated
products of these genes exhibited between 69 to 85% identity with
their P. fluorescens homologs (GenBank Accession number AF005404).
Extrapolating from the role of the phz operon in P. fluorescens and
P. aureofaciens, the isolation of PA14 phzB mutants that are
defective in fast killing strongly suggested that phenazines are
involved in this process. The hypothesis that phenazines, including
pyocyanin, are one of the mediators of fast killing was further
tested by the non-polar disruption of the genes, phnA and phnB,
which encode the two subunits of an anthranilate synthase,
previously shown to be specifically involved in phenazine synthesis
in P. aeruginosa strain PAO1 (Essar et al., J. Bacteriol.
172:884-990, 1990). Consistent with a role in phenazine
biosynthesis, deletion of the phnA and phnB genes in PA14 severely
reduced pyocyanin production. Furthermore, the .DELTA.phnAphnB
mutant was defective in fast killing, demonstrating the critical
role of phenazines in this process.
[0161] The role of phenazines in pathogenesis was also examined in
Arabidopsis and mice. The two independent mutants containing
insertions within the phzB gene, 3E8, and 6A6, were dramatically
reduced in pathogenicity in both the Arabidopsis leaf infiltration
model as well as the mouse full thickness skin bum model (Tables 4
and 5), suggesting that phenazines are multi-host pathogencity
factors. It is interesting to note that many of the other mult-host
pathogenicity factors identified in this and our previous studies
are likely to be involved in the production of several other
virulence factors and are not effectors, or molecules that directly
interact with the host (described infra). Thus, phenazines
represent the only known class of multi-host pathogenicity
effectors that we have identified. These findings are also
significant since despite intensive in vitro analyses of
phenazines, the physiological significance of their production and
their role in P. aeruginosa infections remains controversial, and
prior to this study there has been no demonstration of their role
in vivo.
Fast Killing is Multifactorial
[0162] Analysis of fast-killing mutants that generated wild-type
levels of pigments showed that although phenazines were essential
mediators of fast killing, other factors were involved in this
process. Molecular analysis of one such mutant, 23A2, revealed that
the transposon was inserted into a gene previously identified in P.
aeruginosa strain PAO1 as MexA, which is part of the 3 gene operon
MexA, B, OprM (Poole et al., Mol. Microbiol. 10:529-544, 1993). The
products of these genes are localized to the cytoplasmic (MexA,
MexB) and outer membranes (OprM) where they are proposed to
function as a non-ATPase broad-specificity efflux pump (Li et al.,
Antimicrob. Agents Chemother. 39:1948-1953,1995). Originally
identified due to its contribution to the process of multi-drug
resistance in P. aeruginosa, this pump is thought to play a general
role in the export of secondary metabolites, although its natural
substrates remain unknown (Poole, Antimicrob. Agents Chemother.
34:453-456, 1994). The defect of mexA mutant in fast killing, a
process mediated by diffusible toxins, is most-likely due to the
lack of export of one or more factors involved in this process.
Since the mexA mutant was pigmented, phenazines are not likely to
be a substrate for the pump. In addition to its defect in fast
killing, the mexA mutant was marginally reduced in pathogenicity in
the mouse model and severely debilitated in the Arabidopsis leaf
infiltration model. Although the lack of export of specific
virulence factors could explain these defects, an additional model
is that the mexA mutant bacteria are unable to protect themselves
against host defense factors generated in response to the bacterial
infection. Such a protective function has been demonstrated for the
sap genes, which encode proteins related to ATP binding cassette
(ABC) transporters and mediate resistance to host antimicrobial
peptides in the human pathogen, Salmonella typhimurium, as well as
in the phytopathogen, Erwinia chrysanthemi (Taylor, Plant Cell
10:873-875, 1998).
[0163] A second mutant identified in the screen, 36A4, contained a
transposon insertion into a gene with homology to E. coli MdoH,
which is part of the mdoGH operon. In E. coli, the products of this
operon are involved in the synthesis of membrane-derived
oligosaccharides (MDO) or linear, periplasmic glucans (Loubens et
al., Mol. Microbiol. 10:329-340, 1993). A similar locus, termed
hrpM is present in the plant pathogen Pseudomonas syringae pv.
syringae (Mukhopadhyay et al., J. Bacteriol. 170:5479-5488, 1988),
originally identified since mutations within this locus abolish
both the development of disease symptoms on host plants as well as
the hypersensitive response in non-host plants (Anderson and Mills,
Phytopath.75:104-108, 1985). Periplasmic glucans have also been
found in a wide range of gram-negative bacteria, where diverse,
albeit poorly understood functions have been assigned to them. In
addition to being essential virulence factors in P. syringae, other
functions include the adaptation to hypoosmotic environments, and
cell signaling leading to the recognition of eukaryotic hosts by
species of Rhizobium and Agrobacterium (Kennedy, In: Escherichia
and Salmonella, F. C. Neidardt, ed, American Society for
Microbiology Press, Washington, D.C., pp. 1064-1071, 1996).
However, despite being present in the periplasm of several animal
pathogens such as Salmonella and Klebsiella, until this study,
which shows that P. aeruginosa carrying a mutation in an mdoH-like
locus is severely reduced in pathogenicity in a mouse model,
periplasmic glucans have not been shown to play a role in the
infection of animal hosts.
8TABLE 4 Summary for Pathogenicity of P. aeruginosa strain
UCBPP-PA14 mutants on various hosts Pathogenicity Phenotypes Growth
in % Mouse Strain Isolation Arabidopsis Ability to kill C.
Mortality Number Strain Name Leaf.sup.b elegans.sup.c 5 .times.
10.sup.5d Gene Identity PA14 PA14 5.5 .times. 10.sup.7 + 100 rep
(reduced pathogenicity in plants) 16G12 rep1 2.3 .times. 10.sup.5 +
100 no matches 49H2 rep2 1.2 .times. 10.sup.6 + 63 not sequenced
25A12 rep3 1.7 .times. 10.sup.6 + 75 no matches 33A9 rep4 5.1
.times. 10.sup.6 + 0 no matches 33C7 rep5 8.4 .times. 10.sup.5 + 0
no matches ren (reduced pathogenicity in nematodes) 35A9.sup.g ren1
5.7 .times. 10.sup.7 - 55 mtrR 44B1 ren2 5.4 .times. 10.sup.7 - 56
no matches 1G2.sup.f,g,h NT - NT no matches 8C12.sup.f,g,h NT - NT
no matches 2A8.sup.f,h NT - NT no matches rpn (reduced
pathogenicity in plants and nematodes) 25F1 rpn1 1.5 .times.
10.sup.4 - 20 orfT 35H7.sup.e rpn2 1.2 .times. 10.sup.4 - .sup.
NT.sup.e gacA 41A5 rpn3 1.3 .times. 10.sup.4 - 100 no matches 41C1
rpn4 2.4 .times. 10.sup.5 - 85 aefA 50E12 rpn5 2.0 .times. 10.sup.5
- 0 ptsP pho15 rpn6 3.9 .times. 10.sup.4 - 62 dsbA 12A1 rpn7 1.7
.times. 10.sup.6 - 50 lasR pho23 rpn8 6.4 .times. 10.sup.4 - 5 no
matches 34B12.sup.g,h rpn11 4.0 .times. 10.sup.4 - 50 dst* of phnB
34H4 rpn12 3.8 .times. 10.sup.6 - 50 no matches 25F1 rpn1 1.5
.times. 10.sup.4 - 20 orfT 3E8.sup.g,h rpn13 1 .times. 10.sup.6 -
12.5 phzB 23A2.sup.h rpn14 1.7 .times. 10.sup.5 - 71 mexA
36A4.sup.h rpn15 4 .times. 10.sup.4 - 0 hrpN .sup.bCFU/cm.sup.2
leaf area of bacterial counts at four days after inoculation of
10.sup.3 bacteria: means of four to five samples. Mutants are
defined as less pathogenic when the means of four to five samples.
Mutants are defined as less pathogenic when the mean CFU/cm.sup.2
leaf area of bacterial counts is 2 standard deviation lower
relative to wild-type within the same set of experiments. .sup.cA
mutant is considered attenuated in nematode pathogenicity (-) if
the mean time required to kill 50% of the worms feeding on it
(LT.sub.50 from 3 replicates) is two standard deviations less than
the LT.sub.50 of parental UCBPP-PA14 in the same experiment; for
calculations of LT.sub.50 see Materials and Methods. .sup.dSix-week
old male ARK/J inbred strain mice (from Jackson Laboratories),
weighing between 20 to 30 gm were injected with 5 .times. 10.sup.5
cells as described by Stevens et al., J. of Burn Care and Rehabil.
15: 232-235, 1994. The number of animals that died of sepsis was
monitored each day for ten days. .sup.eTwo other independently
isolated gacA mutants are ID7(rpn9) and 33D11(rpn10). Mutant rpn9
has been tested on mice and showed 50% mortality .sup.ftested only
in nematodes .sup.gphenazine-defective mutants .sup.hmutants
defective in fast killing, not affected in slow killing dst*=
downstream
[0164]
9TABLE 5 Pathogenicity of PA 14 Fast Killing Mutants in Plants and
Mice % Mouse Growth in Arabidopsis Mortality (n) Strain
leaves.sup.a 5 .times. 10.sup.5b Gene Identity PA14 7 .times.
10.sup.8 100 (>16) 1G2 3 .times. 10.sup.7 100 (8) no matches,
contains histidine kinase motif 3E8.sup.c, 6A6 3 .times. 10.sup.5
18 (16) phzB 8C12 5 .times. 10.sup.5 63 (8) no matches 23A2 1.2
.times. 10.sup.4 85 (16) mexA 36A4 2 .times. 10.sup.4 0 (16) hrpM
.sup.aCFU/cm.sup.2 leaf area of bacterial counts at five days
post-inoculation with 10.sup.3 bacteria. Values represent means of
four to five samples. Mutants are defined as less pathogenic when
the mean value of bacterial counts is two standard deviations lower
than the wild type within the same experimental set. .sup.bSix-week
old male AKR/J inbred mice (from Jackson laboratories), weighing
between 20 to 30 gm were injected with 5 .times. 10.sup.5 bacterial
cells. (n) is the total number of mice injected. The number of mice
that died of sepsis was monitored daily for seven days. .sup.c3E8
and 6A6 are independently generated mutants that contain TnphoA
inserted in exactly the same location. The numbers reported are
those obtained using 3E8. Similar results were obtained with 6A6
(data not shown).
Isolation of Additional Virulence Genes
[0165] Based on the nucleotide and amino acid sequences described
herein, the isolation of additional coding sequences of virulence
factors is made possible using standard strategies and techniques
that are well known in the art. Any pathogenic cell can serve as
the nucleic acid source for the molecular cloning of such a
virulence gene, and these sequences are identified as ones encoding
a protein exhibiting pathogenicity-associated structures,
properties, or activities.
[0166] In one particular example of such an isolation technique,
any one of the nucleotide sequences described herein may be used,
together with conventional screening methods of nucleic acid
hybridization screening. Such hybridization techniques and
screening procedures are well known to those skilled in the art and
are described, for example, in Benton and Davis (Science 196:180,
1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961,
1975); Ausubel et al. (Current Protocols in Molecular Biology,
Wiley Interscience, New York, 1997); Berger and Kimmel (supra); and
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, New York. In one particular
example, all or part of the 33A9 sequence (described herein) may be
used as a probe to screen a recombinant plant DNA library for genes
having sequence identity to the 33A9 gene (FIGS. 5 and 6A-B).
Hybridizing sequences are detected by plaque or colony
hybridization according to standard methods.
[0167] Alternatively, using all or a portion of the amino acid
sequence of the 33A9 polypeptide, one may readily design
33A9-specific oligonucleotide probes, including degenerate
oligonucleotide probes (i.e., a mixture of all possible coding
sequences for a given amino acid sequence). These oligonucleotides
may be based upon the sequence of either DNA strand and any
appropriate portion of the 33A9 sequence (FIGS. 5 and 6A-B; SEQ ID
NOs:102 and 103, respectively) of the 33A9 protein. General methods
for designing and preparing such probes are provided, for example,
in Ausubel et al. (supra), and Berger and Kimmel, Guide to
Molecular Cloning Techniques, 1987, Academic Press, New York. These
oligonucleotides are useful for 33A9 gene isolation, either through
their use as probes capable of hybridizing to 33A9 complementary
sequences or as primers for various amplification techniques, for
example, polymerase chain reaction (PCR) cloning strategies. If
desired, a combination of different, detectably-labelled
oligonucleotide probes may be used for the screening of a
recombinant DNA library. Such libraries are prepared according to
methods well known in the art, for example, as described in Ausubel
et al. (supra), or they may be obtained from commercial
sources.
[0168] As discussed above, sequence-specific oligonucleotides may
also be used as primers in amplification cloning strategies, for
example, using PCR. PCR methods are well known in the art and are
described, for example, in PCR Technology, Erlich, ed., Stockton
Press, London, 1989; PCR Protocols: A Guide to Methods and
Applications, Innis et al., eds., Academic Press, Inc., New York,
1990; and Ausubel et al. (supra). Primers are optionally designed
to allow cloning of the amplified product into a suitable vector,
for example, by including appropriate restriction sites at the 5'
and 3' ends of the amplified fragment (as described herein). If
desired, nucleotide sequences may be isolated using the PCR "RACE"
technique, or Rapid Amplification of cDNA Ends (see, e.g., Innis et
al. (supra)). By this method, oligonucleotide primers based on a
desired sequence are oriented in the 3' and 5' directions and are
used to generate overlapping PCR fragments. These overlapping 3'-
and 5'-end RACE products are combined to produce an intact
full-length cDNA. This method is described in Innis et al. (supra);
and Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998, 1988.
[0169] Partial virulence sequences, e.g., sequence tags, are also
useful as hybridization probes for identifying full-length
sequences, as well as for screening databases for identifying
previously unidentified related virulence genes. For example, the
sequences of 36A4, 25A12, and 33C7 were expanded to those
encompassed by contigs 2507, 1126, and 1344, respectively.
[0170] Confirmation of a sequence's relatedness to a pathogenicity
polypeptide may be accomplished by a variety of conventional
methods including, but not limited to, functional complementation
assays and sequence comparison of the gene and its expressed
product. In addition, the activity of the gene product may be
evaluated according to any of the techniques described herein, for
example, the functional or immunological properties of its encoded
product.
[0171] Once an appropriate sequence is identified, it is cloned
according to standard methods and may be used, for example, for
screening compounds that reduce the virulence of a pathogen.
Polypeptide Expression
[0172] In general, polypeptides of the invention may be produced by
transformation of a suitable host cell with all or part of a
polypeptide-encoding nucleic acid molecule or fragment thereof in a
suitable expression vehicle.
[0173] Those skilled in the field of molecular biology will
understand that any of a wide variety of expression systems may be
used to provide the recombinant protein. The precise host cell used
is not critical to the invention. A polypeptide of the invention
may be produced in a prokaryotic host (e.g., E. coli) or in a
eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells,
e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or
preferably COS cells). Such cells are available from a wide range
of sources (e.g., the American Type Culture Collection, Rockland,
Md.; also, see, e.g., Ausubel et al., supra). The method of
transformation or transfection and the choice of expression vehicle
will depend on the host system selected. Transformation and
transfection methods are described, e.g., in Ausubel et al.
(supra); expression vehicles may be chosen from those provided,
e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et
al., 1985, Supp. 1987).
[0174] One particular bacterial expression system for polypeptide
production is the E. coli pET expression system (Novagen, Inc.,
Madison, Wis.). According to this expression system, DNA encoding a
polypeptide is inserted into a pET vector in an orientation
designed to allow expression. Since the gene encoding such a
polypeptide is under the control of the T7 regulatory signals,
expression of the polypeptide is achieved by inducing the
expression of T7 RNA polymerase in the host cell. This is typically
achieved using host strains which express T7 RNA polymerase in
response to IPTG induction. Once produced, recombinant polypeptide
is then isolated according to standard methods known in the art,
for example, those described herein.
[0175] Another bacterial expression system for polypeptide
production is the pGEX expression system (Pharmacia). This system
employs a GST gene fusion system which is designed for high-level
expression of genes or gene fragments as fusion proteins with rapid
purification and recovery of functional gene products. The protein
of interest is fused to the carboxyl terminus of the glutathione
S-transferase protein from Schistosoma japonicum and is readily
purified from bacterial lysates by affinity chromatography using
Glutathione Sepharose 4B. Fusion proteins can be recovered under
mild conditions by elution with glutathione. Cleavage of the
glutathione S-transferase domain from the fusion protein is
facilitated by the presence of recognition sites for site-specific
proteases upstream of this domain. For example, proteins expressed
in pGEX-2T plasmids may be cleaved with thrombin; those expressed
in pGEX-3X may be cleaved with factor Xa.
[0176] Once the recombinant polypeptide of the invention is
expressed, it is isolated, e.g., using affinity chromatography. In
one example, an antibody (e.g., produced as described herein)
raised against a polypetide of the invention may be attached to a
column and used to isolate the recombinant polypeptide. Lysis and
fractionation of polypeptide-harboring cells prior to affinity
chromatography may be performed by standard methods (see, e.g.,
Ausubel et al., supra).
[0177] Once isolated, the recombinant protein can, if desired, be
further purified, e.g., by high performance liquid chromatography
(see, e.g., Fisher, Laboratory Techniques In Biochemistry And
Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
[0178] Polypeptides of the invention, particularly short peptide
fragments, can also be produced by chemical synthesis (e.g., by the
methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984
The Pierce Chemical Co., Rockford, Ill.).
[0179] These general techniques of polypeptide expression and
purification can also be used to produce and isolate useful peptide
fragments or analogs (described herein).
Antibodies
[0180] To generate antibodies, a coding sequence for a polypeptide
of the invention may be expressed as a C-terminal fusion with
glutathione S-transferase (GST) (Smith et al., Gene 67:31-40,
1988). The fusion protein is purified on glutathione-Sepharose
beads, eluted with glutathione, cleaved with thrombin (at the
engineered cleavage site), and purified to the degree necessary for
immunization of rabbits. Primary immunizations are carried out with
Freund's complete adjuvant and subsequent immunizations with
Freund's incomplete adjuvant. Antibody titres are monitored by
Western blot and immunoprecipitation analyses using the
thrombin-cleaved protein fragment of the GST fusion protein. Immune
sera are affinity purified using CNBr-Sepharose-coupled protein.
Antiserum specificity is determined using a panel of unrelated GST
proteins.
[0181] As an alternate or adjunct immunogen to GST fusion proteins,
peptides corresponding to relatively unique immunogenic regions of
a polypeptide of the invention may be generated and coupled to
keyhole limpet hemocyanin (KLH) through an introduced C-terminal
lysine. Antiserum to each of these peptides is similarly affinity
purified on peptides conjugated to BSA, and specificity tested in
ELISA and Western blots using peptide conjugates, and by Western
blot and immunoprecipitation using the polypeptide expressed as a
GST fusion protein.
[0182] Alternatively, monoclonal antibodies which specifically bind
any one of the polypeptides of the invention are prepared according
to standard hybridoma technology (see, e.g., Kohler et al., Nature
256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler
et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In
Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981;
Ausubel et al., supra). Once produced, monoclonal antibodies are
also tested for specific recognition by Western blot or
immunoprecipitation analysis (by the methods described in Ausubel
et al., supra). Antibodies which specifically recognize the
polypeptide of the invention are considered to be useful in the
invention; such antibodies may be used, e.g., in an immunoassay.
Alternatively monoclonal antibodies may be prepared using the
polypeptide of the invention described above and a phage display
library (Vaughan et al., Nature Biotech 14:309-314, 1996).
[0183] Preferably, antibodies of the invention are produced using
fragments of the polypeptide of the invention which lie outside
generally conserved regions and appear likely to be antigenic, by
criteria such as high frequency of charged residues. In one
specific example, such fragments are generated by standard
techniques of PCR and cloned into the pGEX expression vector
(Ausubel et al., supra). Fusion proteins are expressed in E. coli
and purified using a glutathione agarose affinity matrix as
described in Ausubel et al. (supra). To attempt to minimize the
potential problems of low affinity or specificity of antisera, two
or three such fusions are generated for each protein, and each
fusion is injected into at least two rabbits. Antisera are raised
by injections in a series, preferably including at least three
booster injections.
[0184] Antibodies against any of the polypeptides described herein
may be employed to treat bacterial infections.
Screening Assays
[0185] As discussed above, we have identified a number of P.
aeruginosa virulence factors that are involved in pathogenicity and
that may therefore be used to screen for compounds that reduce the
virulence of that organism, as well as other microbial pathogens.
For example, the invention provides methods of screening compounds
to identify those which enhance (agonist) or block (antagonist) the
action of a polypeptide or the gene expression of a nucleic acid
sequence of the invention. The method of screening may involve
high-throughput techniques.
[0186] Any number of methods are available for carrying out such
screening assays. According to one approach, candidate compounds
are added at varying concentrations to the culture medium of
pathogenic cells expressing one of the nucleic acid sequences of
the invention. Gene expression is then measured, for example, by
standard Northern blot analysis (Ausubel et al., supra), using any
appropriate fragment prepared from the nucleic acid molecule as a
hybridization probe. The level of gene expression in the presence
of the candidate compound is compared to the level measured in a
control culture medium lacking the candidate molecule. A compound
which promotes a decrease in the expression of the pathogenicity
factor is considered useful in the invention; such a molecule may
be used, for example, as a therapeutic to combat the pathogenicity
of an infectious organism.
[0187] If desired, the effect of candidate compounds may, in the
alternative, be measured at the level of polypeptide production
using the same general approach and standard immunological
techniques, such as Western blotting or immunoprecipitation with an
antibody specific for a pathogenicity factor. For example,
immunoassays may be used to detect or monitor the expression of at
least one of the polypeptides of the invention in a pathogenic
organism. Polyclonal or monoclonal antibodies (produced as
described above) which are capable of binding to such a polypeptide
may be used in any standard immunoassay format (e.g., ELISA,
Western blot, or RIA assay) to measure the level of the
pathogenicity polypeptide. A compound which promotes a decrease in
the expression of the pathogenicity polypeptide is considered
particularly useful. Again, such a molecule may be used, for
example, as a therapeutic to combat the pathogenicity of an
infectious organism.
[0188] Alternatively, or in addition, candidate compounds may be
screened for those which specifically bind to and inhibit a
pathogenicity polypeptide of the invention. The efficacy of such a
candidate compound is dependent upon its ability to interact with
the pathogenicity polypeptide. Such an interaction can be readily
assayed using any number of standard binding techniques and
functional assays (e.g., those described in Ausubel et al., supra).
For example, a candidate compound may be tested in vitro for
interaction and binding with a polypeptide of the invention and its
ability to modulate pathogenicity may be assayed by any standard
assays (e.g., those described herein).
[0189] In one particular example, a candidate compound that binds
to a pathogenicity polypeptide may be identified using a
chromatography-based technique. For example, a recombinant
polypeptide of the invention may be purified by standard techniques
from cells engineered to express the polypeptide (e.g., those
described above) and may be immobilized on a column. A solution of
candidate compounds is then passed through the column, and a
compound specific for the pathogenicity polypeptide is identified
on the basis of its ability to bind to the pathogenicity
polypeptide and be immobilized on the column. To isolate the
compound, the column is washed to remove non-specifically bound
molecules, and the compound of interest is then released from the
column and collected. Compounds isolated by this method (or any
other appropriate method) may, if desired, be further purified
(e.g., by high performance liquid chromatography). In addition,
these candidate compounds may be tested for their ability to render
a pathogen less virulent (e.g., as described herein). Compounds
isolated by this approach may also be used, for example, as
therapeutics to treat or prevent the onset of a pathogenic
infection, disease, or both. Compounds which are identified as
binding to pathogenicity polypeptides with an affinity constant
less than or equal to 10 mM are considered particularly useful in
the invention.
[0190] In yet another approach, candidate compounds are screened
for the ability to inhibit the virulence of a Pseudomonas cell by
monitoring the effect of the compound on the production of a
phenazine (e.g., pyocyanin). According to one approach, candidate
compounds are added at varying concentrations to a culture medium
of pathogenic cells. Pyocyanin is then measured according to any
standard method, for example, by monitoring its absorbance at 520
nm in acidic solution (Essar et al., J. Bacteriol. 172: 884, 1990).
To maximize pyocyanin production in liquid culture for
quantitation, cells may be cultured in a modified KA broth (King et
al., J. Lab. Clin. Med. 44:301, 1954) by adding 100 .mu.M
FeCl.sub.3. The level of pyocyanin production in the presence of
the candidate compound is compared to the level measured in a
control culture medium lacking the candidate molecule. A compound
which promotes a decrease in the expression of a pyocyanin is
considered useful in the invention; such a molecule may be used,
for example, as a therapeutic to combat the pathogenicity of an
infectious organism. Similar techniques may also be used to screen
for other appropriate phenazines including, without limitation,
pyorubin, aeruginosin A, myxin, and tubermycin A. Other phenazines
are described in Turner and Messenger (Advances In Microbial
Physiology 27:211-1275, 1986), Sorensen and Joseph (In: Pseudomonas
aeruginosa as an Opportunistic Pathogen, Campa, M., ed., Plenum
Press, N.Y., 1993), Ingram and Blackwood (Advances in Applied
Microbiology 13: 267, 1970), and Gerber ( In: CRC Handbook of
Microbiology, Laskin, A. I., and Lechevalier, eds., 2.sup.nd
edition, vol. 5, Chemical Rubber Co., Cleveland, Ohio, 1984, pp.
573-576).
[0191] Potential antagonists include organic molecules, peptides,
peptide mimetics, polypeptides, and antibodies that bind to a
nucleic acid sequence or polypeptide of the invention and thereby
inhibit or extinguish its activity. Potential antagonists also
include small molecules that bind to and occupy the binding site of
the polypeptide thereby preventing binding to cellular binding
molecules, such that normal biological activity is prevented. Other
potential antagonists include antisense molecules.
[0192] Each of the DNA sequences provided herein may also be used
in the discovery and development of antipathogenic compounds (e.g.,
antibiotics). The encoded protein, upon expression, can be used as
a target for the screening of antibacterial drugs. Additionally,
the DNA sequences encoding the amino terminal regions of the
encoded protein or Shine-Delgarno or other translation facilitating
sequences of the respective mRNA can be used to construct antisense
sequences to control the expression of the coding sequence of
interest.
[0193] The invention also provides the use of the polypeptide,
polynucleotide, or inhibitor of the invention to interfere with the
initial physical interaction between a pathogen and mammalian host
responsible for infection. In particular the molecules of the
invention may be used: in the prevention of adhesion and
colonization of bacteria to mammalian extracellular matrix
proteins; to extracellular matrix proteins in wounds; to block
mammalian cell invasion; or to block the normal progression of
pathogenesis.
[0194] The antagonists and agonists of the invention may be
employed, for instance, to inhibit and treat a variety of bacterial
infections.
[0195] Optionally, compounds identified in any of the
above-described assays may be confirmed as useful in conferring
protection against the development of a pathogenic infection in any
standard animal model (e.g., the mouse-burn assay described herein)
and, if successful, may be used as anti-pathogen therapeutics (e.g,
antibiotics).
Test Compounds and Extracts
[0196] In general, compounds capable of reducing pathogenic
virulence are identified from large libraries of both natural
product or synthetic (or semi-synthetic) extracts or chemical
libraries according to methods known in the art. Those skilled in
the field of drug discovery and development will understand that
the precise source of test extracts or compounds is not critical to
the screening procedure(s) of the invention. Accordingly, virtually
any number of chemical extracts or compounds can be screened using
the methods described herein. Examples of such extracts or
compounds include, but are not limited to, plant-, fungal-,
prokaryotic- or animal-based extracts, fermentation broths, and
synthetic compounds, as well as modification of existing compounds.
Numerous methods are also available for generating random or
directed synthesis (e.g., semi-synthesis or total synthesis) of any
number of chemical compounds, including, but not limited to,
saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
Synthetic compound libraries are commercially available from
Brandon Associates (Merrimack, N.H.) and Aldrich Chemical
(Milwaukee, Wis.). Alternatively, libraries of natural compounds in
the form of bacterial, fungal, plant, and animal extracts are
commercially available from a number of sources, including Biotics
(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics
Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge,
Mass.). In addition, natural and synthetically produced libraries
are produced, if desired, according to methods known in the art,
e.g., by standard extraction and fractionation methods.
Furthermore, if desired, any library or compound is readily
modified using standard chemical, physical, or biochemical
methods.
[0197] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their anti-pathogenic activity should be employed whenever
possible.
[0198] When a crude extract is found to have an anti-pathogenic or
anti-virulence activity, or a binding activity, further
fractionation of the positive lead extract is necessary to isolate
chemical constituents responsible for the observed effect. Thus,
the goal of the extraction, fractionation, and purification process
is the careful characterization and identification of a chemical
entity within the crude extract having anti-pathogenic activity.
Methods of fractionation and purification of such heterogenous
extracts are known in the art. If desired, compounds shown to be
useful agents for the treatment of pathogenicity are chemically
modified according to methods known in the art.
Pharmaceutical Therapeutics and Plant Protectants
[0199] The invention provides a simple means for identifying
compounds (including peptides, small molecule inhibitors, and
mimetics) capable of inhibiting the pathogenicity or virulence of a
pathogen. Accordingly, a chemical entity discovered to have
medicinal or agricultural value using the methods described herein
are useful as either drugs, plant protectants, or as information
for structural modification of existing anti-pathogenic compounds,
e.g., by rational drug design. Such methods are useful for
screening compounds having an effect on a variety of pathogens
including, but not limited to, bacteria, viruses, fungi, annelids,
nematodes, platyhelminthes, and protozoans. Examples of pathogenic
bacteria include, without limitation, Aerobacter, Aeromonas,
Acinetobacter, Agrobacterium, Bacillus, Bacteroides, Bartonella,
Bortella, Brucella, Calymmatobacterium, Campylobacter, Citrobacter,
Clostridium, Cornyebacterium, Enterobacter, Escherichia,
Francisella, Haemophilus, Hafnia, Helicobacter, Klebsiella,
Legionella, Listeria, Morganella, Moraxella, Proteus, Providencia,
Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus,
Streptococcus, Treponema, Xanthomonas, Vibrio, and Yersinia.
[0200] For therapeutic uses, the compositions or agents identified
using the methods disclosed herein may be administered
systemically, for example, formulated in a
pharmaceutically-acceptable buffer such as physiological saline.
Treatment may be accomplished directly, e.g., by treating the
animal with antagonists which disrupt, suppress, attenuate, or
neutralize the biological events associated with a pathogenicity
polypeptide. Preferable routes of administration include, for
example, subcutaneous, intravenous, interperitoneally,
intramuscular, or intradermal injections which provide continuous,
sustained levels of the drug in the patient. Treatment of human
patients or other animals will be carried out using a
therapeutically effective amount of an anti-pathogenic agent in a
physiologically-acceptable carrier. Suitable carriers and their
formulation are described, for example, in Remington's
Pharmaceutical Sciences by E. W. Martin. The amount of the
anti-pathogenic agent (e.g., an antibiotic) to be administered
varies depending upon the manner of administration, the age and
body weight of the patient, and with the type of disease and
extensiveness of the disease. Generally, amounts will be in the
range of those used for other agents used in the treatment of other
microbial diseases, although in certain instances lower amounts
will be needed because of the increased specificity of the
compound. A compound is administered at a dosage that inhibits
microbial proliferation. For example, for systemic administration a
compound is administered typically in the range of 0.1 ng - 10 g/kg
body weight.
[0201] For agricultural uses, the compositions or agents identified
using the methods disclosed herein may be used as chemicals applied
as sprays or dusts on the foliage of plants. Typically, such agents
are to be administered on the surface of the plant in advance of
the pathogen in order to prevent infection. Seeds, bulbs, roots,
tubers, and corms are also treated to prevent pathogenic attack
after planting by controlling pathogens carried on them or existing
in the soil at the planting site. Soil to be planted with
vegetables, ornamentals, shrubs, or trees can also be treated with
chemical fumigants for control of a variety of microbial pathogens.
Treatment is preferably done several days or weeks before planting.
The chemicals can be applied by either a mechanized route, e.g., a
tractor or with hand applications. In addition, chemicals
identified using the methods of the assay can be used as
disinfectants.
Other Embodiments
[0202] In general, the invention includes any nucleic acid sequence
which may be isolated as described herein or which is readily
isolated by homology screening or PCR amplification using the
nucleic acid sequences of the invention. Also included in the
invention are polypeptides which are modified in ways which do not
abolish their pathogenic activity (assayed, for example as
described herein). Such changes may include certain mutations,
deletions, insertions, or post-translational modifications, or may
involve the inclusion of any of the polypeptides of the invention
as one component of a larger fusion protein. Also, included in the
invention are polypeptides that have lost their pathogenicity.
[0203] Thus, in other embodiments, the invention includes any
protein which is substantially identical to a polypeptide of the
invention. Such homologs include other substantially pure
naturally-occurring polypeptides as well as allelic variants;
natural mutants; induced mutants; proteins encoded by DNA that
hybridizes to any one of the nucleic acid sequences of the
invention under high stringency conditions or, less preferably,
under low stringency conditions (e.g., washing at 2.times. SSC at
40.degree. C. with a probe length of at least 40 nucleotides); and
proteins specifically bound by antisera of the invention.
[0204] The invention further includes analogs of any
naturally-occurring polypeptide of the invention. Analogs can
differ from the naturally-occurring the polypeptide of the
invention by amino acid sequence differences, by post-translational
modifications, or by both. Analogs of the invention will generally
exhibit at least 85%, more preferably 90%, and most preferably 95%
or even 99% identity with all or part of a naturally-occurring
amino acid sequence of the invention. The length of sequence
comparison is at least 15 amino acid residues, preferably at least
25 amino acid residues, and more preferably more than 35 amino acid
residues. Again, in an exemplary approach to determining the degree
of identity, a BLAST program may be used, with a probability score
between e.sup.-3 and e.sup.-100 indicating a closely related
sequence. Modifications include in vivo and in vitro chemical
derivatization of polypeptides, e.g., acetylation, carboxylation,
phosphorylation, or glycosylation; such modifications may occur
during polypeptide synthesis or processing or following treatment
with isolated modifying enzymes. Analogs can also differ from the
naturally-occurring polypeptides of the invention by alterations in
primary sequence. These include genetic variants, both natural and
induced (for example, resulting from random mutagenesis by
irradiation or exposure to ethanemethylsulfate or by site-specific
mutagenesis as described in Sambrook, Fritsch and Maniatis,
Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989,
or Ausubel et al., supra). Also included are cyclized peptides,
molecules, and analogs which contain residues other than L-amino
acids, e.g., D-amino acids or non-naturally occurring or synthetic
amino acids, e.g., .beta. or .gamma. amino acids.
[0205] In addition to full-length polypeptides, the invention also
includes fragments of any one of the polypeptides of the invention.
As used herein, the term "fragment," means at least 5, preferably
at least 20 contiguous amino acids, preferably at least 30
contiguous amino acids, more preferably at least 50 contiguous
amino acids, and most preferably at least 60 to 80 or more
contiguous amino acids. Fragments of the invention can be generated
by methods known to those skilled in the art or may result from
normal protein processing (e.g., removal of amino acids from the
nascent polypeptide that are not required for biological activity
or removal of amino acids by alternative mRNA splicing or
alternative protein processing events).
[0206] Furthermore, the invention includes nucleotide sequences
that facilitate specific detection of any of the nucleic acid
sequences of the invention. Thus, for example, nucleic acid
sequences described herein or fragments thereof may be used as
probes to hybridize to nucleotide sequences by standard
hybridization techniques under conventional conditions. Sequences
that hybridize to a nucleic acid sequence coding sequence or its
complement are considered useful in the invention. Sequences that
hybridize to a coding sequence of a nucleic acid sequence of the
invention or its complement and that encode a polypeptide of the
invention are also considered useful in the invention. As used
herein, the term "fragment," as applied to nucleic acid sequences,
means at least 5 contiguous nucleotides, preferably at least 10
contiguous nucleotides, more preferably at least 20 to 30
contiguous nucleotides, and most preferably at least 40 to 80 or
more contiguous nucleotides. Fragments of nucleic acid sequences
can be generated by methods known to those skilled in the art.
[0207] The invention further provides a method for inducing an
immunological response in an individual, particularly a human,
which includes inoculating the individual with, for example, any of
the polypeptides (or a fragment or analog thereof or fusion
protein) of the invention to produce an antibody and/or a T cell
immune response to protect the individual from infection,
especially bacterial infection (e.g., a Pseudomonas aeruginosa
infection). The invention further includes a method of inducing an
immunological response in an individual which includes delivering
to the individual a nucleic acid vector to direct the expression of
a polypeptide described herein (or a fragment or fusion thereof) in
order to induce an immunological response.
[0208] The invention also includes vaccine compositions including
the polypeptides or nucleic acid sequences of the invention. For
example, the polypeptides of the invention may be used as an
antigen for vaccination of a host to produce specific antibodies
which protect against invasion of bacteria, for example, by
blocking the production of phenazines. The invention therefore
includes a vaccine formulation which includes an immunogenic
recombinant polypeptide of the invention together with a suitable
carrier.
[0209] The invention further provides compositions (e.g.,
nucleotide sequence probes), polypeptides, antibodies, and methods
for the diagnosis of a pathogenic condition.
[0210] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0211] Other embodiments are within the scope of the claims.
* * * * *