U.S. patent application number 12/816485 was filed with the patent office on 2010-12-23 for anti-bacterial vaccine compositions.
This patent application is currently assigned to PHARMACIA & UPJOHN COMPANY LLC. Invention is credited to Troy E. Fuller, Michael J. Kennedy, David E. Lowery.
Application Number | 20100322975 12/816485 |
Document ID | / |
Family ID | 26826843 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100322975 |
Kind Code |
A1 |
Lowery; David E. ; et
al. |
December 23, 2010 |
Anti-bacterial Vaccine Compositions
Abstract
Gram negative bacterial virulence genes are identified, thereby
allowing the identification of novel anti-bacterial agents that
target these virulence genes and their products, and the provision
of novel gram negative bacterial mutants useful in vaccines.
Inventors: |
Lowery; David E.; (Portage,
MI) ; Fuller; Troy E.; (Ceresco, MI) ;
Kennedy; Michael J.; (Galesburg, MI) |
Correspondence
Address: |
PHARMACIA & UPJOHN
7000 Portage Road, KZO-300-104
KALAMAZOO
MI
49001
US
|
Assignee: |
PHARMACIA & UPJOHN COMPANY
LLC
Kalamazoo
MI
|
Family ID: |
26826843 |
Appl. No.: |
12/816485 |
Filed: |
June 16, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12425599 |
Apr 17, 2009 |
7763262 |
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12816485 |
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11251464 |
Oct 14, 2005 |
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12425599 |
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09545199 |
Apr 6, 2000 |
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11251464 |
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60153453 |
Sep 10, 1999 |
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60128689 |
Apr 9, 1999 |
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Current U.S.
Class: |
424/255.1 ;
435/252.3 |
Current CPC
Class: |
A61K 2039/522 20130101;
C07K 14/285 20130101; A61P 37/04 20180101; A61K 39/102 20130101;
C12N 1/20 20130101; A61P 31/04 20180101 |
Class at
Publication: |
424/255.1 ;
435/252.3 |
International
Class: |
A61K 39/102 20060101
A61K039/102; A61P 31/04 20060101 A61P031/04; C12N 1/21 20060101
C12N001/21 |
Claims
1-76. (canceled)
77. An attenuated Pasteurellaceae bacterium selected from the group
consisting of Pasteurella (Mannheimia) haemolytica, Pasteurella
multocida, and Actinobacillus pleuropneumoniae comprising a
mutation in a polynucleotide sequence that encodes a fhaC
polypeptide comprising an amino acid sequence at least 70%
identical to the fhaC amino acid sequence of SEQ ID NO: 20, wherein
the mutation results in decreased virulence and attenuation of the
bacterium.
78. The bacterium of claim 77, wherein the mutation results in
deletion of all or part of the polynucleotide sequence that encodes
the fhaC polypeptide.
79. The bacterium of claim 77, wherein the mutation results in an
insertion in the polynucleotide sequence that encodes the fhaC
polypeptide.
80. The bacterium of claim 77 that is a Pasteurella multocida
bacteria.
81. An immunogenic composition comprising a bacterium according to
claim 77.
82. A vaccine composition comprising the immunogenic composition
according to claim 81 and a pharmaceutically acceptable
carrier.
83. The vaccine composition according to claim 82, further
comprising an adjuvant.
84. An attenuated Pasteurellaceae bacterium selected from the group
consisting of Pasteurella (Mannheimia) haemolytica, Pasteurella
multocida, and Actinobacillus pleuropneumoniae comprising a
mutation in a polynucleotide sequence that encodes a fhaC
polypeptide, wherein the polynucleotide sequence hybridizes to the
complement of a polynucleotide sequence set forth in SEQ ID NO: 19
under stringent conditions, such conditions comprising a final wash
in buffer comprising 2.times.SSC/0.1% SDS, at 35.degree. C. to
45.degree. C.
85. The bacterium of claim 84, wherein the mutation is in the
polynucleotide sequence set forth in SEQ ID NO: 19.
86. An immunogenic composition comprising the bacterium according
to claim 84 or claim 85.
87. A vaccine composition comprising the immunogenic composition
according to claim 87 and a pharmaceutically acceptable
carrier.
88. The vaccine composition of claim 87, further comprising an
adjuvant.
89. An attenuated Pasteurella multocida bacterium comprising a
mutation in a polynucleotide sequence set forth in SEQ ID NO: 19
that encodes a fhaC polypeptide, wherein the mutation results in
decreased virulence and attenuation of the bacterium.
90. The bacterium of claim 89, wherein the mutation results in
deletion of all or part of the polynucleotide sequence that encodes
the fhaC polypeptide.
91. The bacterium of claim 89, wherein the mutation results in an
insertion in the polynucleotide sequence that encodes the fhaC
polypeptide.
92. An immunogenic composition comprising a bacterium according to
claim 89.
93. A vaccine composition comprising the immunogenic composition
according to claim 92 and a pharmaceutically acceptable carrier.
Description
[0001] This application is a continuation application of U.S.
patent application Ser. No. 09/545,199, filed Apr. 6, 2000, which
in turn claims benefit under 35 U.S.C.sctn.119 of U.S. Provisional
Application Ser. No. 60/153,453, filed Sep. 10, 1999 and
60/128,689, filed Apr. 9, 1999, which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the
identification of genes responsible for virulence of Pasteurella
multocida and Actinobacillus pleuropneumoniae bacteria, thereby
allowing for production of novel attenuated mutant strains useful
in vaccines and identification of new anti-bacterial agents that
target the virulence genes and their products.
BACKGROUND OF THE INVENTION
[0003] The family Pasteurellaceae encompasses several significant
pathogens that infect a wide variety of animals. In addition to P.
multocida, prominent members of the family include Pasteurella
haemolytica, Actinobacillus pleuropneumoniae and Haemophilus
somnus. P. multocida is a gram-negative, nonmotile coccobacillus
which is found in the normal flora of many wild and domestic
animals and is known to cause disease in numerous animal species
worldwide [Biberstein, In M. Kilian, W. Frederickson, and E. L.
Biberstein (ed.), Haemophilus, Pasteurella, and Actinobacillus.
Academic Press, London, p. 61-73 (1981)]. The disease
manifestations following infection include septicemias,
bronchopneumonias, rhinitis, and wound infections [Reviewed in
Shewen, et al., In C. L. Gyles and C. O. Thoen (ed.), Pathogenesis
of Bacterial Infections in Animals. Iowa State University Press,
Ames, p. 216-225 (1993), incorporated herein by reference].
[0004] Infection by P. multocida generally results from invasion
during periods of stress, but transmission may also occur by
aerosol or contact exposure, or via flea and tick vectors. In fowl,
P. multocida infection gives rise to acute to peracute septicemia,
particularly prevalent in domestic turkeys and wild waterfowl under
stress conditions associated with overcrowding, laying, molting, or
severe climatic change. In cattle, a similar hemorrhagic septicemia
follows infection and manifests conditions including high fever and
depression, generally followed by quick death. Transmission is most
likely through aerosol contact, but infection can also arise during
periods of significant climatic change. In rabbits, infection gives
rise to recurring purulent rhinitis, generally followed by
conjunctivitis, otitis media, sinusitis, subcutaneous abscesses,
and chronic bronchopneumonia. In severe infections, rabbit
mortality arises from acute fibrinous bronchopneumonia, septicemia,
or endotoxemia. Disease states normally arise during periods of
stress. In pigs, common P. multocida disease states include
atrophic rhinitis and bacterial pneumonia. Similar pneumonia
conditions are also detected in dogs, cats, goats, and sheep. P.
multocida is commonly detected in oral flora of many animals and is
therefore a common contaminant in bite and scratch wounds.
[0005] P. multocida strains are normally designated by capsular
serogroup and somatic serotype. Five capsular serogroups (A, B, D,
E, and F) and 16 somatic serotypes are distinguished by expression
of characteristic heat-stable antigens. Most strains are host
specific and rarely infect more than one or two animals. The
existence of different serotypes presents a problem for vaccination
because traditional killed whole cell bacteria normally provide
only serotype-specific protection. However, it has been
demonstrated that natural infection with one serotype can lead to
immunological protection against multiple serotypes [Shewen, et
al., In C. L. Gyles and C. O. Thoen (Ed.), Pathogenesis of
Bacterial Infections in Animals. Iowa State University Press, Ames,
p. 216-225 (1993)] and cross protection can also be stimulated by
using inactivated bacteria grown in vivo [Rimler, et al., Am J Vet
Res. 42:2117-2121 (1981)]. One live spontaneous mutant P. multocida
strain has been utilized as a vaccine and has been shown to
stimulate a strong immune response [Davis, Poultry Digest.
20:430-434 (1987), Schlink, et al., Avian Dis. 31(1):13-21 (1987)].
This attenuated strain, however, has been shown to revert to a
virulent state or cause mortality if the vaccine recipient is
stressed [Davis, Poultry Digest. 20:430-434 (1987), Schlink, et
al., Avian Dis. 31(1):13-21 (1987)).
[0006] Another member of the Pasteurella family, A.
pleuropneumoniae exhibits strict host specificity for swine and is
the causative agent of highly contagious porcine pleuropneumonia.
Infection normally arises in intensive breeding conditions, and is
believed to occur by a direct mode of transmission. The disease is
often fatal and, as a result, leads to severe economic loss in the
swine producing industry. A. pleuropneumoniae infection may be
chronic or acute, and infection is characterized by a hemorrhagic,
necrotic bronchopneumonia with accompanying fibrinous pleuritis. To
date, bacterial virulence has been attributed to structural
proteins, including serotype-specific capsular polysaccharides,
lipopolysaccharides, and surface proteins, as well as extracellular
cytolytic toxins. Despite purification and, in some instances
cloning, of these virulence factors, the exact role of these
virulence factors in A. pleuropneumoniae infection is poorly
understood.
[0007] Twelve serotypes of A. pleuropneumoniae have been identified
based on antigenic differences in capsular polysaccharides and
production of extracellular toxins. Serotypes 1, 5, and 7 are most
relevant to A. pleuropneumoniae infection in the United States,
while serotypes 1, 2, 5, 7, and 9 are predominant in Europe. There
are at least three significant extracellular toxins of A.
pleuropneumoniae that are members of the haemolysin family and are
referred to as RTX toxins. RTX toxins are produced by many Gram
negative bacteria, including E. coli, Proteus vulgarisa, and
Pasteurella haemolytica, and the proteins generally share
structural and functional characteristics. Toxins from the various
serotypes differ, however, in host specificity, target cells, and
biological activities.
[0008] The major A. pleuropneumoniae RTX toxins include ApxI,
ApxII, and ApxIII. ApxI and ApxII have haemolytic activity, with
ApxI being more potent. ApxIII shows no haemolytic activity, but is
cytotoxic for alveolar macrophages and neutrophils. Most A.
pleuropneumoniae serotypes produce two of these three toxins. For
example, serotypes 1, 5, 9, and 11 express ApxI and ApxII, and
serotypes 2, 3, 4, 6, and 8 express ApxII and ApxIII. Serotype 10,
however, produces only ApxI, and serotypes 7 and 12 express only
ApxII. Those A. pleuropneumoniae serotypes that produce both ApxI
and ApxII are the most virulent strains of the bacteria.
[0009] The Apx toxins were demonstrated to be virulence factors in
murine models and swine infection using randomly mutated wild type
bacteria [Tascon, et al., Mol. Microbiol. 14:207-216 (1994)]. Other
A. pleuropneumoniae mutants have also been generated with targeted
mutagenesis to inactivate the gene encoding the AopA outer membrane
virulence protein [Mulks and Buysee, Gene 165:61-66 (1995)].
[0010] In attempts to produce vaccine compositions, traditional
killed whole cell bacteria have provided only serotype-specific
protection [MacInnes and Smart, supra], however, it has been
demonstrated that natural infection with a highly virulent serotype
can stimulate strong protective immunity against multiple serotypes
[Nielsen, Nord Vet Med. 31:407-13 (1979), Nielsen, Nord Vet Med.
36:221-234 (1984), Nielsen, Can J Vet Res. 29:580-582 (1988),
Nielsen, ACTA Vet Scand. 15:80-89(1994)). One defined
live-attenuated vaccine strain producing an inactive form of the
ApxII toxin has shown promise for cross protection in swine
[Prideaux, et al., Infection & Immunity 67:1962-1966 (1999)],
while other undefined live-attenuated mutants have also shown
promise [Inzana, et al., Infect Immun. 61:1682-6, (1993),
Paltineanu, et al., In International Pig Veterinary Society, 1992,
p.214, Utrera, et al., In International Pig Veterinary Society,
1992, p. 213].
[0011] Because of the problems associated with vaccine formulations
comprising bacterial strains with undefined, spontaneous mutations,
there exists a need in the art for rational construction of live
attenuated bacterial strains for use in vaccines that will safely
stimulate protective immunity against homologous and heterologous
P. multocida and A. pleuropneumoniae serotypes. There further
exists a need to identify attenuated bacterial strains and genes
required for bacterial virulence, thereby facilitating development
of methods to identify anti-bacterial agents.
SUMMARY OF THE INVENTION
[0012] In general, the present invention provides materials and
methods for production and use of vaccine compositions comprising
attenuated gram negative bacteria. In one aspect, vaccine
compositions of the invention comprise attenuated species in the
Pasteurellaceae family of bacteria, which is known in the art and
described, in part, in Dewhirst, et al., J. Bacteriol.
174:2002-2013 (1992), incorporated herein by reference in its
entirety. Species in the family include, but are not limited to, A.
actinomycetemcomitans, A. capsulatus, A. equuli, A. lignieresii, A.
pleuropneumoniae (H. pleuropneumoniae), A. seminis, A. suis (H.
suis), A. ureae (p. ureae), A. capsulatus, Bisgaard taxon 11, H.
aegyptius, H. aphrophilus, H. aphrophilus (H. parainfluenzae), H.
ducreyi, H. haemoglobinophilus, H. haemolyticus, H. influenzae, H.
paracuniculus, H. paragallinarum, H. parahaemolyticus, H.
parainfluenzae, (H. paraphrophilus), H. paraphrohaemolyticus, H.
paraphrophilus, H. parasuis, H. parasuis type 5, H. segnis, H.
somnus, Haemophilus minor group, Haemophilus taxon C, P. aerogenes,
P. anatis, P. avium (H. avium), P. canis, P. dagmatis, P.
gallinarum, P. haemolytica, P. trehalosi (P. haemolytica biotype
T), P. langaa, P. multocida, P. pneumotropica, P. stomatis, P.
volantium (H. parainfluenzae), P. volantium, Pasteurella species A,
Pasteurella species B, and Haemophilus paraphrohaemolyticus.
Preferably, vaccine compositions comprise attenuated Pasteurella
haemolytica, Actinobacillus pleuropneumoniae, Haemophilus somnus,
or Pasteurella multocida bacteria. In a most preferred embodiment,
vaccine compositions of the invention comprise attenuated
Pasteurella multocida and A. plueropneumoniae bacterial
strains.
[0013] One aspect of the invention provides gram negative bacterial
organisms containing a functional mutation in a gene sequence
represented by any one of SEQ ID NOS: 1, 3, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58, 60, 68,
70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108, 110, 112,
114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 135, 136,
138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,
163, and 164, or species homologs thereof, wherein the mutation
inhibits or abolishes expression and/or biological activity of an
encoded gene product (i.e., the polypeptide encoded by a gene);
said functional mutation resulting in attenuated virulence of the
bacterial strain. As understood in the art, species homologs
include genes found in two or more different species which possess
substantial polynucleotide sequence homology and possess the same,
or similar, biological functions and/or properties. Preferably
polynucleotide sequences which represent species homologs will
hybridize under moderately stringent conditions, as described
herein by example, and possess the same or similar biological
activities and or properties. In another aspect, polynucleotides
representing species homologs will share greater than about 60%
sequence homology, greater than about 70% sequence homology,
greater than about 80% sequence homology, greater than about 90%
sequence homology or greater than about 95% sequence homology.
Functional mutations that modulate (i.e., increase or decrease)
expression and/or biological activity of a gene product include
insertions or deletions in the protein coding region of the gene
itself or in sequences responsible for, or involved in, control of
gene expression. Deletion mutants include those wherein all or part
of a specific gene sequence is deleted. In one aspect, the mutation
results in deletion of at least about 10%, at least about 20%, at
least about 30%, at least about 40% at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about. 98%, or at least about 99%
of said gene. In another aspect, the mutation results in an
insertion in the gene, wherein the insertion causes decreased
expression of a gene product encoded by the mutated gene and/or
expression of an inactive gene product encoded by the mutated gene.
Also contemplated are compositions, and preferably vaccine
compositions, comprising mutated and attenuated gram negative
bacterial organisms, optionally comprising a suitable adjuvant
and/or a pharmaceutically acceptable diluent or carrier. In order
for a modified strain to be effective in a vaccine formulation, the
attenuation must be significant enough to prevent the pathogen from
evoking severe clinical symptoms, but also insignificant enough to
allow limited replication and growth of the bacteria in the
host.
[0014] The invention also provides polynucleotides encoding gene
products that are required for virulence in gram negative bacteria.
Polynucleotides of the invention include DNA, such as complementary
DNA, genomic DNA including complementary or anti-sense DNA, and
wholly or partially synthesized DNA; RNA, including sense and
antisense strands; and peptide nucleic acids as described, for
example in Corey, TIBTECH 15:224-229 (1997). Virulence gene
polynucleotides of the invention include those set forth in SEQ ID
NOs:1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39,
41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 163, and 164, or species
homologs thereof, polynucleotides encoding a virulence gene product
encoded by a polynucleotide of SEQ D NOs: 1, 3, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58, 60,
68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 135,
136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,
162, 163, and 164, or a species homolog thereof, and polynucleotide
that hybridize, under moderately to highly stringent conditions, to
the noncoding strand (or complement) of any one of the
polynucleotides set out in SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58, 60, 68,
70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108, 110, 112,
114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 135, 136,
138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,
163, and 164, species homologs thereof. The invention therefore
comprehends gene sequences from Pasteurellaceae set out in SEQ ID
NOs: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37,
39, 41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 163, and 164, as well as related
gene sequences from other gram negative bacterial organisms,
including naturally occurring (i.e., species homologs) and
artificially induced variants thereof. The invention also
comprehends polynucleotides which encode polypeptides deduced from
any one of the polynucleotides set out in SEQ ID NOs: 1, 3, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55,
57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,
134, 135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, and 164, and species homologs thereof. Knowledge of the
sequence of a polynucleotide of the invention makes readily
available every possible fragment of that polynucleotide. The
invention therefore provides fragments of a polynucleotide of the
invention.
[0015] The invention further embraces expression constructs
comprising polynucleotides of the invention. Host cells
transformed, transfected or electroporated with a polynucleotide of
the invention are also contemplated. The invention provides methods
to produce a polypeptide encoded by a polynucleotide of the
invention comprising the steps of growing a host cell of the
invention uider conditions that permit, and preferably promote,
expression of a gene product encoded by the polynucleotide, and
isolating the gene product from the host cell or the medium of its
growth.
[0016] Identification of polynucleotides of the invention makes
available the encoded polypeptides. Polypeptides of the invention
include full length and fragment, or truncated, proteins; variants
thereof; fusion, or chimeric proteins; and analogs, including those
wherein conservative amino acid substitutions have been introduced
into wild-type polypeptides. Antibodies that specifically recognize
polypeptides of the invention are also provided, and include
monoclonal and polyclonal antibodies, single chain antibodies,
chimeric antibodies, humanized antibodies, human antibodies , and
complementary determining region (CDR)-grafted antibodies, as well
as compounds that include CDR sequences which specifically
recognize a polypeptide of the invention. The invention also
provides anti-idiotype antibodies immunospecific for antibodies of
the invention.
[0017] According to another aspect of the invention, methods are
provided for identifying novel anti-bacterial agents that modulate
the function of gram negative bacteria virulence genes or gene
products. Methods of the invention include screening potential
agents for the ability to interfere with expression of virulence
gene products encoded by the DNA sequences set forth in any one of
SEQ ID NOS: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33,
37, 39, 41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82,
84, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146,
148, 150, 152, 154, 156, 158, 160, 162, 163, and 164, or species
homologs thereof, or screening potential agents for the ability to
interfere with biological function of a bacterial gene product
encoded in whole or in part by a DNA sequence set forth in any one
of SEQ ID NOS: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31,
33, 37, 39, 41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80,
82, 84, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146,
148, 150, 152, 154, 156, 158, 160, 162, 163, and 164, species
homologs thereof, or the complementary strand thereof, followed by
identifying agents that provide positive results in such screening
assays. In particular, agents that interfere with the expression of
virulence gene products include anti-sense polynucleotides and
ribozymes that are complementary to the virulence gene sequences.
The invention further embraces methods to modulate transcription of
gene products of the invention through use of
oligonucleotide-directed triplet helix formation.
[0018] Agents that interfere with the function of virulence gene
products include variants of virulence gene products, binding
partners of the virulence gene products and variants of such
binding partners, and enzyme inhibitors (where the product is an
enzyme).
[0019] Novel anti-bacterial agents identified by the methods
described herein are provided, as well as methods for treating a
subject suffering from infection with gram negative bacteria
involving administration of such novel anti-bacterial agents in an
amount effective to reduce bacterial presence.
[0020] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon consideration
of the following detailed description of the invention which
describes presently prepared embodiments thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0021] "Virulence genes," as used herein, are genes whose function
or products are required for successful establishment and/or
maintenance of bacterial infection in a host animal. Thus,
virulence genes and/or the proteins encoded thereby are involved in
pathogenesis in the host organism, but may not be necessary for
growth.
[0022] "Signature-tagged mutagenesis (STM)," as used herein, is a
method generally described in International Patent Publication No.
WO 96/17951, incorporated herein by reference, and includes, for
example, a method for identifying bacterial genes required for
virulence in a murine model of bacteremia. In this method,
bacterial strains that each have a random mutation in the genome
are produced using transposon integration; each insertional
mutation carries a different DNA signature tag which allows mutants
to be differentiated from each other. The tags comprise 40 by
variable central regions flanked by invariant "arms" of 20 by which
allow the central portions to be co-amplified by polymerase chain
reaction (PCR). Tagged mutant strains are assembled in microtiter
dishes, then combined to form the "inoculum pool" for infection
studies. At an appropriate time after inoculation, bacteria are
isolated from the animal and pooled to form the "recovered pool."
The tags in the recovered pool and the tags in the inoculum pool
are separately amplified, labeled, and then used to probe filters
arrayed with all of the different tags representing the mutants in
the inoculum. Mutant strains with attenuated virulence are those
which cannot be recovered from the infected animal, i.e., strains
with tags that give hybridization signals when probed with tags
from the inoculum pool but not when probed with tags from the
recovered pool. In a variation of this method, non-radioactive
detection methods such as chemiluminescence can be used
[0023] Signature-tagged mutagenesis allows a large number of
insertional mutant strains to be screened simultaneously in a
single animal for loss of virulence. Screening nineteen pools of
mutant P. multocida strains resulted in the identification of more
than 60 strains with reduced virulence, many of which were
confirmed to be attenuated in virulence by subsequent determination
of an approximate LD 50, for the individual mutants. Screening of
A. pleuropneumoniae mutants resulted in identification of more than
100 strains having mutations in 35 different genes. Of these,
mutations in 22 genes results in significantly attenuated A.
pleuropneumoniae strains. The nucleotide sequence of the open
reading frame disrupted by the transposon insertion was determined
by sequencing both strands and an encoded amino acid sequence was
deduced. Novelty of both the polynucleotide and amino acid
sequences was determined by comparison of the sequences with DNA
and protein database sequences.
[0024] The identification of bacterial, and more particularly P.
multocida and A. pleuropneumoniae virulence genes provides for
microorganisms exhibiting reduced virulence (i.e., attenuated
strains), which are useful in vaccines. Such microorganisms include
Pasteurellaceae mutants containing at least one functional mutation
inactivating a gene represented by any one of SEQ ID NOS: 1, 3, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53,
55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104,
106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, 135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,
156, 158, 160, 162, 163, and 164. The worker of ordinary skill in
the art will realize that a "functional mutation" may occur in
protein coding regions of a gene of the invention, as well as in
regulatory regions that modulate transcription of the virulence
gene RNA.
[0025] The worker of ordinary skill will also appreciate that
attenuated P. multocida and A. pleuropneumoniae strains of the
invention include those bearing more than one functional mutation.
More than one mutation may result in additive or synergistic
degrees of attenuation. Multiple mutations can be prepared by
design or may fortuitously arise from a deletion event originally
intended to introduce a single mutation. An example of an
attenuated strain with multiple deletions is a Salmonella
typhimurium strain wherein the cya and crp genes are functionally
deleted. This mutant S. typhimurium strain has shown promise as a
live vaccine.
[0026] Identification of virulence genes in P. multocida and A.
pleuropneumoniae can provide information regarding similar genes,
i.e., species homologs, in other pathogenic species. As an example,
identification of the aroA gene led to identification of conserved
genes in a diverse number of pathogens, including P. haemolytica,
Aeromonas hydrophila, Aeromonas salmonicida, Salmonella
typhimurium, Salmonella enteritidis, Salmonella dublin, Salmonella
gallanerum, Bordella pertussis, Yersinia entericolitica, Neisseria
gonorrhoeae, and Bacillus anthracis. In many of these species,
attenuated bacterial strains bearing mutations in the aroA gene
have proven to be effective in vaccine formulations. Using the
virulence genes sequences identified in P. multocida, similar or
homologous genes can be identified in other organisms, particularly
within the Pasteurella family, as well as A. pleuropneumoniae and
Haemophilus somnus. Likewise, identification of A. pleuropneumoniae
virulence genes can permit identification of related genes in other
organisms. Southern hybridization using the P. multocida and A.
pleuropneumoniae genes as probes can identify these related genes
in chromosomal libraries derived from other organisms.
Alternatively, PCR can be equally effective in gene identification
across species boundaries. As still another alternative,
complementation of, for example, a P. multocida mutant with a
chromosomal library from other species can also be used to identify
genes having the same or related virulence activity. Identification
of related virulence genes can therefore lead to production of an
attenuated strain of the other organism which can be useful as
still another vaccine formulation. Examples of P. multocida genes
that have been demonstrated to exist in other species (e.g. P.
haemolytica, A. pleuropneumoniae and H. somnus) include genes exbB,
atpG, andpnp
[0027] Attenuated P. multocida strains identified using STM are
insertional mutants wherein a virulence gene has been rendered
non-functional through insertion of transposon sequences in either
the open reading frame or regulatory DNA sequences. In one aspect,
therefore, the attenuated P. multocida strains, as well as other
gram-negative mutant bacterial strains of the invention can bear
one or more mutations which result in an insertion in the gene,
with the insertion causing decreased expression of a gene product
encoded by the mutated gene and/or expression of an inactive gene
product encoded by the mutated gene. These insertional mutants
still contain all of the genetic information required for bacterial
virulence and can possibly revert to apathogenic state by deletion
of the inserted transposon. Therefore, in preparing a vaccine
formulation, it is desirable to take the information gleaned from
the attenuated strain and create a deletion mutant strain wherein
some, most, or all of the virulence gene sequence is removed,
thereby precluding the possibility that the bacteria will revert to
a virulent state. The attenuated P. multocida strains, as well as
other gram-negative mutant bacterial strains of the invention
therefore include those bearing one or more mutation which results
in deletion of at least about 10%, at least about 20%, at least
about 30%, at least about 40% at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, at least about 98%, or at least about 99% of the
virulence gene.
[0028] The vaccine properties of an attenuated insertional mutant
identified using STM are expected to be the same or similar to
those of a bacteria bearing a deletion in the same gene. However,
it is possible that an insertion mutation may exert "polar" effects
on adjoining gene sequences, and as a result, the insertion mutant
may possess characteristic distinct from a mutant strain with a
deletion in the same gene sequence. Deletion mutants can be
constructed using any of a number oftechniques well known and
routinely practiced in the art.
[0029] In one example, a strategy using counterselectable markers
can be employed which has commonly been utilized to delete genes in
many bacteria. For a review, see, for example, Reyrat, et al.,
Infection and Immunity 66:4011-4017 (1998), incorporated herein by
reference. In this technique, a double selection strategy is often
employed wherein a plasmid is constructed encoding both a
selectable and counterselectable marker, with flanking DNA
sequences derived from both sides of the desired deletion. The
selectable marker is used to select for bacteria in which the
plasmid has integrated into the genome in the appropriate location
and manner. The counterselecteable marker is used to select for the
very small percentage of bacteria that have spontaneously
eliminated the integrated plasmid. A fraction of these bacteria
will then contain only the desired deletion with no other foreign
DNA present. The key to the use of this technique is the
availability of a suitable counterselectable marker.
[0030] In another technique, the cre-lox system is used for site
specific recombination of DNA. The system consists of 34 base pair
lox sequences that are recognized by the bacterial cre recombinase
gene. If the lox sites are present in the DNA in an appropriate
orientation, DNA flanked by the lox sites will be excised by the
cre recombinase, resulting in the deletion of all sequences except
for one remaining copy of the lox sequence. Using standard
recombination techniques, it is possible to delete the targeted
gene of interest in the P. multocida or A. pleuropneumoniae genome
and to replace it with a selectable marker (e.g., a gene coding for
kanamycin resistance) that is flanked by the lox sites. Transient
expression (by electroporation of a suicide plasmid containing the
cre gene under control of a promoter that functions in P. multocida
or A. pleuropneumoniae) of the cre recombinase should result in
efficient elimination of the lox flanked marker. This process would
result in a mutant containing the desired deletion mutation and one
copy of the lox sequences.
[0031] In another approach, it is possible to directly replace a
desired deleted sequence in the P. multocida or A. pleuropneumoniae
genome with a marker gene, such as green fluorescent protein (GFP),
.beta.-galactosidase, or luciferase. In this technique, DNA
segments flanking a desired deletion are prepared by PCR and cloned
into a suicide (non-replicating) vector for P. multocida or A.
pleuropneumoniae. An expression cassette, containing a promoter
active in P. multocida or A. pleuropneumoniae and the appropriate
marker gene, is cloned between the flanking sequences. The plasmid
is introduced into wild-type P. multocida or A. pleuropneumoniae.
Bacteria that incorporate and express the marker gene (probably at
a very low frequency) are isolated and examined for the appropriate
recombination event (i.e., replacement of the wild type gene with
the marker gene).
[0032] The reduced virulence of these organisms and their
immunogenicity may be confirmed by administration to a subject
animal. While it is possible for an avirulent microorganism of the
invention to be administered alone, one or more of such mutant
microorganisms are preferably administered in a vaccine composition
containing suitable adjuvant(s) and pharmaceutically acceptable
diluent(s) or carrier(s). The carrier(s) must be "acceptable" in
the sense of being compatible with the avirulent microorganism of
the invention and not deleterious to the subject to be immunized.
Typically, the carriers will be water or saline which will be
sterile and pyrogen free. The subject to be immunized is a subject
needing protection from a disease caused by a virulent form of P.
multocida, A. pleuropneumoniae, or other pathogenic
microorganisms.
[0033] It will be appreciated that the vaccine of the invention may
be useful in the fields of human medicine and veterinary medicine.
Thus, the subject to be immunized may be a human or other animal,
for example, farm animals including cows, sheep, pigs, horses,
goats and poultry (e.g., chickens, turkeys, ducks and geese)
companion animals such as dogs and cats; exotic and/or zoo animals;
and laboratory animals including mice, rats, rabbits, guinea pigs,
and hamsters.
[0034] The invention also provides polypeptides and corresponding
polynucleotides required for P. multocida or A. pleuropneumoniae
virulence. The invention includes both naturally occurring and
non-naturally occurring polynucleotides and polypeptide products
thereof. Naturally occurring virulence products include distinct
gene and polypeptide species as well as corresponding species
homologs expressed in organisms other than P. multocida or A.
pleuropneumoniae strains. Non-naturally occurring virulence
products include variants of the naturally occurring products such
as analogs and virulence products which include covalent
modifications. In a preferred embodiment, the invention provides
virulence polynucleotides comprising the sequences set forth in SEQ
ID NOs: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37,
39, 41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 163, and 164, and species
homologs thereof, and polypeptides having amino acids sequences
encoded by the polynucleotides.
[0035] The present invention provides novel purified and isolated
P. multocida and A. pleuropneumoniae polynucleotides (e.g., DNA
sequences and RNA transcripts, both sense and complementary
aritisense strands) encoding the bacterial virulence gene products.
DNA sequences of the invention include genomic and cDNA sequences
as well as wholly or partially chemically synthesized DNA
sequences. Genomic DNA of the invention comprises the protein
coding region for a polypeptide of the invention and includes
variants that may be found in other bacterial strains of the same
species. "Synthesized," as used herein and is understood in the
art, refers to purely chemical, as opposed to enzymatic, methods
for producing polynucleotides. "Wholly" synthesized DNA sequences
are therefore produced entirely by chemical means, and "partially"
synthesized DNAs embrace those wherein only portions of the
resulting DNA were produced by chemical means. Preferred DNA
sequences encoding P. multocida virulence gene products are set out
in SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31,
33, 37, 39, 41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80,
82, 84, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120,
122, 124, 126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144,
146, 148, 150, 152, 154, 156, 158, 160, 162, 163, and 164, and
species homologs thereof. Preferred A. pleuropneumoniae DNA
sequences encoding virulence gene products are set out in SEQ ID
NOs: 122, 124, 126, 128, 130, 132, 134, 135, 136, 138, 140, 142,
144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 163, and 164, and
species homologs thereof. The worker of skill in the art will
readily appreciate that the preferred DNA of the invention
comprises a double stranded molecule, for example, molecules having
the sequences set forth in SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58, 60, 68,
70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108, 110, 112,
114, 116, 118, and 120, 122, 124, 126, 128, 130, 132, 134, 135,
136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,
162, 163, and 164, and species homologs thereof, along with the
complementary molecule (the "non-coding strand" or "complement")
having a sequence deducible from the sequence of SEQ ID NO: 1, 3,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51,
53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102,
104, 106, 108, 110, 112, 114, 116, 118, and 120, 122, 124, 126,
128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146, 148, 150,
152, 154, 156, 158, 160, 162, 163,and 164, according to
Watson-Crick base pairing rules for DNA. Also preferred are
polynucleotides encoding the gene products encoded by any one of
the polynucleotides set out in SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58, 60,
68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, and 120, 122, 124, 126, 128, 130, 132, 134,
135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162, 163, and 164, and species homologs thereof. The invention
further embraces species, preferably bacterial, homologs of the P.
multocida and A. pleuropneumoniae DNA.
[0036] The polynucleotide sequence information provided by the
invention makes possible the identification and isolation of
polynucleotides encoding related bacterial virulence molecules by
well known techniques including Southern and/or Northern
hybridization, and polymerase chain reaction (PCR). Examples of
related polynucleotides include polynucleotides encoding
polypeptides homologous to a virulence gene product encoded by any
one of the polynucleotides set out in SEQ ID NOs: 1, 3, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57,
58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, and 120, 122, 124, 126, 128, 130,
132, 134, 135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,
156, 158, 160, 162, 163, and 164, and species homologs thereof, and
structurally related polypeptides sharing one or more biological
and/or physical properties of a virulence gene product of the
invention.
[0037] The invention also embraces DNA sequences encoding bacterial
gene products which hybridize under moderately to highly stringent
conditions to the non-coding strand, or complement, of any one of
the polynucleotides set out in SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58, 60,
68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, and 120, 122, 124, 126, 128, 130, 132, 134,
135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162, 163, and 164, and species homologs thereof DNA sequences
encoding virulence polypeptides which would hybridize thereto but
for the degeneracy of the genetic code are contemplated by the
invention. Exemplary high stringency conditions include a final
wash in buffer comprising 0.2..times..SSC/0.1% SDS, at 65.degree.
C. to 75.degree. C., while exemplary moderate stringency conditions
include a final wash in buffer comprising 2..times..SSC/0.1% SDS,
at 35.degree. C. to 45.degree. C. It is understood in the art that
conditions of equivalent stringency can be achieved through
variation of temperature and buffer, or salt concentration as
described in Ausubel, et al. (Eds.), Protocols in Molecular
Biology, John Wiley and & Sons (1994), pp. 6.0.3 to 6.4.10.
Modifications in hybridization conditions can be empirically
determined or precisely calculated based on the length and the
percentage of guanosine/cytosine (GC) base pairing of the probe.
The hybridization conditions can be calculated as described in
Sambrook, et al., (Eds.), Molecular Cloning: A Labratory Manual,
Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.
(1989), pp. 9;47 to 9.51.
[0038] Autonomously replicating recombinant expression
constructions such as plasmid and viral DNA vectors incorporating
virulence gene sequences are also provided. Expression constructs
wherein virulence polypeptide-encoding polynucleotides are
operatively linked to an endogenous or exogenous expression control
DNA sequence and a transcription terminator are also provided. The
virulence genes may be cloned by PCR, using P. multocida genomic
DNA as the template. For ease of inserting the gene into expression
vectors, PCR primers are chosen so that the PCR-amplified gene has
a restriction enzyme site at the 5' end preceding the initiation
codon ATG, and a restriction enzyme site at the 3' end after the
termination codon TAG, TGA or TAA. If desirable, the codons in the
gene are changed, without changing the amino acids, according to E.
coli codon preference described by Grosjean and Fiers, Gene,
18:199-209 (1982), and Konigsberg and Godson, Proc. Natl. Acad.
Sci. (USA), 80:687-691 (1983). Optimization of codon usage may lead
to an increase in the expression of the gene product when produced
in E. coli. If the gene product is to be produced extracellularly,
either in the periplasm of E. coli or other bacteria, or into the
cell culture medium, the gene is cloned without its initiation
codon and placed into an expression vector behind a signal
sequence.
[0039] According to another aspect of the invention, host cells are
provided, including procaryotic and eukaryotic cells, either stably
or transiently transformed, transfected, or electroporated with
polynucleotide sequences of the invention in a manner which permits
expression of virulence polypeptides of the invention. Expression
systems of the invention include bacterial, yeast, fungal, viral,
invertebrate, and mammalian cells systems. Host cells of the
invention are a valuable source of immunogen for development of
antibodies specifically immunoreactive with the virulence gene
product. Host cells of the invention are conspicuously useful in
methods for large scale production of virulence polypeptides
wherein the cells are grown in a suitable culture medium and the
desired polypeptide products are isolated from the cells or from
the medium in which the cells are grown by, for example,
immunoaffinity purification or any of the multitude of purification
techniques well known and routinely practiced in the art. Any
suitable host cell may be used for expression of the gene product,
such as E. coli, other bacteria, including P. multocida, Bacillus
and S. aureus, yeast, including Pichia pastoris and Saccharomyces
cerevisiae, insect cells, or mammalian cells, including CHO cells,
utilizing suitable vectors known in the art. Proteins may be
produced directly or fused to a peptide or polypeptide, and either
intracellularly or extracellularly by secretion into the
periplasmic space of a bacterial cell or into the cell culture
medium. Secretion of a protein requires a signal peptide (also
known as pre-sequence); a number of signal sequences from
prokaryotes and eukaryotes are known to function for the secretion
of recombinant proteins. During the protein secretion process, the
signal peptide is removed by signal peptidase to yield the mature
protein.
[0040] To simplify the protein purification process, a purification
tag may be added either at the 5' or 3' end of the gene coding
sequence. Commonly used purification tags include a stretch of six
histidine residues (U.S. Pat. Nos. 5,284,933 and 5,310,663), a
streptavidin-affinity tag described by Schmidt and Skerra, Protein
Engineering, 6:109-122 (1993), a FLAG peptide [Hopp et al.,
Biotechnology, 6:1205-1210 (1988)], glutathione S-transferase
[Smith and Johnson, Gene, 67:31-40 (1988)], and thioredoxin
[LaVallie et al., Bio/Technology, 11:187-193 (1993)]. To remove
these peptide or polypeptides, a proteolytic cleavage recognition
site may be inserted at the fusion junction. Commonly used
proteases are factor Xa, thrombin, and enterokinase.
[0041] The invention also provides purified and isolated P.
multocida and A. pleuropneumoniae virulence polypeptides encoded by
a polynucleotide of the invention. Presently preferred are
polypeptides comprising the amino acid sequences encoded by any one
of the polynucleotides set out in SEQ ID NOs: 1, 3, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41 51, 53, 55, 57, 58,
60, 68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, and 120, 122, 124, 126, 128, 130, 132,
134, 135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, and 164, and species homologs thereof. The invention
embraces virulence polypeptides encoded by a DNA selected from the
group consisting of: a) the DNA sequence set out in any one of SEQ
ID NOs: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37,
39, 41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84,
100, 102, 104, 106 108, 110, 112, 114, 116, 118, and 120, 122, 124,
126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, and 164, and species homologs
thereof; b) DNA molecules encoding P. multocida or A.
pleuropneumoniae polypeptides encoded by any one of SEQ ID NOs: 1,
3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41,
51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84, 100,
102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, 122, 124,
126, 128, 130, 132, 134, 135, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, and 164, and species homologs
thereof; and c) a DNA molecule, encoding a virulence gene product,
that hybridizes under moderately stringent conditions to the DNA of
(a) or (b).
[0042] The invention also embraces polypeptides, i.e., species
homologs and orthologs, that have at least about 99%, at least
about 95%, at least about 90%, at least about 85%, at least about
80%, at least about 75%, at least about 70%, at least about 65%, at
least about 60%, at least about 55%, and at least about 50%
identity and/or homology to the preferred polypeptides of the
invention. Percent amino acid sequence "identity" with respect to
the preferred polypeptides of the invention is defined herein as
the percentage of amino acid residues in the candidate sequence
that are identical with the residues in the virulence gene product
sequence after aligning both sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Percent sequence "homology" with respect to the
preferred polypeptides of the invention is defined herein as the
percentage of amino acid residues in the candidate sequence that
are identical with the residues in one of the virulence polypeptide
sequences after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
also considering any conservative substitutions as part of the
sequence identity. Conservative substitutions can be defined as set
out in Tables A and B.
TABLE-US-00001 TABLE A Conservative Substitutions I SIDE CHAIN
CHARACTERISTIC AMINO ACID Aliphatic Non-polar G, A, P I, L, V
Polar- uncharged C, S, T, M N, Q Polar - charged D, E K, R Aromatic
H, F, W, Y Other N, Q, D, E
[0043] Polypeptides of the invention may be isolated from natural
bacterial cell sources or may be chemically synthesized, but are
preferably produced by recombinant procedures involving host cells
of the invention. Virulence gene products of the invention may be
full length polypeptides, biologically active fragments, or
variants thereof which retain specific biological or immunological
activity. Variants may comprise virulence polypeptide analogs
wherein one or more of the specified (i.e., naturally encoded)
amino acids is deleted or replaced or wherein one or more
non-specified amino acids are added: (1) without loss of one or
more of the biological activities or immunological characteristics
specific for the virulence gene product; or (2) with specific
disablement of a particular biological activity of the virulence
gene product. Deletion variants contemplated also include fragments
lacking portions of the polypeptide not essential for biological
activity, and insertion variants include fusion polypeptides in
which the wild-type polypeptide or fragment thereof have been fused
to another polypeptide.
[0044] Variant virulence polypeptides include those wherein
conservative substitutions have been introduced by modification of
polynucleotides encoding polypeptides of the invention.
Conservative substitutions are recognized in the art to classify
amino acids according to their related physical properties and can
be defined as set out in Table A (from WO 97/09433, page 10,
published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996).
Alternatively, conservative amino acids can be grouped as defined
in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc.
NY:N.Y. (1975), pp.71-T7] as set out in Table B.
TABLE-US-00002 TABLE B Conservative Substitutions II SIDE CHAIN
CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: A,
L, I, V, P B. Aromatic: F, W C. Sulfur-containing: M D. Borderline:
G Uncharged-polar A. Hydroxyl: S, T, Y B. Amides: N, Q C.
Sulfhydryl: C D. Borderline: G Positively Charged (Basic): K, R, H
Negatively Charged (Acidic): D, E
[0045] Variant virulence products of the invention include mature
virulence gene products, i.e., wherein leader or signal sequences
are removed, having additional amino terminal residues. Virulence
gene products having an additional methionine residue at position-1
are contemplated, as are virulence products having additional
methionine and lysine residues at positions-2 and -1. Variants of
these types are particularly useful for recombinant protein
production in bacterial cell types. Variants of the invention also
include gene products wherein amino terminal sequences derived from
other proteins have been introduced, as well as variants comprising
amino terminal sequences that are not found in naturally occurring
proteins.
[0046] The invention also embraces variant polypeptides having
additional amino acid residues which result from use of specific
expression systems. For example, use of commercially available
vectors that express a desired polypeptide as a fusion protein with
glutathione-S-transferase (GST) provide the desired polypeptide
having an additional glycine residue at position-1 following
cleavage of the GST component from the desired polypeptide.
Variants which result from expression using other vector systems
are also contemplated.
[0047] Also comprehended by the present invention are antibodies
(e.g., monoclonal and polyclonal antibodies, single chain
antibodies, chimeric antibodies, humanized, human, and CDR-grafted
antibodies, including compounds which include CDR sequences which
specifically recognize a polypeptide of the invention) and other
binding proteins specific for virulence gene products or fragments
thereof. The term "specific for" indicates that the variable
regions of the antibodies of the invention recognize and bind a
virulence polypeptide exclusively (i.e., are able to distinguish a
single virulence polypeptides from related virulence polypeptides
despite sequence identity, homology, or similarity found in the
family of polypeptides), but may also interact with other proteins
(for example, S. aureus protein A or other antibodies in ELISA
techniques) through interactions with sequences outside the
variable region of the antibodies, and in particular, in the
constant region of the molecule. Screening assays to determine
binding specificity of an antibody of the invention are well known
and routinely practiced in the art. For a comprehensive discussion
of such assays, see Harlow et al. (Eds), Antibodies A Laboratory
Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.
(1988), Chapter 6. Antibodies that recognize and bind fragments of
the virulence polypeptides of the invention are also contemplated,
provided that the antibodies are first and foremost specific for,
as defined above, a virulence polypeptide of the invention from
which the fragment was derived.
[0048] The DNA and amino acid sequence information provided by the
present invention also makes possible the systematic analysis of
the structure and function of the virulence genes and their encoded
gene products. Knowledge of a polynucleotide encoding a virulence
gene product of the invention also makes available anti-sense
polynucleotides which recognize and hybridize to polynucleotides
encoding a virulence polypeptide of the invention. Full length and
fragment anti-sense polynucleotides are provided. The worker of
ordinary skill will appreciate that fragment anti-sense molecules
of the invention include (i) those which specifically recognize and
hybridize to a specific RNA (as determined by sequence comparison
of DNA encoding a virulence polypeptide of the invention to DNA
encoding other known molecules) as well as (ii) those which
recognize and hybridize to RNA encoding variants of the family of
virulence proteins. Antisense polynucleotides that hybridize to RNA
encoding other members of the virulence family of proteins are also
identifiable through sequence comparison to identify
characteristic, or signature, sequences for the family of
molecules.
[0049] The invention further contemplates methods to modulate gene
expression through use of ribozymes. For a review, see Gibson and
Shillitoe, Mol. Biotech. 7:125-137 (1997). Ribozyme technology can
be utilized to inhibit translation of mRNA in a sequence specific
manner through (i) the hybridization of a complementary RNA to a
target mRNA and (ii) cleavage of the hybridized mRNA through
nuclease activity inherent to the complementary strand. Ribozymes
can be identified by empirical methods but more preferably are
specifically designed based on accessible sites on the target mRNA
[Bramlage, et al., Trends in Biotech 16:434-438 (1998)]. Delivery
of ribozymes to target cells can be accomplished using either
exogenous or endogenous delivery techniques well known and
routinely practiced in the art. Exogenous delivery methods can
include use of targeting liposomes or direct local injection.
Endogenous methods include use of viral vectors and non-viral
plasmids.
[0050] Ribozymes can specifically modulate expression of virulence
genes when designed to be complementary to regions unique to a
polynucleotide encoding a virulence gene product. "Specifically
modulate" therefore is intended to mean that ribozymes of the
invention recognizes only a single polynucleotide. Similarly,
ribozymes can be designed to modulate expression of all or some of
a family of proteins. Ribozymes of this type are designed to
recognize polynucleotide sequences conserved in all or some of the
polynucleotides which encode the family of proteins.
[0051] The invention further embraces methods to modulate
transcription of a virulence gene of the invention through use of
oligonucleotide-directed triplet helix formation. For a review, see
Lavrovsky, et al., Biochem. Mol. Med. 62:11-22 (1997). Triplet
helix formation is accomplished using sequence specific
oligonucleotides which hybridize to double stranded DNA in the
major groove as defined in the Watson-Crick model. Hybridization of
a sequence specific oligonucleotide can thereafter modulate
activity of DNA-binding proteins, including, for example,
transcription factors and polymerases. Preferred target sequences
for hybridization include transcriptional regulatory regions that
modulate virulence gene product expression. Oligonucleotides which
are capable of triplet helix formation are also useful for
site-specific covalent modification of target DNA sequences.
Oligonucleotides useful for covalent modification are coupled to
various DNA damaging agents as described in Lavrovsky, et al.
[supra].
[0052] The identification of P. multocida and A. pleuropneumoniae
virulence genes renders the genes and gene products useful in
methods for identifying anti-bacterial agents. Such methods include
assaying potential agents for the ability to interfere with
expression of virulence gene products represented by the DNA
sequences set forth in any one of SEQ ID NOS: 1, 3, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58,
60, 68, 70, 72, 74, 76, 78, 80, 82 84, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, and 120, 122, 124, 126, 128, 130, 132,
134, 135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, 162, 163, and 164, and species homologs thereof (i.e.,
the genes represented by DNA sequences of SEQ ID NOS: 1, 3, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55,
57, 58, 60, 68, 70, 72, 74, 76, 78, 80, 82, 84 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, and 120, 122, 124, 126, 128, 130,
132, 134, 135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,
156, 158, 160, 162, 163, and 164, encode the virulence gene
product, or the DNA sequences of SEQ ID NOS: 1, 3, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58,
60, 68, 70, 72, 74, 76, 78, 80, 82, 84, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, and 120, 122, 124, 126, 128, 130, 132,
134, 135, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, 162, 163, and 164, are adjacent the gene encoding the
virulence gene product, or are involved in regulation of expression
of the virulence gene product), or assaying potential agents for
the ability to interfere with the function of a bacterial gene
product encoded in whole or in part by a DNA sequence set forth in
any one of SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
29, 31, 33, 37, 39, 41, 51, 53, 55, 57, 58, 60, 68, 70, 72, 74, 76,
78, 80, 82, 84, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,
and 120, 122, 124, 126, 128, 130, 132, 134, 135, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 163, and
164, species homologs thereof, or the complementary strand thereof,
followed by identifying agents that are positive in such assays.
Polynucleotides and polypeptides useful, in these assays include
not only the genes and encoded polypeptides as disclosed herein,
but also variants thereof that have substantially the same activity
as the wild-type genes and polypeptides.
[0053] The virulence gene products produced by the methods
described above are used in high throughput assays to screen for
inhibitory agents. The sources for potential agents to be screened
are chemical compound libraries, fermentation media of
Streptomycetes, other bacteria and fungi, and cell extracts of
plants and other vegetations. For proteins with known enzymatic
activity, assays are established based on the activity, and a large
number of potential agents are screened for ability to inhibit the
activity. For proteins that interact with another protein or
nucleic acid, binding assays are established to measure such
interaction directly, and the potential agents are screened for
ability to inhibit the binding interaction.
[0054] The use of different assays known in the art is contemplated
according to this aspect of the invention. When the function of the
virulence gene product is known or predicted by sequence similarity
to a known gene product, potential inhibitors can be screened in
enzymatic or other types of biological and/or biochemical assays
keyed to the function and/or properties of the gene product. When
the virulence gene product is known or predicted by sequence
similarity to a known gene product to interact with another protein
or nucleic acid, inhibitors of the interaction can be screened
directly in binding assays. The invention contemplates a multitude
of assays to screen and identify inhibitors of binding by the
virulence gene product. In one example, the virulence gene product
is immobilized and interaction with a binding partner is assessed
in the presence and absence of a putative inhibitor compound. In
another example, interaction between the virulence gene product and
its binding partner is assessed in a solution assay, both in the
presence and absence of a putative inhibitor compound. In both
assays, an inhibitor is identified as a compound that decreases
binding between the virulence gene product and its binding partner.
Other assays are also contemplated in those instances wherein the
virulence gene product binding partner is a protein. For example,
variations of the di-hybrid assay are contemplated wherein an
inhibitor of protein/protein interactions is identified by
detection of a positive signal in a transformed or transfected host
cell as described in PCT publication number WO 95/20652, published
Aug. 3, 1995.
[0055] Candidate inhibitors contemplated by the invention include
compounds selected from libraries of potential inhibitors. There
are a number of different libraries used for the identification of
small molecule modulators, including: (1) chemical libraries, (2)
natural product libraries, and (3) combinatorial libraries
comprised of random peptides, oligonucleotides or organic
molecules. Chemical libraries consist of structural analogs of
known compounds or compounds that are identified as "hits" or
"leads" via natural product screening. Natural product libraries
are collections of microorganisms, animals, plants, or marine
organisms which are used to create mixtures for screening by: (1)
fermentation and extraction of broths from soil, plant or marine
microorganisms or (2) extraction of plants or marine organisms.
Natural product libraries include polyketides, non-ribosomal
peptides, and variants (non-naturally occurring) thereof. For a
review, see Science 282:63-68 (1998). Combinatorial libraries are
composed of large numbers of peptides, oligonucleotides, or organic
compounds as a mixture. They are relatively easy to prepare by
traditional automated synthesis methods, PCR, cloning, or
proprietary synthetic methods. Of particular interest are peptide
and oligonucleotide combinatorial libraries. Still other libraries
of interest include peptide, protein, peptidomimetic, multiparallel
synthetic collection, recombinatorial, and polypeptide libraries.
For a review of combinatorial chemistry and libraries created
therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).
Identification of modulators through use of the various libraries
described herein permits modification of the candidate "hit" (or
"lead") to optimize the capacity of the "hit" to modulate activity.
Still other candidate inhibitors contemplated by the invention-can
be designed and include soluble forms of binding partners, as well
as binding partners as chimeric, or fusion, proteins. Binding
partners as used herein broadly encompasses antibodies, antibody
fragments, and modified compounds comprising antibody domains that
are immunospecific for the expression product of the identified
virulence gene.
[0056] Other assays may be used when a binding partner (i.e.,
ligand) for the virulence gene product is not known, including
assays that identify binding partners of the target protein through
measuring direct binding of test binding partner to the target
protein, and assays that identify binding partners of target
proteins through affinity ultrafiltration with ion spray mass
spectroscopy/HPLC methods or other physical and analytical methods.
Alternatively, such binding interactions are evaluated indirectly
using the yeast two-hybrid system described in Fields and Song,
Nature, 340:245-246 (1989), and Fields and Sternglanz, Trends in
Genetics, 10:286-292 (1994), both of which are incorporated herein
by reference. The two-hybrid system is a genetic assay for
detecting interactions between two proteins or polypeptides. It can
be used to identify proteins that bind to a known protein of
interest, or to delineate domains or residues critical for an
interaction. Variations on this methodology have been developed to
clone genes that encode DNA-binding proteins, to identify peptides
that bind to a protein, and to screen for drugs. The two-hybrid
system exploits the ability of a pair of interacting proteins to
bring a transcription activation domain into close proximity with a
DNA-binding domain that binds to an upstream activation sequence
(UAS) of a reporter gene, and is generally performed in yeast. The
assay requires the construction of two hybrid genes encoding (1) a
DNA-binding domain that is fused to a first protein and (2) an
activation domain fused to a second protein. The DNA-binding domain
targets the first hybrid protein to the UAS of the reporter gene;
however, because most proteins lack an activation domain, this
DNA-binding hybrid protein does not activate transcription of the
reporter gene. The second hybrid protein, which contains the
activation domain, cannot by itself activate expression of the
reporter gene because it does not bind the UAS. However, when both
hybrid proteins are present, the noncovalent interaction of the
first and second proteins tethers the activation domain to the UAS,
activating transcription of the reporter gene. When the virulence
gene product (the first protein, for example) is already known to
interact with another protein or nucleic acid, this assay can be
used to detect agents that interfere with the binding interaction.
Expression of the reporter gene is monitored as different test
agents are added to the system; the presence of an inhibitory agent
results in lack of a reporter signal.
[0057] When the function of the virulence gene product is unknown
and no ligands are known to bind the gene product, the yeast
two-hybrid assay can also be used to identify proteins that bind to
the gene product. In an assay to identify proteins that bind to the
first protein (the target protein), a large number of hybrid genes
each encoding different second proteins are produced and screened
in the assay. Typically, the second protein is encoded by a pool of
plasmids in which total cDNA or genomic DNA is ligated to the
activation domain. This system is applicable to a wide variety of
proteins, and it is not even necessary to know the identity or
function of the second binding protein. The system is highly
sensitive and can detect interactions not revealed by other
methods; even transient interactions may trigger transcription to
produce a stable mRNA that can be repeatedly translated to yield
the reporter protein.
[0058] Other assays may be used to search for agents that bind to
the target protein. One such screening method to identify direct
binding of test ligands to a target protein is described in U.S.
Pat. No. 5,585,277, incorporated herein by reference. This method
relies on the principle that proteins generally exist as a mixture
of folded and unfolded states, and continually alternate between
the two states. When a test ligand binds to the folded form of a
target protein (i.e., when the test ligand is a ligand of the
target protein), the target protein molecule bound by the ligand
remains in its folded state. Thus, the folded target protein is
present to a greater extent in the presence of a test ligand which
binds the target protein, than in the absence of a ligand. Binding
of the ligand to the target protein can be determined by any method
which distinguishes between the folded and unfolded states of the
target protein. The function of the target protein need not be
known in order for this assay to be performed. Virtually any agent
can be assessed by this method as a test ligand, including, but not
limited to, metals, polypeptides, proteins, lipids,
polysaccharides, polynucleotides and small organic molecules.
[0059] Another method for identifying ligands for a target protein
is described in Wieboldt et al., Anal. Chem., 69:1683-1691 (1997),
incorporated herein by reference. This technique screens
combinatorial libraries of 20-30 agents at a time in solution phase
for binding to the target protein. Agents that bind to the target
protein are separated from other library components by centrifugal
ultrafiltration. The specifically selected molecules that are
retained on the filter are subsequently liberated from the target
protein and analyzed by HPLC and pneumatically assisted
electrospray (ion spray) ionization mass spectroscopy. This
procedure selects library components with the greatest affinity for
the target protein, and is particularly useful for small molecule
libraries.
[0060] The inhibitors/binders identified by the initial screens are
evaluated for their effect on virulence in in vivo mouse models of
P. multocida infections. Models of bacteremia, endocarditis, septic
arthritis, soft tissue abscess, or pneumonia may be utilized.
Models involving use of other animals are also comprehended by the
invention. For example, rabbits can be challenged with a wild type
P. multocida strain before or after administration of varying
amounts of a putative inhibitor/binder compound. Control animals,
administered only saline instead of putative inhibitor/binder
compound provide a standard by which deterioration of the test
animal can be determined. Other animal models include those
described in the Animal and Plant Health Inspection Service, USDA,
Jan. 1, 1994 Edition,.sctn.sctn.113.69-113.70; Panciera and
Corstvet, Am. J. Vet. Res. 45:2532-2537; Ames, et al., Can. J.
Comp. Med. 49:395-400 (1984); and Mukkur, Infection and Immunity
18:583-585 (1977). Inhibitors/binders that interfere with bacterial
virulence are can prevent the establishment of an infection or
reverse the outcome of an infection once it is established.
[0061] Any adjuvant known in the art may be used in the vaccine
composition, including oil-based adjuvants such as Freund's
Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based
adjuvants (e.g., trehalose dimycolate), bacterial
lipopolysaccharide (LPS), peptidoglycans (i.e., mureins,
mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide
[MDP], or MDP analogs), proteoglycans (e.g., extracted from
Klebsiella pneumoniae), streptococcal preparations (e.g. OK432),
Biostim..TM.. (e.g., 01K2), the "Iscoms" of EP 109 942, EP 180 564
and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral
oils (such as miglyol), vegetable oils (such as arachis oil),
liposomes, Pluronic polyols, the Ribi adjuvant system (see, for
example GB-A-2 189 141), or interleukins, particularly those that
stimulate cell mediated immunity. An alternative adjuvant
consisting of extracts of Amycolata, a bacterial genus in the order
Actinomycetales, has been described in U.S. Pat. No. 4,877,612.
Additionally, proprietary adjuvant mixtures are commercially
available. The adjuvant used will depend, in part, on the recipient
organism. The amount of adjuvant to administer will depend on the
type and size of animal. Optimal dosages may be readily determined
by routine methods.
[0062] The vaccine compositions optionally may include
vaccine-compatible pharmaceutically acceptable (i.e., sterile and
non-toxic) liquid, semisolid, or solid diluents that serve as
pharmaceutical vehicles, excipients, or media. Any diluent known in
the art may be used. Exemplary diluents include, but are not
limited to, polyoxyethylene sorbitan monolaurate, magnesium
stearate, methyl- and propylhydroxybenzoate, talc, alginates,
starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum
acacia, calcium phosphate, mineral oil, cocoa butter, and oil of
the obroma.
[0063] The vaccine compositions can be packaged in forms convenient
for delivery. The compositions can be enclosed within a capsule,
caplet, sachet, cachet, gelatin, paper, or other container. These
delivery forms are preferred when compatible with entry of the
immunogenic composition into the recipient organism and,
particularly, when the immunogenic composition is being delivered
in unit dose form. The dosage units can be packaged, e.g., in
tablets, capsules, suppositories or cachets.
[0064] The vaccine compositions may be introduced into the subject
to be immunized by any conventional method including, e.g., by
intravenous, intradermal, intramuscular, intramammary,
intraperitoneal, or subcutaneous injection; by oral, sublingual,
nasal, anal, or vaginal, delivery. The treatment may consist of a
single dose or a plurality of doses over a period of time.
[0065] The invention also comprehends use of an attenuated
bacterial strain of the invention for manufacture of a vaccine
medicament to prevent or alleviate bacterial infection and/or
symptoms associated therewith. The invention also provides use of
inhibitors of the invention for manufacture of a medicament to
prevent or alleviate bacterial infection and/or symptoms associated
therewith.
[0066] The present invention is illustrated by the following
examples. Example 1 describes constructions of P. multocida
mutants. Example 2 relates to screening for P. multocida mutants.
Example 3 addresses methods to determine virulence of the P.
multocida mutants. Example 4 describes cloning of P. multocida
virulence genes. Example 5 addresses identification of genes in
other species related to P. multocida virulence genes. Example 6
describes construction of A. pleuropneumoniae mutants. Example 7
addresses screening for attenuated A. pleuropneumoniae mutants.
Example 8 relates to identification of A. pleuropneumoniae
virulence genes. Example 9 describes competition challenge of A.
pleuropneumoniae mutants and wild type bacteria. Example 10
characterizes A. pleuropneumoniae genes identified. Example 11
addresses efficacy of A. pleuropneumoniae mutant to protect against
wild type bacterial challenge.
Example 1
Construction of a Library of Tagged-Transposon P. multocida
Mutants
[0067] A library of tagged-transposon mutants was constructed in
parental vector pLOF/Km [Herrero, et al., J Bacteriol. 172:6557-67
(1990)] which has previously been demonstrated to be functional and
random in P. multocida [Lee, et al., Vet Microbiol. 50:143-8
(1996)]. Plasmid pLOF/Km was constructed as a modification of
suicide vector pGP704 and included a transposase gene under control
of the Tac promoter as well as the mini-Tn10 transposable element
encoding kanamycin resistance. Plasmid pTEF-1 was constructed as
described below by modifying pLOF/Km to accept sequence tags which
contained a semi-random [NK].sub.35 sequence.
[0068] Plasmid pLOF/Km was first modified to eliminate the unique
KpnI restriction site in the multiple cloning region and then to
introduce a new KpnI site in the mini-Tn10 region. The plasmid was
digested with KpnI and the resulting overhanging ends Were filled
in with Klenow polymerase according to manufacturer's suggested
protocol. Restriction digests and ligations described herein were
performed according to manufacturer's suggested protocols (Gibco
BRL, Gaithersburg, Md. and Boehringer Mannheim, Indianapolis,
Ind.). The blunt end product was self-ligated to produce a plasmid
designated pLOF/Km-KpnI which was transformed into E. coli DH5
alpha:.lamda.pir for amplification. E. coli DH5.alpha.: (.lamda.pir
.PHI.80dlacZ.DELTA.M15, recA1, endA1, gyrA96, thi-1,
hsdR17(r.sub.k-, m.sub.k, supE44, relA1, deoR,
.DELTA.(lacZYA-argF)U169, was propagated at 37.degree. C. in
Luria-Bertani (LB) medium. Plasmids were prepared using QIAGEN
SpinPreps from QIAGEN Inc. (Santa Clarita, Calif.) and digested
with SfiI which cuts at a unique site within the mini-Tn10
transposable element. A SfiI-KpnI-SfiI adaptor was prepared by
annealing oligonucleotides TEF1 (SEQ ID NO: 86) and TEF3 (SEQ ID
NO: 87) and the resulting double-stranded adapter was ligated into
the SfiI site to create plasmid pTEF-1. Oligonucleotides TEF1 and
TEF3 (as well as all other oligonucleotides described herein) were
synthesized by Genosys Biotechnologies (The Woodlands, Tex.).
TABLE-US-00003 TEF1 5'-AGGCCGGTACCGGCCGCCT SEQ ID NO: 86 TEF3
5'-CGGCCGGTACCGGCCTAGG SEQ ID NO: 87
[0069] Unique sequence tags for insertion into the KpnI site of
pTEF-1 were prepared as follows. PCR was carried out to generate
double stranded DNA tags using a GeneAmp XL PCR Kit (PE Applied
Biosystems, Foster City, Calif.) under conditions including 250
.mu.M each dNTP, 1.5 mM Mg(OAc).sub.2, 100 pmol each primer TEF14
(SEQ ID NO: 88) and TEF15 (SEQ ID NO: 89), 1 ng TEF26 (SEQ ID NO:
90) as template DNA and 2.5 units recombinant Tth DNA Polymerase
XL.
TABLE-US-00004 TEF14 SEQ ID NO: 88 5'-CATGGTACCCATTCTAAC TEF15 SEQ
ID NO: 89 5'-CTAGGTACCTACAACCTC TEF26 SEQ ID NO: 90
5'-CTAGGTACCTACAACCTCAAGCTT-[NK].sub.35-AAGCTTGGTTAG
AATGGGTACCATG
Reaction conditions included an initial incubation at 95.degree. C.
for one minute, followed by thirty cycles of 30 seconds at
95.degree. C., 45 seconds at 45.degree. C., and 15 seconds at
72.degree. C., followed by a final incubation at 72.degree. C. for
two minutes. The PCR products were digested with KpnI and purified
using a QIAGEN Nucleotide Removal Kit (QIAGEN, Inc., Chatsworth,
Ga.) according to the manufacturer's suggested protocol. The unique
tag sequences were ligated into the mini-Tn10 element of linearized
pTEF-1, previously digested with KpnI and dephosphorylated with
calf intestinal alkaline phosphatase (Boehringer Mannheim) using
standard procedures. The resulting plasmid library was transformed
into E. coli DH5.alpha.:.lamda.pir. Colony blot analysis was
performed according to the DIG User's Guide (Boehringer-Mannheim)
with hybridization and detection performed as follows.
[0070] Hybridizations were essentially performed according to the
Genius Non-Radioactive User's Guide (Boehringer Mannheim
Biochemicals), the product sheet for the DIG-PCR labeling kit
(Boehringer Mannheim Biochemicals), and the product sheet for CSPD
(Boehringer Mannheim Biochemicals). For preparation of probes, a
100 .mu.l primary PCR reaction was set up using Amplitaq PCR buffer
(PE Applied Biosystems), 200 .mu.M dNTPs, 140 pmol each of primers
TEFS (SEQ ID NO: 91) and TEF6 (SEQ ID NO: 92),2 mM MgCl.sub.2, 2.5
units Amplitaq (PE Applied Biosystems) and 1 ng of plasmid DNA.
TABLE-US-00005 TEF5 5'-TACCTACAACCTCAAGCT SEQ ID NO: 91 TEF6
5'-TACCCATTCTAACCAAGC SEQ ID NO: 92
[0071] Cycle conditions included an initial incubation at
95.degree. C. for two minutes, followed by 35 cycles of 95.degree.
C. for 30 seconds, 50.degree. C. for 45 seconds, 72.degree. C. for
15 seconds and a final incubation at 72.degree. C. for three
minutes. The amplification products were separated using
electrophoresis on a 2%-3:1 NuSieve GTG (FMC BioProducts, Rockland,
Me., USA):Agarose gel and the 109 by product was excised and
purified. Gel extractions were carried out using a QIAGEN Gel
Extraction kit (QIAGEN). Approximately 15 ng of the primary product
was labeled in a 50 .mu.l PCR reaction using the DIG PCR Kit, 50
pmol each of primers TEF24 and TEF25, and a 1:1 mix of DIG Probe
Synthesis Mix with 2 mM dNTP stock solution.
TABLE-US-00006 TEF24 5'-TACCTACAACCTCAAGCTT SEQ ID NO: 93 TEF25
5'-TACCCATTCTAACCAAGCTT SEQ ID NO: 94
PCR conditions included an initial incubation at 95.degree. C. for
four minutes, followed by 25 cycles of 95.degree. C. for 30
seconds, 50.degree. C. for 45 seconds, 72.degree. C. for 15 seconds
and a final incubation at 72.degree. C. for three minutes. The
labeled PCR product was digested with HindIll in a total reaction
volume of 90 .mu.l and purified from the constant primer arms using
a 2%-3:1 NuSieve GTG (FMC BioProducts):Agarose gel. The region
containing the labeled variable tag was excised and the entire gel
slice was dissolved and denatured in 10 ml of DIG EasyHyb at
95.degree. C. for ten minutes.
[0072] Dot blots were prepared using a Hybond.RTM.-N.sup.+ membrane
(Amersham-Pharmacia Biotech). Target DNA for each tag was prepared
in 96 well plates using approximately 30 ng of PCR product. An
equal volume of 0.1 N NaOH was added to denature the sample and
each sample was applied to the membrane with minimal vacuum using a
Minifold I.TM. Dot-Blot Apparatus from Schleicher and Schuell
(Keene, N.H., USA). Each well was washed with 150 .mu.l of
Neutralization Solution (0.5 M Tris/3 M NaCl, pH 7.5) and 150 .mu.l
of 2.times.SSC. Membranes were UV-crosslinked in a Stratalinker
(Stratagene, La Jolla, Calif., USA) and prehybridized for one hour
in 20 mls DIG EasyHyb Buffer at 42.degree. C. The denatured probe
was added and hybridization carried out overnight at 42.degree. C.
The membrane was washed two times in 2.times.SSC containing 0.1%
SDS for five minutes each wash. Two high stringency washes were
performed in 50 ml of pre-warmed 0.1.times.SSC buffer containing
0.1% SDS at 68.degree. C. for 15 minutes before proceeding with
standard Genius Detection protocols (Genius Manual).
[0073] It is desirable to use a non-radioactive detection system
for safety, lower cost, ease of use, and reduction of hazardous
materials. In initial experiments using similar procedures
previously described [Mei, et al., Mol Microbiol. 26:399-407
(1997)], unacceptable background levels of hybridization were
obtained in negative controls. In order to decrease background, tag
length was increased by 30 by to a total of 70, amplification
primers were lengthened to include all sequence flanking the
variable region, a lower concentration of dig-dUTP was used, and
the conserved sequences flanking the sequence tag region were
removed by gel purification. Most significantly, PCR was used to
generate [NK].sub.35 sequence tags as the target DNA in dot blots
rather than the entire plasmids containing the tagged transposons
after detecting background hybridization from the transposon itself
Using these modifications background was eliminated making
chemiluminescent/non-radioactive screening more effective.
[0074] Approximately four hundred different transformants resulting
from the ligation of pTEF-1 with the PCR generated sequence tags
were screened by colony blot and the 96 strongest hybridizing
colonies were assembled into microtiter plates for further use.
Even though the likelihood of duplicated tags was very low, half of
the plate of master tags was probed against the other to confirm
that no tags were duplicated. The plasmids containing these tags
were purified and transformed into E. coli S17-1:.lamda.pir(pir,
recA, thi, pro, hsd, (r-m.sup.+), RP4-2, (Tc::Mu), (Km::Tn7),
[TmpR], [SmR]), and the transformed bacteria propagated at
37.degree. C. in Luria-Bertani (LB) medium. Each of the 96 E. coli
S17-1:.lamda.pir transformants containing the tagged plasmid pTEF-1
was used in conjugative matings to generate transposon mutants of
P. muitocida. P. muitocida strain TF5 is a spontaneous nalidixic
acid resistant mutant derived from UC6731, a bovine clinical
isolate. P. muitocida strains were grown on brain heart infusion
(BHI) media (Difco Laboratories, Detroit, Mich., USA) at 37.degree.
C. and in 5% CO.sub.2 when grown on plates. Matings were set up by
growing each E. coli S 17-1:.lamda.pir/pTEF1:[NK].sub.35 clone and
the TF5 strain to late log phase. Fifty .mu.l of culture for each
tagged-pTEF-1 clone, was mixed with 200 .mu.l of the TF5 culture
and 50 .mu.l of each mating mixture was spotted onto 0.22 TM
filters previously placed on BHI plates containing 100 mM IPTG and
10 mM MgSO.sub.4. Following overnight incubation at 37.degree. C.
with 5% CO.sub.2, mating mixtures were washed off of each filter
into 3 ml of PBS and 25 .mu.l of each was plated onto
BHIN.sup.50K.sup.100 plates. Following selective overnight growth,
colonies were assembled into microtiter plates by toothpick
transfer into 200 .mu.l BHIN.sup.50K.sup.50 making sure that each
well in a microtiter plate always contained a transposon mutant
with the same sequence tag. Following overnight growth, 50 .mu.l of
75% glycerol was added to each. well and plates were stored frozen
at -80.degree. C.
[0075] Nineteen pools were assembled by transferring the transposon
mutants to microtiter plates making sure that each well contained a
transposon mutant with the appropriate tag for that well. In other
words, a specific well in each microtiter plate always contained a
transposon mutant with the same sequence tag even though the
location of the transposon within those mutants may be
different.
Example 2
Murine Screening for Attenuated P. multocida Mutants
[0076] Nineteen pools of Pasteurella multocida transposon mutants
were screened using a murine model of septicemia. Frozen plates of
pooled P. multocida transposon mutants were removed from
-80.degree. C. storage and subcultured by transferring 10 .mu.l
from each well to a new 96 well round bottom plate (Corning Costar,
Cambridge, Mass., USA) containing 200 .mu.l of brain heart infusion
(DIFCO) with 50 .mu.g/ml nalidixic acid (Sigma) and 50 .mu.g/ml
kanamycin (Sigma)) (BHIN.sup.50K.sup.50. Plates were incubated
without shaking overnight at 37.degree. C. in 5% CO.sub.2.
Overnight plates were subcultured by transferring 10 .mu.l from
each well to a new flat bottomed 96-well plate (Corning Costar)
containing 100 .mu.l of BHI per well and incubating at 37.degree.
C. with shaking at approximately 150 rpm. The OD.sub.540 was
monitored using a micro-titer plate reader. At an OD.sub.540 of
approximately 0.2 to 0.25, each plate was pooled to form the "input
pool" by combining 100 .mu.l from each of the wells of the
micro-titer plate. The culture was diluted appropriately in BHI to
doses of approximately 10.sup.4, 10.sup.5, 10.sup.6 CFU/ml and 0.2
ml of each dilution was used to infect female 14-16 g BALB/c mice
by intraperitoneal administration. At two days post-infection, one
or two surviving mice were euthanized and the spleens harvested.
The entire spleen was homogenized in 1.0 ml sterile 0.9% saline.
Dilutions of the homogenate from 10-2 to 10-5 were prepared and
plated onto BHIN.sup.50K.sup.50 plates. Following overnight growth,
at least 20,000 colonies were pooled in 10 mls BHI broth to form
the "recovered pool" and 0.5 ml of the recovered pool was
centrifuged at 3,500.times.g and the pellet used to prepare genomic
DNA according to a previously described protocol [Wilson, In F. M.
Ausubel, et al., (ed.), Current Protocols in Molecular Biology,
vol. 1. John Wiley and Sons, New York, p. 2.4.1-2.4.5. (1997)].
[0077] Initial experiments with virulent wild-type P. multocida
indicated that organisms could be recovered from the spleen, lungs,
kidneys, and liver indicating a truly septicemic model of
infection. Dot blots for both the "input" and "recovered" pools
were performed as described in Example 1 and evaluated both by
visual inspection and by semi-quantitative analysis. Hybridization
was carried out as described in Example 1 except that 5 .mu.g of
genomic DNA from input and recovered pools was used as template.
Semi-quantitative analysis indicates whether a significant
reduction in a single clone has occurred. If a mutant is unable to
survive within the host, then the recovered signal should be very
low compared to the input signal yielding a high input/recovered
ratio. Most mutants will grow as well in vivo as in vitro and
therefore a ratio of their signals should be approximately equal to
1. Clones selected by quantitative analysis as being highly reduced
in the recovered pool were selected for further study. Additional
clones with questionable input/recovered ratios were also selected
after visually evaluating films made from the dot blots.
Example 3
Determination of Virulence for P. multocida Candidate Mutants
[0078] Each potential mutant which exhibited reduced recovery from
splenic tissue was isolated from the original pool plate and used
individually in a challenge experiment to verify and roughly
estimate the attenuation caused by the transposon mutation.
Individual candidate mutants from in vivo screens were grown on
Sheep Blood Agar plates overnight in 5% CO.sub.2 at 37.degree. C.
Approximately six colonies of each mutant were inoculated into BHI
broth and allowed to grow for six hours, Dilutions were prepared
and five mice each were infected as described above with 10.sup.2,
10.sup.3, 10.sup.4 and 10.sup.5 CFU each. Attenuation was
determined by comparing mortality after six days relative to the
wild type. Surviving mice were presumed to be protected and then
challenged with a dose of wild type P. multocida at a concentration
approximately 200-fold greater than the LD.sub.50 for the wild type
strain. Survival rate was then determined for each challenged group
of mice.
[0079] Results indicated that 62 of 120 potential transposon
mutants were attenuated having an approximate LD.sub.50 of at
least. 10 fold higher than the wild type strain. The clones and
their approximate LD.sub.50 values are listed in Table 1. A control
experiment with the wild type strain was run in parallel with each
set of challenges and in all cases mortality in wild
type-challenged groups was 100%.
[0080] In addition to LD.sub.50 values, Table 1 also provides data
from vaccination and challenge experiments. Briefly, groups of mice
(n=5 to 10) were vaccinated by intraperitoneal injection with the
individual P. multocida strains shown in Table 1 at a dose that was
approximately 200 times greater than the LD.sub.50 of the virulent,
wild type strain. Animals were observed for 28 days after which
mortality figures were calculated.
TABLE-US-00007 TABLE 1 P. multocida Virulence Genes Nucleotide
Representative PossibleGene Vaccination Challenge SEQ ID NO:
Isolate Function # survivors/total # survivors/total LD.sub.50 --
wild type -- 0/10 -- <10 23 PM1B1 guaB 10/10, 10/10, 10/10 9/10,
9/10 4.3 .times. 10E6 11 PM1D1 dsbB 10/10, 5/10 10/10, 5/5 8.4
.times. 10E4 3 PM1BD7 atpG 5/5, 10/10 10/10 >3 .times. 10E5 74
PM1BE11 yhcJ (HI0145) 10/10 5/10 >2 .times. 10E5 70 PM1BF6 yabK
3/5, 8/10 9/9 >2 .times. 10E5 (HI1020) 19 PM2G8 fhaC 4/5, 9/10
9/9 >4 .times. 10E5 76 PM3C9 yiaO (HI0146) 3/5 >6 .times.
10E5 118 PM3G11 UnkO 4/5, 10/10 10/10 >3 .times. 10E5 31 PM7B4
iroA (UnkB) 0/5 17 PM4C6 fhaB (fhaB2) 2/5, 10/10, 9/10 10/10, 9/9
>3 .times. 10E6 9 PM4G10-T9 dnaA 4/5 >5 .times. 10E5 1
PM4D5-T5 atpB 5/5 >4 .times. 10E5 53 PM4D5-T1 UnkC2 5/5 >4
.times. 10E5 15 PM4F2 fhaB (fhaB1) 3/5, 6/10, 10/10 6/6, 10/10
>3 .times. 10E5 41 PM5F7 mreB 4/5 1 .times. 10E3 7 PM5E2 devB
0/5, 3/10 2/3 ? 68 PM6H5-T1 xylA 5/5 >3 .times. 10E5 78 PM6H8
yigF (HI0719) 5/5, 9/10 9/9 >3 .times. 10E5 108 PM7D12 pnp 5/5,
9/10 9/9 51 PM8C1R1-T2 UnkC1 5/5 ~6 .times. 10E5 37 PM8C1-T3 mglB
5/5 ~6 .times. 10E5 58 PM8C1R1-T6 UnkD1 5/5 ~6 .times. 10E5 45
PM10H7 purF (HI1207) 3/5, 8/10, 8/10 8/8, 8/8 >3 .times. 10E5 25
PM10H10-T2 HI1501 5/5 >1 .times. 10E4 72 PM11G8-T2 ygiK 5/5
>2.4 .times. 10E3 21 PM11G8-T4 greA 5/5 >2.4 .times. 10E3 84
PM12H6 yyam 3/5, 0/10 ~2.2 .times. 10E3 (HI0687) 33 PM15G8-T2 kdtB
5/5 >1.2 .times. 10E5 116 PM15G8-T1 UnkK 5/5 >1.2 .times.
10E5 104 PM16G11-T1 hmbR 3/5 >1.9 .times. 10E5 29 PM16G11-T2
hxuC 3/5 >1.9 .times. 10E5 35 PM16H8 lgtC 5/5, 10/10 10/10
>2.4 .times. 10E5 80 PM16H3 yleA (HI0019) 5/5, 10/10 >2.0
.times. 10E5 49 PM17H6-T1 sopE 4/5 ~6 .times. 10E5 120 PM17H6 UnkP
4/5 ~6 .times. 10E5 5 PM18F5-T8 cap5E 5/5 >2.4 .times. 10E5 82
PM18F5-T10 yojB (HI0345) 5/5 >2.4 .times. 10E5 13 PM19A1 exbB
5/5, 10/10 10/10 >1.2 .times. 10E5 112 PM19D4 rci 5/5, 8/10 8/8
~1.6 .times. 10E5 39 PM20A12 mioC 3/5, 8/10 8/8 ~2 .times. 10E4
(HI0669) 60 PM20C2 UnkD2 5/5, 10/10 10/10 >8.2 .times. 10E6
Example 4
Cloning and Identification of Genes Required for P. multocida
Virulence
[0081] Each transposon mutant which was verified to be attenuated
was analyzed further to determine the identity of the disrupted
open reading frame. DNA from each mutant was amplified, purified,
and digested with restriction enzymes that were known not to cut
within the transposon and generally produced 4-8 kb fragments that
hybridized with the transposon. Using selection for kanamycin
resistance encoded by the transposon, at least one fragment for
each transposon mutant was cloned.
[0082] Southern hybridization with multiple restriction enzymes was
performed for each attenuated mutant using a labeled 1.8 kb M/uI
fragment from pLOF/Km as a probe to identify a suitably sized
fragment for cloning. The mini-Tn10 element and flanking DNA from
each mutant was cloned into pUC19 and the flanking sequence
determined using internal primers TEF32 and TEF40, primer walking
and in some cases universal pUC-19 primers.
TABLE-US-00008 TEF-32 GGCAGAGCATTACGCTGAC SEQ ID NO: 95 TEF-40
GTACCGGCCAGGCGGCCACGCGTATTC SEQ ID NO: 96
[0083] Sequencing reactions were performed using the BigDye.TM. Dye
Terminator Chemistry kit from PE Applied Biosystems (Foster City,
Calif.) and run on an ABI Prism 377 DNA Sequencer. Double stranded
sequence for putative interrupted open reading frames was obtained
for each clone. Sequencer3.0 software (Genecodes, Corp., Ann Arbor,
Mich.) was used to assemble and analyze sequence data. GCG programs
[Devereux, et al., 1997. Wisconsin Package Version 9.0, 9.0 ed.
Genetics Computer Group, Inc., Madison] were used to search for
homologous sequences in currently available databases.
[0084] In 37% of the clones that were identified as being
attenuated, there were multiple insertions of the mini-Tn10
transposable element. Each insertion including its flanking
sequence was cloned individually into pGP704 and mated into the
wild-type strain to produce new mutants of P. multocida, each
carrying only one of the multiple original insertions. Individual
mutants were retested individually to determine the insertion
responsible for the attenuated phenotype. The nucleotide sequence
of the disrupted, predicted open reading frame was determined by
sequencing both strands, and the predicted amino acid sequence was
used to search currently available databases for similar sequences.
Sequences either matched known genes, unknown genes, and
hypothetical open reading frames previously sequenced or did not
match any previously identified sequence. For those genes having
homology to previously identified sequences, potential functions
were assigned as set out in Table 1.
Example 5
Identification of Related Genes in Other Species
[0085] In separate experiments, STM was also performed using
Actinobacillus pleuropneumoniae (App). One of the App strains
contained an insertion in a gene that was sequenced (SEQ ID NO: 97)
and identified as a species homolog of the P. multocida atpG gene.
This result suggested the presence in other bacterial species of
homologs to previously unknown P. multocida genes that can also be
mutated to produce attenuated strains of the other bacterial
species for use in vaccine compositions. In order to determine if
homologs of other P. multocida genes exists in other bacterial
species, Southern hybridization was performed on genomic DNA from
other species using the A. pleuropneumoniae atpG gene as a
probe.
[0086] Actinobacillus pleuropneumoniae, Pasteurella haemolytica
(Ph), P. multocida, and Haemophilus somnus (Hs) genomic DNA was
isolated using the CTAB method and digested with EcoRI and HindIll
for two hours at 37.degree. C. Digested DNA was separated on a 0.7%
agarose gel at 40V in TAE buffer overnight. The gel was immersed
sequentially in 0.1 M HCL for 30 minutes, twice in 0.5 M NaOH/1.5 M
NaCl for 15 minutes each, and twice in 2.5 M NaCl/1 M Tris, pH 7.5.
The DNA was transferred to nitrocellulose membranes (Amersham
Hybond N.sup.+) overnight using 20.times.SSC buffer (3 M NaCl/0.3 M
sodium citrate). The DNA was crosslinked to the membrane using a UV
Stratalinker on autocrosslink setting (120 millijoules). The
membrane was prehybridized in 5.times.SSC/1% blocking solution/0.1%
sodium lauroyl sarcosine/0.02% SDS at 50.degree. C. for
approximately seven hours and hybridized overnight at 50.degree. C.
in the same solution containing a PCR generated atgG probe.
[0087] The probe was prepared using primers DEL-1 389 (SEQ ID NO:
98) and TEF-46 (SEQ ID NO: 99) in a with a GeneAmp XL PCR kit in a
GeneAmp PCR System 2400. Template was genomic A. pleuropneumoniae
DNA.
TABLE-US-00009 DEL-1389 TCTCCATTCCCTTGCTGCGGCAGGG SEQ ID NO: 98
TEF-46 GGAATTACAGCCGGATCCGGG SEQ ID NO: 99
The PCR was performed with an initial heating step at 94.degree. C.
for five minutes, 30 cycles of denaturation t 94.degree. C. for 30
sec, annealing at 50.degree. C. for 30 sec, and elongation at
72.degree. C. for three minutes, and a final extension step at
72.degree. C. for five minutes. The amplification products were
separated on an agarose gel, purified using a QIAquick gel
purification kit (QIAGEN), and labeled using a DIG-High Primer kit
(Boehringer Mannheim). The blot was removed from the hybridization
solution and rinsed in 2.times.SSC and washed two times for five
minutes each wash in the same buffer. The blot was then washed two
times for 15 minutes each in 0.5.times.SSC at 60.degree. C.
Homologous bands were visualized using a DIG Nucleic Acid Detection
Kit (Boehringer Mannheim).
[0088] Single bands were detected in Pasteurella haemolytica,
Haemophilus somnus and A. pleuropneumoniae using EcoRI digested
DNA. Two bands were detected using EcoRi digested DNA from
Pasteurella multocida.
Example 6
Construction of a Library of Tagged-Transposon P. multocida
Mutants
[0089] Transposon mutagenesis using pLOF/Km has previously been
reported to be functional and random in A. pleuropneumoniae
[Tascon, et al., J Bacteriol. 175:5717-22(1 993)]. To construct
tagged transposon mutants of A. pleuropneumoniae, each of 96 E.
coli S17-1:.lamda.pir transformants containing pre-selected tagged
plasmids (pTEF-1:[NK].sub.35) was used in conjugative matings to
generate transposon mutants of A. pleuropneumoniae strain AP225, a
serotype 1 spontaneous nalidixic acid resistant mutant derived from
an in vivo passaged ATCC 27088 strain. A. pleuropneumoniae strains
were grown on Brain Heart Infusion (BHI) (Difco Laboratories,
Detroit, Mich.) media with 10 .mu.g/ml B-nicotinamide adenine
dinucleotide)(V.sup.10), (Sigma, St. Louis, Mo.) at 37.degree. C.
and in 5% CO.sub.2 when grown on plates. E. coli S17-1:.lamda.pir
(.lamda.pir, recA, thi, pro, hsdR(r.sub.k-,m.sub.k+), RP4-2,
(Tc.sup.R::Mu), (Km.sup.R::Tn7), [Tmp.sup.R], [Sm.sup.R]) was
propagated at 37.degree. C. in Luria-Bertani (LB) medium.
Antibiotics when necessary were used at 100 .mu.g/ml ampicillin
(Sigma), 50 .mu.g/ml nalidixic acid (N.sup.50)(Sigma), and 50
(K.sup.50) or 100 (K.sup.100).mu.g/ml of kanamycin (Sigma).
[0090] Matings were set up by growing each E. coli
S17-1:.lamda.pir/pTEF1:[NK].sub.35 clone and the AP225 strain to
late log phase. A 50 .mu.l aliquot of culture for each
tagged-pTEF-1 clone was mixed with 150 .mu.l of the APP225 culture,
and then 50 .mu.l of each mating mixture was spotted onto 0.22
.mu.M filters previously placed onto BHIV.sup.10 plates containing
100 .mu.M IPTG and 10 mM MgSO.sub.4. Following overnight incubation
at 37.degree. C. with 5% CO.sub.2, mating mixtures were washed off
of each filter into 2 ml of PBS and 200 .mu.l of each was plated
onto BHIV.sup.10N.sup.50K.sup.100 plates. After selective overnight
growth, colonies were assembled into microtiter plates by toothpick
transfer into 200 .mu.l BHIV.sup.10N.sup.50K.sup.50 making sure
that each well in a microtiter plate always contained a transposon
mutant with the same sequence tag. Following overnight growth, 50
.mu.l of 75% glycerol was added to each well and plates were stored
frozen at -80.degree. C.
[0091] APP does not appear to have as much bias towards multiple
insertions of the mini-Tn10 element as did P. multocid. Only
approximately 3% of the mutants were determined to contain multiple
insertions, which is in agreement with the 4% previously reported
[Tascon, et al., J Bacteriol. 175:5717-22(1993)]. A problem in APP
consisted of identifying numerous mutants (discussed below)
containing insertions into 23S RNA regions: 28 total mutants with
insertions into 13 unique sites. This may indicate that 23S RNA
contains preferential insertion sites and that the growth of APP is
affected by these insertions enough to result in differential
survival within the host. Southern blot analysis using an APP 23S
RNA probe suggests that APP may contain only three ribosomal
operons as compared to five in H. influenzae [Fleischmann, et al.,
Science 269:496-512 (1995)] and seven complete operons in E. coli
[Blattner, et al., Science 277:1453-1474 (1997)]. This site
preference and its effect on growth rate may be a significant
barrier to "saturation mutagenesis" since a significant number of
clones will contain insertions into these rRNAs and large volume
screening will be necessary to obtain additional unique attenuating
mutations.
Example 7
Porcine Screening for Attenuated A. pleuropneumoniae Mutants
[0092] Twenty pools of A. pleuropneumoniae transposon mutants,
containing a total of approximately 800 mutants, were screened
using a porcine intratracheal infection model. Each pool was
screened in two separate animals.
Frozen plates of pooled A. pleuropneumoniae transposon mutants were
removed from -80.degree. C. storage and subcultured by transferring
20 .mu.l from each well to a new 96 well round bottom plate
(Corning Costar, Cambridge, Mass., USA) containing 180 .mu.l of
BHIV.sup.10N.sup.50K.sup.50. Plates were incubated without shaking
overnight at 37.degree. C. in 5% CO.sub.2. Overnight plates were
then subcultured by transferring 10 .mu.l from each well to a new
flat bottomed 96 well plate (Coming Costar) containing 100 .mu.l of
BHIV.sup.10 per well and incubating at 37.degree. C. with shaking
at 150 rpm. The OD.sub.562 was monitored using a microtiter plate
reader. At an OD.sub.562 of approximately 0.2 to 0.25, each plate
was pooled to form the "input pool" by combining 100 .mu.l from
each of the wells of the microtiter plate. The culture was diluted
appropriately in BHI to approximately 2.times.10.sup.6 CFU/ml. For
each diluted pool, 4.0 ml was used to infect 10-20 kg SPF pigs
(Whiteshire-Hamroc, Albion, Ind.) by intratracheal administration
using a tracheal tube. At approximately 20 hours post-infection,
all surviving animals were euthanized and the lungs removed. Lavage
was performed to recover surviving bacteria by infusing 150 mls of
sterile PBS into the lungs, which were then massaged to distribute
the fluid. The lavage fluid was recovered, and the process was
repeated a second time. The lavage fluid was centrifuged at
450.times.g for 10 minutes to separate out large debris.
Supernatants were then centrifuged at 2,800.times.g to pellet the
bacteria. Pellets were resuspended in 5 mls BHI and plated in
dilutions ranging from 10.sup.-2 to10.sup.-5 onto
BHIV.sup.10N.sup.50K.sup.50 plates. Following overnight growth, at
least 100,000 colonies were pooled in 10 mls //BHlbroth to form the
"recovered pools". A 0.7 ml portion of each recovered pool was used
to prepare genomic DNA by the CTAB method [Wilson, In Ausubel, et
al., (eds.), Current Protocols in Molecular Biology, vol. 1. John
Wiley and Sons, New York, p. 2.4.1-2.4.5 (1997)].
[0093] Recovery from the animals routinely was in the 10.sup.8 CFU
range from lung lavage.
[0094] Dot blots were performed and evaluated both by visual
inspection and by semi-quantitative analysis as described
previously. All hybridizations and detections were performed as
described. Briefly, probes were prepared by a primary PCR
amplification, followed by agarose gel purification of the desired
product and secondary PCR amplification incorporating dig-dUTP.
Oligonucleotides including TEF5, TEF6, TEF24, TEF25, TEF48 and
TEF62, were synthesized by Genosys Biotechnologies (The Woodlands,
Tex.). Primers TEF69, TEF65, and TEF66 were also used for inverse
PCR reactions and sequencing.
TABLE-US-00010 TEF69 GACGTTTCCCGTTGAATATGGCTC SEQ ID NO: 166 TEF65
GCCGGATCCGGGATCATATGACAAGA SEQ ID NO: 167 TEF66
GACAAGATGTGTATCCACCTTAAC SEQ ID NO: 168
[0095] The labeled PCR product was then digested with HindIII to
separate the constant primer arms from the unique tag region. The
region containing the labeled variable tag was excised and the
entire gel slice was then dissolved and denatured in DIG EasyHyb.
Dot blots were prepared and detected using the standard CSPD
detection protocol. Film exposures were made for visual evaluation,
and luminescent counts per second (LCPS) were determined for each
dot blot sample. The LCPS.sub.input/LCPS.sub.recovered ratio for
each mutant was used to determine mutants likely to be
attenuated.
[0096] Clones selected as being present in the input pool but
highly reduced in the recovered pool were selected for further
study. Additional clones with questionable input/recovered ratios
were also selected after visually evaluating films made from the
dot blots. A total of 110 clones were selected.
Example 8
Identification of A. pleuropneumoniae Virulence Genes
[0097] A partial flanking sequence was determined for each of the
110 mutants by inverse PCR and direct product sequencing. Inverse
PCR was used to generate flanking DNA products for direct
sequencing as described above. Sequencing reactions were performed
using the BigDye.TM. Dye Terminator Chemistry kit from PE Applied
Biosystems (Foster City, Calif.) and run on an ABI Prism 377 DNA
Sequencer. Sequencher 3.0 software (Genecodes, Corp., Ann Arbor,
Mich.) was used to assemble and analyze sequence data. GCG programs
[Devereux and Haeberli, Wisconsin Package Version 9.0, 9.0 ed.
Genetics Computer Group, Inc., Madison (1997)] were used to search
for homologous sequences in currently available databases.
[0098] Table 2 shows the A. pleuropneumoniae genes identified and
extent to which open reading frames were determinable. Sequence
identification numbers are provided for nucleotide sequences as
well as deduced amino acid sequences where located.
TABLE-US-00011 TABLE 2 A. pleuropneumoniae Open Reading Frames
Complete Open Reading Frame atpH SEQ ID NO: 134 aptG SEQ ID NO: 132
exbB SEQ ID NO: 140 OmpP5 SEQ ID NO: 152 OmpP5-2 SEQ ID NO: 150 tig
SEQ ID NO: 160 fkpA SEQ ID NO: 142 hupA SEQ ID NO: 146 rpmF SEQ ID
NO: 158 Start Codon - NO Stop Codon lpdA SEQ ID NO: 148 potD SEQ ID
NO: 156 yaeE SEQ ID NO: 164 apvC SEQ ID NO: 128 NO Start Codon -
Stop Codon dksA SEQ ID NO: 136 dnaK SEQ ID NO: 138 HI0379 SEQ ID
NO: 144 NO Start Codon - NO Stop Codon pnp SEQ ID NO: 154 apvA-or 1
SEQ ID NO: 122 apvA-or 2 SEQ ID NO: 124 apvB SEQ ID NO: 126 apvD
SEQ ID NO: 130 RNA or Noncoding Sequences tRNA-leu SEQ ID NO: 162
tRNA-glu SEQ ID NO: 163
[0099] The putative identities listed in Table 3 (below, Example 9)
were assigned by comparison with bacterial databases. The 110
mutants represented 35 groups of unique transposon insertions. The
number of different mutations per loci varied, with some clones
always containing an insertion at a single site within an ORF to
clones containing insertions within different sites of the same
ORF. Three multiple insertions were detected in the 110 mutants
screened as determined by production of multiple PCR bands and
generation of multiple sequence electropherograms.
Example 9
Competition Challenge of A. pleuropneumoniae Mutants with Wild Type
APP225
[0100] A representative clone from each of the unique attenuated
mutant groups identified above that was absent or highly reduced in
the recovered population was isolated from the original pool plate
and used in a competition challenge experiment with the wild type
strain (AP225) to verify the relative attenuation caused by the
transposon mutation. Mutant and wild type strains were grown in
BHIV.sup.10 to an OD.sub.590 of 0.6-0.9. Approximately
5.0.times.10.sup.6 CFU each of the wild type and mutant strains
were added to. 4 mls BHI. The total 4 ml dose was used infect a
10-20 kg SPF pig by intratracheal administration with a tracheal
tube. At approximately 20 hours post-infection, all surviving
animals were euthanized and the lungs removed. Lung lavages were
performed as described above. Plate counts were carried out on
BHIV.sup.10N.sup.50 and BHIV.sup.10N.sup.50K.sup.100 to determine
the relative numbers of wild type to mutant in both the input
cultures and in the lung lavage samples. A Competitive Index (CI)
was calculated as the [mutant CFU/wild type CFU].sub.input/[mutant
CFU/wild type CFU].sub.recovered.
[0101] Of the 35 potential transposon mutants, 22 were
significantly attenuated, having a competitive index (CI) of less
than 0.2. A transposon mutant that did not seem to be attenuated
based on the STM screening results was chosen from one of the pools
as a positive control. This mutant had a CI in vivo of
approximately 0.6. An in vitro competition was also done for this
mutant resulting in a CI of 0.8. The mutant was subsequently
determined to contain an insertion between 2 phenylalanine
tRNA's.
[0102] Competitive indices for unique attenuated single-insertion
mutants are listed in Table 3. Competitive indices for atpG, pnp,
and exbB App mutants indicated that the mutants were unable to
compete effectively with the wild type strains and were therefore
attenuated.
TABLE-US-00012 TABLE 3 Virulence and Proposed Function of A.
pleuropneumoniae Mutants Mutant Similarity Putative or Known
Functions C.I. AP20A6 atpH ATP synthase .009 AP7F10 atpG ATP
synthase .013 AP17C6 lpdA dihydrolipoamide dehydrogenase .039
AP11E7 exbB transport of iron compounds .003, .003, .006 AP3H7 potD
Spermidine/putrescine transport .308 AP8H6 OmpP5 Adhesin/OmpA
homolog .184 AP18H8 OmpP5-2 Adhesin/OmpA homolog .552 AP13E9 tig
Peptidyl-prolyl isomerase .050 AP13C2 fkpA Peptidyl-prolyl
isomerase <.001 AP15C11 pnp Polynucleotide phosphorylase .032
AP18F12 hupA Histone - like protein .001 AP20F8 dksA Dosage
dependent suppressor .075 of dnaK mutations AP5G4 dnaK Heat shock
protein - .376 molecular chaperone AP17C9 tRNA-leu Protein
Synthesis .059 (gene regulation?) AP5D6 tRNA-glu Protein Synthesis
.055 AP18B2 rpmF Protein Synthesis .112 AP10E7 yaeA Unknown .001
AP19A5 HI0379 Unknown .061 AP10C10 apvA Unknown .157 AP18F5 apvB
Unknown .103 AP2A6 apvC Unknown .091 AP2C11 apvD Unknown .014
[0103] Accuracy of the CI appeared to be very good as the exbB
mutant was competed within three different animals yielding CI's of
0.003, 0.003 and 0.006. The use of a Competitive Index number to
assign attenuation based upon one competition in a large animal
study was further confirmed based on preliminary vaccination
results in pigs with 7 mutants (n=8) described below in Example
11.
Example 10
Characterization of Attenuated A. pleuropneumoniae Virulence
Genes
[0104] The A. pleuropneumoniae genes identified represent four
broad functional classes: biosynthetic enzymes, cellular transport
components, cellular regulation components and unknowns.
[0105] The atpG gene, encoding the F1-.gamma. subunit of the
F.sub.0F.sub.1 H+-ATPase complex, can function in production of ATP
or in the transport of protons by hydrolyzing ATP. A related atpG
attenuated mutant was also identified in P. multocida. Another atp
gene, atpH, that encodes the F.sub.1 .delta. subunit was also
identified. Phenotypes of atp mutants include non-adaptable
acid-sensitivity phenotype [Foster, J Bacteriol. 173:6896-6902
(1991)], loss of virulence in Salmonella typhimurium [Garcia del
Portillo, et al., Infect Immun. 61:4489-4492 (1993)] and P.
multocida (above) and a reduction in both transformation
frequencies and induction of competence regulatory genes in
Haemophilus influenzae Rd [Gwinn, et al., J Bacteriol. 179:7315-20
(1997)].
[0106] LpdA is a dihydrolipoamide dehydrogenase that is a component
of two enzymatic complexes: pyruvate dehydrogenase and
2-oxoglutarate dehydrogenase. While the relationship to virulence
is unknown production of LpdA is induced in Salmonella typhimurium
when exposed to a bactericidal protein from human which may suggest
that this induction may be involved in attempts to repair the outer
membrane [Qi, et al., Mol Microbiol. 17:523-31 (1995)].
[0107] Transport of scarce compounds necessary for growth and
survival are critical in vivo. ExbB is a part of the TonB transport
complex [Hantke, and Zimmerman, Microbiology Letters. 49:31-35
(1981)], interacting with TonB in at least two distinct ways
[Karlsson, et al., Mol Microbiol. 8:389-96 (1993), Karlsson, et
al., Mol Microbiol. 8:379-88 (1993)]. Iron acquisition is essential
for pathogens. In this work, attenuated exbB mutants in both APP
and P. multocida have been identified. Several TonB-dependent iron
receptors have been identified in other bacteria [Biswas, et al.,
Mol. Microbiol. 24:169-179 (1997), Braun, FEMS Microbiol Rev.
16:295-307 (1995), Elkins, et al., Infect Immun. 66:151-160 (1998),
Occhino, et al., Mol Microbiol. 29:1493-507 (1998), Stojiljkovic
and Srinivasan, J Bacteriol. 179:805-12 (1997)]. A.
pleuropneumoniae produces 2 transferrin-binding proteins, which
likely depend on the ExbB/ExbD/TonB system, for acquisition of
iron. PotD is a periplasmic binding protein that is required for
spermidine (a polyamine) transport [Kashiwagi, et al., J Biol Chem.
268:19358-63 (1993)]. Another member of the Pasteurellaceae family,
Pasteurella haemolytica, contains a homologue of potD (Lpp38) that
is a major immunogen m convalescent or outer membrane protein
vaccinated calves [Pandher and Murphy, Vet Microbiol 51:33141
(1996)]. In P. haemolytica, PotD appeared to be associated with
both the inner and outer membranes. The role of PotD in virulence
or in relationship to protective antibodies is unknown although
previous work has shown potD mutants of Streptococcus pneumoniae to
be attenuated [Polissi, et al., Infect. Immun. 66:5620-9
(1998)]
[0108] Relatively few "classical virulence factors," such as
adhesins or toxins with the exception of homologues to OMP P5 of
Haemophilus influenzae, were identified. H. influenzae OMP P5 is a
major outer membrane protein that is related to the OmpA porin
family of proteins [Munson, et al., M Infect Immun. 61:4017-20
(1993)]. OMP P5 in nontypeable Haemophilus influenzae has been
shown to encode a fimbrial subunit protein expressed as a
filamentous structure [Sirakova, et al., Infect Immun. 62:2002-20
(1994)] that contributes to virulence and binding of both mucin and
epithelial cells [Miyamoto and Bakaletz, Microb Pathog. 21:343-56
(1996), Reddy, et al., Infect. Immun. 64:1477-9 (1996), Sirakova,
et al., Infect Immun. 62:2002-20 (1994)]. A significant finding was
identification of two distinct ORF's that appear to encode OMP P5
homologues. This is also the case with two very similar proteins,
MOMP and OmpA2 from Haemophilus ducreyi. It remains to be
determined whether both are functionally involved in the production
of fimbriae and whether the presence of two such ORFs represents a
divergent duplication with redundant or complementing functions.
Interestingly, the two OMP P5 mutants seem to have disparate CI
values, suggesting a difference in essentiality or functionality
for only one copy. OMP P5 has been shown to undergo molecular
variation during chronic infections [Duim, et al., Infect Immun.
65:1351-1356 (1997)], however, this appears to be restricted to a
single gene undergoing point mutations resulting in amino acid
changes rather than "type switching" due to differential expression
of multiple genes.
[0109] Protein folding enzymes are important accessories for the
efficient folding of periplasmic and extracellular proteins, and
two genes were identified whose products have peptidyl-prolyl
isomerase activity:fkpA and tig (trigger factor). FkpA is a
periplasmic protein that is a member of the FK506-binding protein
family [Home and Young, Arch Microbiol. 163:357-65 (1995);
Missiakas, et al., Mol Microbiol. 21:871-84 (1996)]. FkpA has been
shown to contribute to intracellular survival of Salmonella
typhimurium [Home, et al., Infect Immun. 65:806-10 (1997)) and a
Legionella pneumophila homolog, mip [Engleberg, et al., Infect
Immun. 57:1263-1270 (1989)], is responsible for virulence and
infection of macrophages [Cianciotto, et al., J. Infect. Dis.
162:121-6 (1990); Cianciotto, et al., Infect. Immun. 57:1255-1262
(1989)]. Tig, or trigger factor [Crooke and Wickner, Proc. Natl.
Acad. Sci. USA. 84:5216-20 (1987), Guthrie, and Wickner, J
Bacteriol. 172:5555-62 (1990), reviewed in Hesterkamp, and Bukau.,
FEBS Lett. 389:32-4 (1996)], is a peptidyl prolyl isomerase
containing a typical FKBP region [Callebaut and Momon, FEBS Lett.
374:211-215 (1995)], but is unaffected by FK506 [Stoller, et al.,
EMKO J. 14:4939-48 (1995)]. Tig has been shown to associate with
the ribosomes and nascent polypeptide chains [Hesterkamp, et al.,
Proc Natl Acad Sci USA 93:443741 (1996), Stoller, et al., EMBO J.
14:4939-48 (1995)]. Possible roles include an unknown influence on
cell, division [Guthrie, and Wickner, J Bacteriol. 172:5555-62
(1990)] in E. coli, a role in the secretion and activation of the
Streptococcus pyogenes cysteine proteinase [Lyon, et al., EMBO J.
17:6263-75 (1998)] and survival under starvation conditions in
Bacillus subtilis [Gothel, et al, Biochemistry 37:13392-9
(1998)].
[0110] Bacterial pathogens employ many mechanisms to coordinately
regulate gene expression in order to survive a wide variety of
environmental conditions within the host. Differences in mRNA
stability can modulate gene expression in prokaryotes [Belasco and
Higgins, Gene 72:15-23 (1988)]. For example, rnr (vacB) is required
for expression of plasmid borne virulence genes in Shigella
flexneri [Tobe, et al., J Bacteriol. 174:6359-67 (1992)] and
encodes the RnaseR ribonuclease [Cheng, et al., J. Biol. Chem.
273:14077-14080 (1998)]. PNP is a polynucleotide phosphorylase that
is involved in the degradation of mRNA. Null pnp/rnr mutants are
lethal, suggesting a probable overlap of function. It therefore is
possible that both rnr and pnp are involved in the regulation of
virulence gene expression. A pnp mutant of P. multocida is a
virulent in a mouse septicemic model (Example 2)]. Other
pnp-associated phenotypes include competence deficiency and cold
sensitivity in Bacillus subtilis [Wang and Bechhofer, J Bacteriol.
178:2375-82 (1996)].
[0111] HupA is a bacterial histone-like protein, which in
combination with HupB constitute the HU protein in E. coli. Reports
have suggested that hupA and hupB single mutants do not demonstrate
any observable phenotype [Huisman, et al., J Bacteriol. 171:3704-12
(1989), Wada, et al., J Mol Biol. 204:581-91 (1988)], however,
hupA-hupB double mutants have been shown to be cold sensitive,
sensitive to heat shock and blocked in many forms of site-specific
DNA recombination [Wada, et al., J Mol Biol. 204:581-91 (1988),
Wada, et al., Gene. 76:345-52 (1989)]. One limited data previously
indicated that hupA is directly involved in virulence [Turner, et
al., Infect Immun. 66:2099-106 (1998)]. The mechanism of hupA
attenuation remains unknown.
[0112] DnaK is a well known and highly conserved heat shock protein
involved in regulatory responses to various stressful environmental
changes [reviewed in Lindquist and Craig, Annu Rev Genet. 22:631-77
(1988)]. DnaK is also one of the most significantly induced stress
proteins in Yersinia enterocolitica after being phagocytosed by
macrophages [Yamamoto, et al., Microbiol Immunol. 38:295-300
(1994)] and a Brucella suis dnaK mutant failed to multiply within
human macrophage-like cells [Kohler, et al., Mol Microbiol.
20:701-12 (1996)]. In contrast, another intracellular pathogen,
Listeria monocytogenes, did not show induction of dnaK after
phagocytosis [Hanawa, et al., Infect Immun. 63:4595-9 (1995)]. A
dnaK mutant of Vibrio cholera affected the production of ToxR and
its regulated virulence factors in vitro but similar results were
not obtained from in vivo grown cells [Chakrabarti, et al., Infect
Immun. 67:1025-1033 (1999)]. The CI of A. pleuropneumonia dnaK
mutant was higher than most of the attenuated mutants although
still approximately half of the positive control strain.
[0113] DksA is a dosage dependent suppressor of filamentous and
temperature-sensitive growth in a dnak mutant of E. coli [Kang and
Craig, J Bacteriol. 172:2055-64 (1990)]. There is currently no
defined molecular function for DksA, but the gene has been
identified as being critical for the virulence of Salmonella
typhimurium in chickens and newly hatched chicks [Turner, et al.,
Infect Immun. 66:2099-106 (1998)]. In that work, it was noted that
the dksA mutant did not grow well with glucose or histidine but did
grow well with glutamine or glutamate as the sole carbon source.
This observation may indicate that the dksA mutant is somehow
impaired in the biosynthesis of glutamate [Turner, et al., Infect
Immun. 66:2099-106 (1998)].
[0114] Three genes were identified that have roles in protein
synthesis: tRNA-leu, tRNA-glu and rpmF. Excluding protein
synthesis, tRNA's also have a wide variety of functional roles in
peptidoglycan synthesis [Stewart, et al., Nature 230:36-38 (1971)],
porphyrin ring synthesis [Jahn, et al., Trends Biochem Sci.
17:215-8 (1992)], targeting of proteins for degradation [Tobias, et
al., Science 254:1374-7(1991)], post-translational addition of
amino acids to proteins [Leibowitz and Soffer, B.B.R.C. 36:47-53
(1969)] and mediation of bacterial-eukaryotic interactions [Gray,
et al., J Bacteriol. 174:1086-98 (1992), Hromockyj, et al., Mol
Microbiol. 6:2113-24 (1992)]. More specifically, tRNA-leu is
implicated in transcription attenuation [Carter, et al., Proc.
Natl. Acad. Sci. USA 83:8127-8131 (1986)], lesion formation by
Pseudomonas syringae [Rich and Willis, J Bacteriol. 179:2247-58
(1997)] and virulence of uropathogenic E. coli [Dobrindt, et al.,
FEMS Microbiol Lett. 162:135-141 (1998), Ritter, et al., Mol
Microbiol. 17:109-21 (1995)]. It is unknown whether the tRNA that
we have identified represents a minor species of tRNA-leu in A.
pleuropneumoniae. Regardless, it is possible that tRNA-leu may have
any one of a wide range of functions. RpmF is a ribosomal protein
whose gene is also part of an operon containing fatty acid
biosynthesis enzymes in E. coli. Further work will be required to
indicate if this is the case in A. pleuropneumoniae, although the
same clustering of fab genes and rpmF occurs in Haemophilus
influenzae [Fleischmann, et al., Science 269:496-512 (1995)). The
expression of the fab genes is not necessarily dependent on
transcripts originating upstream of rpmF as there has been a
secondary promoter identified within rpmF [Zhang and Cronan, Jr., J
Bacteriol. 180:3295-303 The final class of attenuated mutants
includes mutations within genes of unknown function or genes that
have not been previously identified. Homologs of yaeA and HI0379
have previously been identified in Escherichia coli [Blattner, et
al., Science 277:1453-1474 (1997)] and Haemophilus influenzae
[Fleischmann, et al., Science 269:496-512 (1995)], respectively.
The remaining unknowns have been designated Actinobacillus
pleuropneumoniae virulence genes (apv). The apvC gene shows
significant similarity to HI0893, however, the proposed similarity
of HI0893 as a transcriptional repressor similar to the fatty acid
response regulator Bm3R1 [Palmer, J Biol Chem. 273:18109-16 (1998)]
is doubtful. The apvD gene is also most similar to a putative
membrane protein (b0878) with unknown function from E. coli
[Blattner, et al., Science 277:1453-1474(1997)]. Two other
unknowns, apvA and apvB had no significant matches in the public
databases.
Example 11
Safety and Efficacy of A. pleuropneumoniae Mutants
[0115] Nine groups (n=8) of SPF pigs (4-5 weeks old, 3-10 kg) were
used to determine the safety and efficacy of seven A.
pleuropneumoniae mutants as live attenuated vaccine strains. Seven
groups were infected intranasally with 10.sup.10 CFU of each mutant
on day 1. One group was vaccinated on days 1 and 15 with the
commercially available vaccine Pleuromune (Bayer), and one naive
group was not vaccinated. On day 29, all groups were challenged
intranaslally with 1-5.times.10.sup.5 CFU per pig of wild type
APP225. All surviving animals were euthanized and necropsied on day
42 of the study. Results are shown in Table 4.
TABLE-US-00013 TABLE 4 Efficacy of A. pleuropneumoniae Mutants %
Mortality following intranasal challenge Vaccine Vaccination
Challenge Pleuromune 0 37.5 exbB 0 0 tig 12.5 0 fkpA 12.5 0 HI0385
50.0 0 pnp 0 0 yaeE 0 0 atpG 0 0 None N/A 50.0
[0116] The exbB, atpG, pnp, and yaeA mutants caused no mortality
when administered at a dosage of 10.sup.10 CFU intranasally. The
fkpA and tig mutant groups had one death each and the HI0379 group
(highest Apr. 6, 2000CI of the 7 mutants tested shown in Example 9)
had four deaths. Wildtype LD.sub.50 using this model was generally
1.times.10.sup.7 CFU, indicating that each of these mutants is at
least 100 fold attenuated and that there is a reasonable
correlation between CI and attenuation.
[0117] Numerous modifications and variations in the invention as
set forth in the above illustrative examples are expected to occur
to those skilled in the art. Consequently only such limitations as
appear in the appended claims should be placed on the invention.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100322975A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100322975A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References