U.S. patent application number 10/147299 was filed with the patent office on 2004-03-25 for proteins with repetitive bacterial-ig-like (big) domains present in leptospira species.
Invention is credited to Barocchi, Michele A., Croda, Julio Henrique, Ko, Albert I., Matsunaga, James, Reis, Mitermayer Galvao, Riley, Lee W., Siqueira, Isadora Cristina, Young, Tracy Ann.
Application Number | 20040058323 10/147299 |
Document ID | / |
Family ID | 31990111 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040058323 |
Kind Code |
A1 |
Ko, Albert I. ; et
al. |
March 25, 2004 |
Proteins with repetitive bacterial-Ig-like (big) domains present in
leptospira species
Abstract
The invention relates to three isolated DNA molecules that
encode for proteins, BigL1, BigL2 and BigL3, in the Leptospira sp
bacterium which have repetitive Bacterial-Ig-like (Big) domains and
their use in diagnostic, therapeutic and vaccine applications.
According to the present invention, the isolated molecules encoding
for BigL1, BigL2 and BigL3 proteins are used for the diagnosis and
prevention of infection with Leptospira species that are capable of
producing disease in humans and other mammals, including those of
veterinary importance.
Inventors: |
Ko, Albert I.; (Salvador,
BR) ; Reis, Mitermayer Galvao; (Salvador, BR)
; Croda, Julio Henrique; (Salvador, BR) ;
Siqueira, Isadora Cristina; (Salvador, BR) ;
Matsunaga, James; (Los Angeles, CA) ; Riley, Lee
W.; (Berkeley, CA) ; Barocchi, Michele A.;
(San Francisco, CA) ; Young, Tracy Ann; (Berkeley,
CA) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201
US
|
Family ID: |
31990111 |
Appl. No.: |
10/147299 |
Filed: |
September 19, 2002 |
Current U.S.
Class: |
435/6.18 ;
424/190.1; 435/252.3; 435/320.1; 435/69.1; 530/350; 536/23.7 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 37/04 20180101; A61K 2039/505 20130101; C07K 16/1207 20130101;
C12Q 1/68 20130101; G01N 2469/20 20130101; C12Q 1/689 20130101;
Y02A 50/30 20180101; G01N 33/56911 20130101; C07H 21/04 20130101;
C07K 14/20 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/252.3; 435/320.1; 530/350; 536/023.7; 424/190.1 |
International
Class: |
C07K 014/20; C12Q
001/68; C07H 021/04; A61K 039/02; C12P 021/02; C12N 001/21 |
Claims
We claim:
1. A substantially purified polypeptide having an amino acid
sequence as set forth in SEQ ID NO:2 or functionally equivalent
sequences thereof.
2. An isolated polynucleotide segment encoding an amino acid
sequence as set forth in SEQ ID NO:2 or functionally equivalent
sequences thereof.
3. An isolated polynucleotide selected from SEQ ID NO:1 or
functionally equivalent sequences thereof.
4. An substantially purified polypeptide having a amino acid
sequence as set forth in SEQ ID NO:4 or functionally equivalent
sequences thereof.
5. An isolated polynucleotide encoding an amino acid sequence as
set forth in SEQ ID NO:4 or functionally equivalent sequences
thereof.
6. An isolated polynucleotide selected from SEQ ID NO:3 or
functionally equivalent sequences thereof.
7. A substantially purified polypeptide having a amino acid
sequence as set forth in SEQ ID NO:6 or functionally equivalent
sequence thereof.
8. An isolated polynucleotide encoding an amino acid sequence as
set forth in SEQ ID NO:6 or functionally equivalent sequences
thereof.
9. An isolated polynucleotide selected from SEQ ID NO:5 or
functionally equivalent sequences thereof.
10. The polynucleotide of claims 2, 5 or 8 wherein the
polynucleotide sequence is from Leptospira species.
11. An substantially purified polypeptide sequence selected from
SEQ ID NO: 8 or functionally equivalent sequences thereof.
12. An isolated polynucleotide segment encoding an amino acid
sequence as set forth in SEQ ID NO: 8 or functionally equivalent
sequences thereof.
13. An isolated polynucleotide selected from SEQ ID NO: 7 or
functionally equivalent sequences thereof.
14. An substantially purified polypeptide sequence selected from
SEQ ID NO: 10 or functionally equivalent sequences thereof.
15. An isolated polynucleotide segment encoding an amino acid
sequence as set forth in SEQ ID NO: 10 or functionally equivalent
sequence thereof.
16. An isolated polynucleotide selected from SEQ ID NO: 9 or
functionally equivalent sequences thereof.
17. Antibodies that binds to the substantially purified polypeptide
in claims 1, 4, 7, 11 and 14, or functionally equivalent
sequences.
18. The pharmaceutical composition used to induce an immune
response to pathogenic spirochete in a mammalian subject comprising
of an immunogenically effective amount of the substantially
purified polypeptides in claims 1, 4, 7, 11 and 14 or functionally
equivalent sequences thereof in a pharmaceutically acceptable
carrier.
19. The pharmaceutical composition of claim 18 wherein the
pharmaceutically acceptable carrier contains an adjuvant.
20. The pharmaceutical composition of claim 18 wherein the
pathogenic spirochete is selected from Treponema, Borrelia and
Leptospira.
21. The pharmaceutical composition useful for providing an immune
response to pathogenic spirochetes in a mammalian subject
comprising an immunologically effective amount of antibody that
binds polypeptides in claims 1, 4, 7, 11 and 14 or functionally
equivalent sequences thereof, in a pharmaceutically acceptable
carrier.
22. The method of inducing an immune response against infection
with a pathogenic Leptospira in mammalian subjects comprising of
administering to a pharmaceutical composition of claim 18.
23. The method of inducing an immune response to a pathogenic
spirochete in mammalian subject comprising administering to the
individual a pharmaceutical composition of claim 19.
24. A method for detecting pathogens in a sample which comprises
contacting a sample suspected of containing a pathogenic spirochete
with a reagent that binds to the pathogen-specific cell component
and detecting binding of the reagent to the component.
25. The method according to claim 24 wherein the reagent that binds
to the pathogen-specific cell component is an oligonucleotide for
the identification of bigL1, bigL2 and bigL3 polynucleotide.
26. The method according to claim 24 wherein the reagent that binds
to the pathogen-specific cell component is an antibody against the
BigL1, BigL2 or BigL3 polypeptide or polypeptides with functionally
equivalent sequences.
27. The method of claim 22 wherein the spirochete-specific
component is a polynucleotide that encodes for polypeptides in
claims 1, 4, 7, 11 and 14 or functionally equivalent sequences
thereof.
28. The method of claim 22 wherein the spirochete-specific
component is polypeptides in claims 1, 4, 7, 11 and 14 or
functionally equivalent sequences thereof.
29. The method of detecting antibodies to polypeptides in claims 1,
4, 7, 11 and 14 or functionally equivalent sequences thereof in a
sample, which comprises of contacting the sample with polypeptides
in claims 1, 4, 7, 11 and 14 or functionally equivalent sequences
thereof under conditions which allow the antibody to bind with
polypeptides in claims 1, 4, 7, 11 and 14 or functionally
equivalent sequences thereof, and detecting the binding of the
antibody to polypeptides in claims 1, 4, 7, 11 and 14 or
functionally equivalent sequences thereof.
30. A method of detecting polypeptides in claims 1, 4, 7, 11 and 14
or functionally equivalent sequences thereof in a sample,
comprising of contacting the sample with antibodies to polypeptides
in claims 1, 4, 7, 11 and 14 or functionally equivalent sequences
thereof, which allow the polypeptides in the sample to bind to the
antibodies and measuring the binding of the polypeptides to the
said antibodies.
31. A method of detecting nucleic acid encoding BigL1, BigL2, BigL3
or functionally equivalent sequences thereof in a sample, which
uses polynucleotides that encodes for polypeptides in claims 1, 4,
7, 11 and 14 or functionally equivalent sequence thereof.
32. A kit useful for the detection of BigL1, BigL2, and BigL3
polypeptides or polypeptides with functionally equivalent
sequences; bigL1, bigL2 and bigL3 polynucleotides; or antibodies
that bind to BigL1, BigL2, BigL3, polypeptides or polypeptides with
functionally equivalent sequences.
33. A kit useful for the detection of nucleic acid encoding BigL1,
BigL2, BigL3 or functionally equivalent sequence thereof, the kit
comprising carrier means containing one or more containers
comprising a first container containing a polynucleotide that
hybridizes to BigL1, BigL2, BigL3 nucleic acid or functionally
equivalent sequence thereof.
34. A kit useful for the detection of antibody to BigL1, BigL2,
BigL3 nucleic acid or functionally equivalent sequence thereof, the
kit comprising carrier means containing one or more containers
comprising a first container containing BigL1, BigL2 or BigL3
polypeptide or functionally equivalent sequence thereof.
Description
FIELD OF THE INVENTION
[0001] The invention relates to three isolated DNA molecules that
encode for proteins, BigL1, BigL2 and BigL3, in the Leptospira sp
bacterium which have repetitive Bacterial-Ig-like (Big) domains and
their use in diagnostic, therapeutic and vaccine applications.
According to the present invention, the isolated molecules encoding
for BigL1, BigL2 and BigL3 proteins are used for the diagnosis and
prevention of infection with Leptospira species that are capable of
producing disease in humans and other mammals, including those of
veterinary importance.
BACKGROUND OF THE INVENTION
[0002] Spirochetes are motile, helically shaped bacteria and
include three genuses, Leptospira, Borrelia and Treponema, which
are pathogens of humans and other animals. Borrelia and Treponema
are the causative agents of diseases that include Lyme disease,
relapsing fever, syphilis and yaws. Leptospira consists of a
genetically diverse group of eight pathogenic and four
non-pathogenic, saprophytic species (1, 2). Leptospires are also
classified according to serovar status--more than 200 pathogenic
serovars have been identified. Structural heterogeneity in
lipopolysaccharide moieties appears to be the basis for the large
degree of antigenic variation observed among serovars (1, 2).
[0003] Leptospirosis is a zoonotic disease: transmission to humans
occurs through contact with domestic or wild animal reservoirs or
an environment contaminated by their urine. Infection produces a
wide spectrum of clinical manifestations. The early-phase of
illness is characterized by fever, chills, headache and severe
myalgias. Disease progresses in 5 to 15% of the clinical infections
to produce severe multisystem complications such as jaundice, renal
insufficiency and hemorrhagic manifestations (1-4). Severe
leptospirosis is associated with mortality rates of 5-40%.
[0004] Leptospirosis has a world-wide distribution. Because of the
large spectrum of animal species that serve as reservoirs, it is
considered to be the most widespread zoonotic disease (1).
Leptospirosis is traditionally an important occupational disease
among risk groups such as military personnel, farmers, miners,
sewage and refuse removal workers, veterinarians and abattoir
workers (1-3). However, new patterns of disease transmission have
emerged recently that emphasize the growing importance of
leptospirosis as a public health problem. In developed countries,
leptospirosis has become the cause of outbreaks associated with
recreational activities (1) and sporting events (1, 4, 5). In
Brazil and other developing countries, underlying conditions of
poverty have produced large urban epidemics of leptospirosis
associated with high mortality (4, 5).
[0005] In addition to its public health impact, leptospirosis is a
major economic burden as the cause of disease in livestock and
domestic animals (2). Leptospirosis produces abortions,
stillbirths, infertility, failure to thrive, reduced milk
production and death in animals such as cows, pigs, sheep, goats,
horses and dogs and induces chronic infection and shedding of
pathogenic leptospires in livestock (2) and therefore represents an
additional source of economic loss for the animal husbandry
industry because of current international and national quarantine
regulations.
[0006] The control of human and animal leptospirosis is hindered by
the current lack of adequate diagnostic tools. The standard
serologic test, the microscopic agglutination test (MAT), is
inadequate for rapid case identification since it can only be
performed in few reference laboratories and requires analyses of
paired sera to achieve sufficient sensitivity (1, 2). Dependence
upon the MAT results in delays in establishing the cause of
outbreaks as seen in several investigations (1, 2). Enzyme-linked
immunosorbent assays (ELISA), and other rapid serologic tests based
on whole-cell leptospiral antigen preparations have been developed
for use as an alternative method to screen for leptospiral
infection, although the MAT is still required for case confirmation
(1, 2). Recombinant antigen-based serologic tests are widely used
in screening for spirochetal infections such as Lyme disease and
syphilis, but the use of recombinant proteins for serodiagnosis of
leptospirosis has not been widely investigated. Recently, a
recombinant flagellar-antigen immuno-capture assay was described
for serodiagnosis of bovine leptospirosis (6). A recombinant heat
shock protein, Hsp58, showed a high degree of ELISA reactivity with
serum samples from a small number of human cases (7). However, the
utility of recombinant antigens for the serodiagnosis of
leptospirosis has not been investigated in large validation
studies.
[0007] Furthermore, there are no effective interventions presently
available, which control or prevent leptospirosis. Environmental
control measures are difficult to implement because of the
long-term survival of pathogenic leptospires in soil and water and
the abundance of wild and domestic animal reservoirs (1, 3).
Efforts have focused on developing protective immunization as an
intervention against leptospirosis. Currently-available vaccines
are based on inactivated whole cell or membrane preparations of
pathogenic leptospires and appear to induce protective responses
through induction of antibodies against leptospiral
lipopolysaccharide (1, 3). However, these vaccines do not induce
long-term protection against infection. Furthermore, they do not
provide cross-protective immunity against leptospiral serovars that
are not included in the vaccine preparation. The large number of
pathogenic serovars (>200) and the cost of producing a
multi-serovar vaccine have been major limitations in developing
efficacious vaccines through strategies based on whole cell or
membrane preparations.
[0008] The mechanism of pathogenesis in leptospirosis, as in
spirochetal disease such as Lyme disease and syphilis, relies on
the pathogen's ability to widely disseminate within the host during
the early stage of infection (2). Membrane-associated leptospiral
proteins are presumed to mediate interactions that enable entry and
dissemination through host tissues. Putative surface-associated
virulence factors serve as candidates for vaccine strategies that
induce responses to these factors which block dissemination in the
host. Furthermore, membrane-associated proteins would be accessible
to the immune response during host infection and therefore,
constitute targets for immune protection through mechanisms such
antibody-dependent phagocytosis and complement-mediated killing.
Production of these antigen targets as recombinant proteins offers
a cost-effective approach for protective immunization for
leptospirosis as a sub-unit based vaccine. In addition, selection
of surface-associated targets that are conserved among pathogenic
leptospires can avoid the limitations encountered with currently
available whole-cell vaccine preparations.
[0009] A major limitation in the field of leptospirosis has been
identifying surface-associated and host-expressed proteins with
conventional biochemical and molecular methods. From the genome
sequence of the spirochete, Borrelia burgdorferi, more than 100
surface associated lipoproteins were identified. Based on genome
size and the biology of its lifecycle, Leptospira are expected to
have a significantly greater number of surface-associated targets.
At present, less than 10 surface-associated proteins have been
characterized though isolation of membrane extracts, purification
and characterization of proteins in these extracts and molecular
cloning of these protein targets (8-14) (12). Immunization with
recombinant proteins for several identified targets, LipL32, OmpL1
and LipL41, induce partial, but not complete, protective responses
(11, 12).
[0010] To develop a more comprehensive understanding of leptospiral
protein expression we have used the humoral immune response during
human leptospirosis as a reporter of protein antigens expressed
during infection. The identification of leptospiral antigens
expressed during infection has potentially important implications
for the development of new serodiagnostic and immunoprotective
strategies. Sera from patients with leptospirosis was used to
identify clones from a genomic Leptospira DNA phage library which
express immunoreactive polypeptides. A proportion of these clones
were found to encode a novel family of membrane-associated
Leptospira proteins. The identification of these polynucleotides
and polypeptides and their application for diagnosis of
leptospirosis and inducing an immune response to pathogenic
spirochetes is the basis for this invention.
SUMMARY OF THE INVENTION
[0011] The invention relates to DNA molecules in Leptospira and the
polypeptides they encode which have repetitive bacterial Ig-like
domains. The invention describes the isolation of three DNA
molecules, originally derived from L. kirschneri and L.
interrogans, which encode proteins, herein designated "BigL1",
"BigL2" and "BigL3", that have molecular masses of approximately
110, 205 and 205 kDa, respectively, based on the predicted amino
acid sequence of the polypeptides. The three proteins have 12-13
tandem repeat sequences of approximately 90 amino acids. Repeats
sequence from BigL1, BigL2 and BigL3 are highly related (>90%
amino acid sequence identify) to each other and belong to the
family of bacteria Ig-like (Big) domains, moieties which are found
in virulence factors of bacterial pathogens.
[0012] The DNA molecules that encode for Leptospira proteins with
Big domains, herein called "bigL1", "bigL2" and "bigL3", can be
inserted as heterologous DNA into an expression vector for
producing peptides and polypeptides. Recombinant polypeptides can
be purified from surrogate hosts transformed with such expression
vectors. BigL1, BigL2 and BigL3-derived polypeptides are
serological markers for active and past infection since sera from
leptospirosis patients and animals infected or immunized with
pathogenic Leptospira recognize isolated polypeptides.
[0013] Furthermore, BigL1, BigL2 and BigL3 polypeptides from
recombinant or native antigen preparations are immunogenic.
Antibodies obtained from experimental animals immunized with
purified recombinant BigL1, BigL2 and BigL3 polypeptides recognize
native antigen from Leptospira, and are useful for detecting
pathogenic spirochetes in samples from subjects with a suspected
infection.
[0014] In addition, BigL1, BigL2 and BigL3 polypeptides induce an
immune response against pathogenic spirochetes. BigL1, BigL2 and
BigL3-derived polypeptides; antibodies to these polypeptides; and
polynucleotides that encode for BigL1, BigL2 and BigL3 may be used
alone or combined with pharmaceutically acceptable carrier to treat
or prevent infection with Leptospira. Since Big domains are present
in proteins associated with virulence in other bacterial pathogens,
these moieties may be used to treat or prevent infections unrelated
to those caused by Leptospira.
[0015] In a first embodiment, the invention provides isolated DNA
molecules for bigL1, bigL2 and bigL3 and the polypeptides that are
encoded by these DNA molecules or have functionally equivalent
sequences. In addition, a method is provided for producing an
expression vector containing bigL1, bigL2 and bigL3 polynucleotides
and obtaining substantially purified polypeptides derived from
these sequences.
[0016] A second embodiment of the present invention is to provide
pharmaceutical composition for inducing immune responses in
subjects to pathogenic spirochetes, comprising of an
immunogenically effective amount of one or more selected antigens
among the group consisting of BigL1, BigL2, BigL3 and polypeptides
with functionally equivalent sequences in a pharmaceutically
acceptable vehicle.
[0017] In a third embodiment, the invention provides a method for
identifying a compound which binds to BigL1, BigL2, BigL3
polypeptides or polypeptides with functionally equivalent sequences
that includes incubating components comprising of the compound and
BigL1, BigL2 or BigL3 polypeptide or polypeptides with functionally
equivalent sequences under conditions sufficient to allow the
components to interact and measuring the binding of the compound to
the BigL1, BigL2 or BigL3 polypeptide or polypeptides with
functionally equivalent sequences. Preferably, the inventive method
is a serodiagnostic method utilizing sera from a subject with a
suspected active or past infection with Leptospira or other related
bacterial pathogen.
[0018] In a fourth embodiment, the invention provides a method for
detecting pathogens in a sample which includes contacting a sample
suspected of containing a pathogenic spirochete with a reagent that
binds to the pathogen-specific cell component and detecting binding
of the reagent to the component. In one aspect, the reagent that
binds to the pathogen-specific cell component is an oligonucleotide
for the identification of bigL1, bigL2 and bigL3 polynucleotide. In
another aspect, the reagent that binds to the pathogen-specific
cell component is an antibody against the BigL1, BigL2 or BigL3
polypeptide or polypeptides with functionally equivalent
sequences.
[0019] A fifth embodiment, the invention provides a kit useful for
the detection of BigL1, BigL2, and BigL3 polypeptides or
polypeptides with functionally equivalent sequences; bigL1, bigL2
and bigL3 polynucleotides; or antibodies that bind to BigL1, BigL2,
BigL3, polypeptides or polypeptides with functionally equivalent
sequences.
[0020] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A and B show a Southern blot analysis of bigL gene
sequences in Leptospira. Genomic DNA (3 mcg/lane) from L.
interrogans strain Fiocruz L1-130 (lanes 1), L. kirschneri strain
Rm52 (lanes 2) and L. biflexi strain Patoc I (lanes 3) digested
with NsiI and subject to agarose gel electrophoresis. After
transfer to nitrocellulose membranes, blots were probed with DNA
fragments that encode for BigL repetitive domains
(4.sup.th-6.sup.th repetitive domain of BigL3, FIG. 1A) and
C-terminal regions of bigL1, bigL2 and bigL3, which are unique to
each of these genes, respectively (FIG. 1B).
[0022] FIG. 2 shows a schematic diagram of the genomic organization
of the region encoding the BigL1 and BigL3 proteins in L.
kirschneri. The BigL1 protein would contain a signal peptide
(hatched box) and thirteen 90-amino-acid bacterial
immunoglobulin-like domains (solid boxes). The BigL3 protein would
contain a signal peptide, twelve 90-amino-acid bacterial
immunoglobulin-like domains, and a 793 amino acidcarboxyterminal
(C-terminal) domain. The locations of the 2156 bp region of 100%
DNA sequence identity are shown. The organization of the region
depicted was conserved in L. interrogans and L. kirschneri.
[0023] FIG. 3 shows the polymerase chain reaction (PCR)
amplification of DNA fragments from strains of five pathogenic
species of Leptospira. Degenerate primers were designed based on
the L. kirschneri and L. interrogans sequence encoding for the
BigL3 region corresponding to positions 46-65 aa. PCR reactions
were performed from purified DNA from five pathogenic (L.
kirschneri, borgpetersenii, interrogans, santarosai, and noguchi)
and two non-pathogenic species (L. biflexi and wolbachii).
[0024] FIG. 4 shows amplified products from RT-PCR of RNA extracts
of L. kirschneri with bigL1, bigL2 and bigL3 specific primers.
Reverse transcription reactions (lanes "+") were performed on RNA
extracts of cultured leptospires and then subject to a polymerase
chain reaction (PCR) amplification step with primers that bind to
unique sequences within bigL1, bigL2 and bigL3. Amplification with
primers based on sequences within lipL45 was performed as a control
reaction as was PCR reactions for which samples were not subjected
to the reverse transcription step.
[0025] FIG. 5 shows the immunoblot reactivity of pooled sera from
patients and animal reservoirs infected with pathogenic Leptospira
and laboratory animals immunized with whole L. interrogans antigen
preparation to recombinant BigL3 protein (rBigL3). Western blot
analysis was performed with purified rBigL3 (1 mcg per lane, lanes
3). Membranes were probed with sera from patients with
leptospirosis (lane A), healthy individuals (lane B), captured rats
that are colonized with L. interrogans (lane C), captured rats that
are not colonized with L. interrogans (lane D), laboratory rats
immunized with whole antigen preparations of in vitro cultured L.
interrogans (lane E) and pre-immune sera from the laboratory rats
collected prior to immunization (lane F). Reactivity to whole L.
interrogans antigen preparation (lanes 1) and recombinant LipL32
protein (rLipL32, lanes 2) is shown for comparison. The numbers on
the left indicate the positions and relative mobilities (kDa) for
molecular mass standards (Invitrogen).
[0026] FIG. 6 shows an ELISA evaluation of individual patient
seroreactivity to rBigL3 during the acute (lanes A) and
convalescent (lanes B) phase of illness with leptospirosis. Sera
from 4 leptospirosis patients (unbroken lines) and 4 health
individuals (broken lines), at dilutions of 1:50, 1:100 and 1:200,
were incubated with RBigL3 (25-200 ng/well). Mu and gamma chain
specific antibodies conjugated to horse radish peroxidase was used
to determine IgM and IgG seroreactivity, respectively. Mean
absorbance values (OD 450 nm) and standard deviations are
represented in the graphs.
[0027] FIG. 7 shows the rBigL3 IgM (Column A) and IgG (Column B)
reactivity of sera from 29 individual patients with leptospirosis
during the acute (lanes 2) and convalescent (lanes 3) phase of
illness and 28 health individuals (lanes 1). Sera (1:50 dilutions)
and Mu and gamma chain specific antibodies conjugated to horse
radish peroxidase were used to determine reactivity. Solid bars
represent mean absorbance (OD 450 nm) values.
[0028] FIG. 8 shows the immunoblot reactivity of individual
patients with leptospirosis to rBigL3 during the acute (lanes 6-9)
and convalescent (lanes 10-13) phase of illness. Western blot
analysis was performed with purified rBigL3 (1 mcg per lane, lanes
3). Membranes were probed with sera diluted 1:100. A gamma
chain-specific antibodies conjugated to alkaline phosphatase were
used to determine reactivity to the recombinant 58 kD protein of
region 1 of BigL3 (2.sup.nd to 6.sup.th Big repeat domains).
Reactivity to rLipL32 (1 mcg per lane) was performed as a
comparison. The mobility of purified rBigL32 and rLipL32 (lane 14)
and molecular mass standards (lane 15) are shown after staining
with Ponceau-S and coomassie blue, respectively.
[0029] FIG. 9 shows the immunoblot reactivity of rat anti-rBigL3
antisera to rBigL3 and native angigen from L. interrogans lysates.
Immunoblots were prepared with purified rBigL3 (1 mg/lane; lanes 3,
5, 7, 9) and whole antigen preparations (10.sup.8 leptospira per
lane; lanes 2, 4, 6 and 8) from cultured leptospires. Membranes
were probed with pooled sera (dilutions 1:500 [lanes 4 and 5],
1:100 [lanes 6 and 7] and 1:2500 [lanes [8 and 9]] from rats
immunized with rBigL3 from E. coli ewpressing a cloned DNA fragment
of bigL3 from L. interrogans. Pre-immune sera was obtained prior to
the first immunization and used in the immunoblot analysis as a
control (lanes 2 and 3). The mobility (kDa) of molecular mass
standards are shown on the left side of the figure
[0030] FIG. 10 shows the immunoblot reactivity of rabbit
anti-rBigL3 antisera to native antigen from Leptospira strain
lysates. Immunoblots were prepared with whole antigen preparations
(10.sup.8 leptospira per lane) of the following cultured strains:
lane 1, L interrogans sv pomona (type kennewicki) strain RM211,
low-passage; lane 2, L. interrogans sv canicola strain CDC Nic
1808, low passage; lane 3, L. interrogans sv pomona strain PO-01,
high passage; lane 4, L. interrogans sv bratislava strain AS-05,
high passage; lane 5, L. kirschneri sv grippotyphosa strain RM52,
low passage; lane 6, L. kirschneri sv grippotyphosa strain P8827-2,
low passage; lane 7, L. kirschneri sv grippotyphosa strain 86-89,
low passage; lane 8, L. kirschneri sv grippotyphosa strain Moskva
V, high passage; lane 9, L. kirschneri sv mozdok strain 5621, high
passage; lane 10, L. kirschneri sv grippotyphosa strain RM52, high
passage. Membranes were probed with sera from rabbits immunized
with rBigL3 from E. coli expressing a cloned DNA fragment of bigL3
from L. kircshneri and, as a control measure, sera from rabbits
immunized with recombinant L. kirschneri GroEL protein. The
positions of native antigens corresponding to BigL3 and GroEL and
the mobility (kDa) of molecular mass standards are shown on the
left and right sides, respectively, of the figure.
DETAILED DESCRIPTION OF THE INVENTION
[0031] For convenience, the meaning of certain terms and phrases
employed in the specification, examples, and appended claims are
provided below. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs.
[0032] BigL--are polypeptides of Leptospira sp. having tandem
repeat sequences each of which are similar, according to their
sequence homology, to bacterial immunoglobulin-like (Big) domains.
Big domains are present in bacterial proteins, expressed in
bacterial pathogens such as E. coli, Yersinia and Bordetella, which
have virulence functions such as host cell adhesion.
[0033] Reference sequence--is a new sequence obtained by isolation
from a natural organism or through genetic engineering and presents
an accurate biological function, which is characteristic of the
present invention.
[0034] Functionally equivalent sequences--are the sequences,
related to a reference sequence, that are the result of
variability, i.e. all modification, spontaneous or induced, in a
sequence, being substitution and/or deletion and/or insertion of
nucleotides or amino acids, and/or extension and/or shortening of
the sequence in one of their ends, without resulting in
modification of the characteristic function of the reference
sequence. Functionally equivalent sequences emcompass fragment and
analog therof. In other words, sequences functionally equivalent
are sequences that are "substantially the same" or "substantially
identical" to the reference sequence, such as polypeptides or
nucleic acids that have at least 80% homology in relation to the
sequence of amino acids or reference nucleic acids. The homology
usually is measured by a software system that performs sequence
analyses (Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710,
University Avenue, Madison, Wis., 53705).
[0035] As we mentioned before, Leptospira antigens expressed during
the host infection are important in the identification of targets
for diagnosis tests and vaccines. The LipL32 protein is one of
these targets and was identified as immunodominant antigen by the
immune humoral response during the natural infection. However the
sensitivity of serologic tests based upon detection of antibodies
against LipL32 in patient sera during acute-phase illness with
leptospirosis detection is limited (see Flannery, B: "Evaluation of
recombinant Leptospira antigen-based Enzyme-linked Immunosorbent
Assays for the serodiagnosis of Leptospirosis" J. Clin.
Microbiology 2001;39(9): 3303-3310; WO9942478).
[0036] The present invention is based on the identification of the
family of proteins BigL associated with species of spirochetal
bacteria, including those belonging to Leptospira.
[0037] According to the present invention, the BigL protein family
was identified as targets of the host humoral immune response,
generated during infection with pathogenic Leptospira or
immunization with pathogenic Leptospira or recombinant BigL
polypeptides. BigL polypeptides and polynucleotides that encode
these polypeptides are useful as in diagnostic tests to identify
naturally occurring infection in different species including humans
and animal reservoirs. The diagnostic test based on those proteins
presents improved sensitivity and specificity in relation to
standard diagnostic tests or those that are have been used in the
published literature. the identification of leptospirosis in the
initial phase. In addition BigL polypeptides can induce immune
responses when used in a pharmaceutical composition for
immunization.
[0038] In the present invention, the three BigL polypeptides are
characterized with molecular weights 128.4 kD, 201.3 kD and 200.4
kD, based on the deduced amino acid sequence of the isolated
polynucleotides, bigL1, bigL2 and bigL3, which encode for these
polypeptides. The amino acid sequence of the BigL polypeptides has
a signal sequence and a putative signal peptidase cleavage site
largely conforming to the spirochetal lipobox; therefore BigL
polypeptides are membrane-associated lipoproteins. The polypeptides
of 128.4 kD, 201.3 kD and 200.4 kD are designated "BigL1", "BigL2"
and "BigL3", respectively.
[0039] Although the BigL polypeptides of the present invention have
been isolated originally of Leptospira sp, they are useful not just
for induction of the immune response against the pathogenic
organisms Leptospira sp., but also against other spirochetes
bacteria and pathogens that have factors with Big domains.
Additionally, BigL polypeptides can be used for the diagnosis of
infections due to Leptospira sp., other pathogenic spirochetes and
bacterial pathogens.
[0040] Several processes that incorporate state-of-the-art
methodologies can be used to obtain polynucleotide sequences that
encode for BigL polypeptides. These processes include, but they are
not limited to, the isolation of DNA using hybridization of genomic
libraries with probes to detect homologous sequences of
nucleotides; screening of antibodies of expression libraries to
detect fragments of cloned DNA with shared structural aspects;
polymerase chain reaction (PCR) in genomic DNA using initiators
able to recombine sequence of DNA of interest; and computer-based
searches of sequence databases for similar sequences to that of the
bigL polynucleotides.
[0041] In the present invention the identification of the antigens
was based on knowledge that there is differential expression of
Leptospira antigens during culture (in vitro) and during host
infection (in vivo). Differential expression of Leptospira antigens
is presumed to be important in host adaptation during infection. We
used a strategy to identify immunoreactive antigens and therefore
antigens expressed during host infection. Sera from patients
infected with pathogenic Leptospira were used to select
polynucleotide sequences from genomic Leptospira DNA library in
lambda phage that encode for immunoreactive polypeptides.
[0042] The present invention identified and isolated three
polynucleotides with nucleotide sequences corresponding to SEQ ID
No:1, SEQ ID No:3 and SEQ ID No:5, as well as the amino acid
sequences of the respective polypeptides, BigL1, BigL2 and BigL3,
encoded by such nucleotides.
[0043] Step 1--The screening the positive clones consisted
basically of the following steps:
[0044] (a) The DNA of a pathogenic Leptospira was cut with an
appropriate enzyme and ligated into a specific site in the lambda
phage genome. Host bacteria were infected with phage and the
resulting clones, expressing recombinant polypeptides after
induction with IPTG, were submitted to immunoblot protocol where a
membrane of colony lysates was incubated with sera from patients
with laboratory confirmed leptospirosis and then with a secondary
antibody conjugated to horseradish peroxidase, which recognized
human Ig. Positive clones were detected through an indicator
reaction, for antigen-antibody complexes based on the production of
color.
[0045] (b) The sequence of cloned and isolated polynucleotides was
determined using phage vector-specific sequences as initiators of
the sequencing reaction. Analysis of the clone sequences and the
use of a primer walking strategy identified the complete nucleotide
sequence for the genes that encode for BigL1, BigL2, and BigL3.
[0046] (c) Most of the obtained positive clones contains genes
encoding proteins of thermal shock Hsp58 and DnaK and the protein
of outer membrane LipL41. However, it was found clones containing
genes encoding repetitions in tandem of 90 amino acids compared by
Database of proteins family (Pfam) as proteins of bacterium, type
immunoglobulin (Big). With the analysis of the clone sequences,
were identified 3 genes containing 12 tandem repeats for bigL1 and
13 tandem repeats in bigL2 and bigL3.
[0047] Step 2--Subcloning expression and purification of the
protein
[0048] Drawing of two oligonucleotides with base in sequences of
two proteins BigL
[0049] Amplification by PCR of the initial BigL portion encoding
for part of the repetitive region, from those oligonucleotides
[0050] Sequencing of the product of the amplification
[0051] Subcloning of the region-encoding by the product
sequenced
[0052] Expression of the recombinant protein.
[0053] Purification of the recombinant protein.
[0054] Immunoblot analyses demonstrate that sera from leptospirosis
patient and rodent reservoirs infected with pathogenic Leptospira
produce antibodies primarily to the BigL domain repeats of the BigL
polypeptides, indicating that they are the main antigenic regions
recognized during infection.
[0055] In relation to the polypeptides of the present invention
they consist of sequences of DNA, cDNA or RNA (and sequences of
nucleic acids which are complementary), as well as their
functionally equivalent sequence, i.e., those sequences that encode
the whole or a part, of the polypeptides designated as BigL1, BigL2
and BigL3, but are non-identical due to variability.
[0056] The polypeptides and polynucleotides in the present
invention consist of BigL1, BigL2 and BigL3 and the polynucleotides
that encode these polypeptides; however they include, in addition,
polypeptides and polynucleotides that have functionally equivalent
sequence.
[0057] In the present invention, both polynucleotides and
polypeptides may be of natural, synthetic or recombinant origin,
having the necessary purity degree to grant to their biological
activities.
[0058] The present invention also refers to the polynucleotides
encoding for BigL1, BigL2 and BigL3 which are used in PCR reactions
to obtain either complete or partial amplified DNA fragments of the
bigL polynucleotides, for the purpose of detection of Leptospira in
samples or expression of recombinant BigL polypeptides. In the case
of initiators used for the polynucleotide amplification in the
present invention, they are oligonucleotides made of two or more
deoxyribonucleotides or ribonucleotides, natural or synthetic.
[0059] Each initiator is preferably constructed in order to be
substantially similar to a flanking region of the sequence strand
that is the target for amplification. In this sense, an initiator
can be designated functionally equivalent if corresponding polymers
can produce the same process, without being identical, facing the
utilization or application considered.
[0060] Polynucleotide sequences of this invention can also be
inserted in an expression vector, such as a plasmid, virus or other
vehicle used for recombinant cloning, which is used by inserting or
incorporating whole or partial nucleotide sequences that encode for
BigL1, BigL2 and BigL3 or their functionally equivalent sequences.
Such expression vectors contain a promoter sequence that
facilitates the efficient transcription from genetic sequence in
the host in which the vector is inserted. Such hosts can include
prokaryotes or eukaryotes, including microorganisms such as yeast
or insects and mammals. Such processes for the use of expression
vectors construction and for the expression of recombinant
sequences, properly so-called, are well known by experts in
technique.
[0061] The present invention provides for a method to produce
antibodies that bind to complete or partial polypeptides of BigL1,
BigL2 and BigL3 or their functionally equivalent sequences. Such
antibodies are useful as research and diagnostic tools in the study
and diagnosis of spirochete infections in general, and more
specifically in the development of diagnostics and therapeutics
whether treatment or prevention, for leptospirosis. Such antibodies
can be administered alone or as part of a pharmaceutical
composition that use these antibodies and a pharmaceutically
acceptable carrier as part of anti-spirochetal therapeutic.
[0062] The invention is relates to the use of pharmaceutical
compositions of BigL polypeptides or the polynucleotides that
encode for these polypeptides as vaccines, either as a vaccine for
prevention of disease which induces an immunoprotective response to
infection or colonization with pathogenic spirochetes or as
therapeutic vaccine that provides a beneficial impact in reducing
the duration or severity of the clinical course of illness in an
subject due infected with a pathogenic spirochete or in reducing
the reservoir state of a carrier of pathogenic spirochete such as
in pigs, cows, rats or dogs that harbor and excrete pathogenic
spirochetes for prolonged periods of time. Such compositions may be
prepared with an immunogenically effective quantity of an antibody
against BigL1, BigL2 and BigL3 respectively, or with one or more of
BigL1, BigL2 and BigL3 isolated from the leptospiral pathogen or
recombinant BigL polypeptides, or its functionally equivalent
sequences, in excipients and additives or auxiliaries.
[0063] Another embodiment of present invention relates to the
pharmaceutical composition used to induce an immune response to a
pathogenic spirochete in an individual, particularly Leptospira
sp., including a immunologically effective quantity of BigL1, BigL2
and BigL3 or of their functionally equivalent sequence in a
pharmaceutically acceptable vehicle. As "individual" we refer to
any mammal, including humans, rodents, domesticated and laboratory
animals and livestock. As "quantity immunologically effective" we
refer to quantity of BigL polypeptide antigen necessary to induce,
in an individual, an immunological response against Leptospira or
any other pathogenic spirochete or bacterial pathogen. The
invention further provides a kit for:
[0064] 1--detecting one of polypeptides, BigL1, BigL2 and BigL3, or
their functionally equivalent sequences;
[0065] 2--detecting nucleic acid encoding for BigL1, BigL2 and
BigL3 or their functionally equivalent sequences;
[0066] 3--detecting antibodies for such polypeptides, BigL1, BigL2
and BigL3, or their functionally equivalent sequences.
[0067] The kit used for detection of BigL polypeptides includes
those that use a vehicle containing one or more receptacles with a
first receptacle containing a linking reagent to BigL1, BigL2 and
BigL3 or to their functionally equivalent sequences.
[0068] The kit used for detection of polynucleotides that encode
BigL polypeptides includes those that use a vehicle containing one
or more receptacles with a first receptacle containing a
polynucleotide that hybridizes to the nucleic acid sequence that
encodes BigL1, BigL2 and BigL3 or to their functionally equivalent
sequences.
[0069] The kit useful for detecting antibodies against BigL
polypeptides includes those that use a vehicle containing one or
more receptacles with a first receptacle containing a polypeptide
of BigL1, BigL2 and BigL3 or of their functionally equivalent
sequences.
[0070] The present invention will be now described with reference
to the Examples, which are should not be considered as limitative
of the present invention.
EXAMPLE 1
Example 1A
Bacterial Strains, Plasmids and Media
[0071] Leptospira kirschneri serovar grippotyphosa, strain RM52,
was isolated during an outbreak of porcine abortion in 1983). L.
interrogans serovar copenhageni, strain Fiocruz (L1-130), was
isolated from the bloodstream of a human leptospirosis patient. L.
kirschneri serovar grippotyphosa strain RM52 and other leptospiral
strains were obtained from the National Leptospirosis Reference
Center (National Animal Disease Center, Agricultural Research
Service, U.S. Department of Agriculture, Ames, Iowa). Leptospiral
strains were cultivated at 30.degree. C. in Johnson-Harris bovine
serum albumin-Tween80 medium (Bovuminar PLM-5 Microbiological
Media, Intergen (2). Low-passage samples of the RM52 isolate were
either stored in liquid nitrogen or passaged in liquid medium at
least 200 times to generate a high-passage form. The high-passage
strain was unable to produce a lethal infection in hamsters at any
dose and was only able to infect hamsters at a dose of 10.sup.7 by
intraperitoneal inoculation.
[0072] Escherichia coli XL1-Blue
MRF'mcrA)183.DELTA.mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1
gyrA96 relA1 lac [F'proAB lacI.sup.qZ.DELTA.M15 Tn10 (Tetr)]
(Stratagene) and E. coli PLK-F' (endA1 gyrA96 hsdR17 lac recA1
relA1 supE44 thi-1 [F' lacIqZ.DELTA.M15]) were used as the host
strains for infection with the .lambda.Zap II (Stratagene) and
.lambda.Trip1Ex (Clontech) vectors, respectively. E. coli SOLR
(e14.sup.- [mcrA], A[mcrCB-hsdSMR-mrr]171 sbcC recB recJ
umuC::Tn5[Kan.sup.r ] uvrC lac gyrA96 relA1 thi-1 endA1
.lambda..sup.r, [F' proAB lacI.sup.qZ.DELTA.M15],
Su-[non-suppressing]] and E. coli BM25.8 (supE44 thi
.DELTA.lac-proAB [F' traD36 proAB.sup.+ lacI.sup.qZ.DELTA.M15]
.lambda.imm434(kan.sup.r) P1 (cam.sup.r) hsdR
(r.sup.K12-m.sup.K12-)) were used for in vivo excision of the
pBluescript and pTrip1Ex phagemids, respectively. BLR(DE3)pLysS
[F.sup.- ompT hsdS.sub.B (r.sub.B-m.sub.B-) gal dcm
_(sr1-recA)306::Tn10(TcR) (DE3) pLysS(CmR)] (Novagen) was used as
the host strain for the pRSET expression vector (Invitrogen). E.
coli strains were grown in LB supplemented with 100 .mu.g/ml
ampicillin, 100 .mu.g/ml carbenicillin, or 25 .mu.g/ml
chloramphenicol where appropriate. Antibiotics were purchased from
Sigma.
Example 1B
Isolation and Characterization of bigL Genes
[0073] This example illustrates the identification and isolation of
the bigL genes. Genomic DNA was prepared from virulent, low-passage
L. kirschneri, serovar grippotyphosa, strain RM52 by the method of
Yelton and Charon (15). Genomic DNA was prepared from a clinical
isolate of L. interrogans, serovar copenhageni, strain Fiocruz
L1-130, using a kit to genomic DNA (Qiagen). The QIAquick PCR
Purification Kit (Qiagen) was used to obtain purified DNA. The
genomic DNA was partially digested with Tsp509I and ligated to
XTrip1Ex arms following the instructions provided (Clontech). The
Gigapack III Gold Packaging Extract (Stratagene) was used to
packaged ligated, digested genomic DNA into lambda heads. The phage
titer of the library was determined by infection E. coli
XL1-Blue.
[0074] For screening of genomic library, approximately 103 pfu
approximately were plated on a lawn of E. coli XL1-Blue,
transferred to nitrocellulose membrane (Schleicher & Schuell),
sensibilized with IPTG and processed as recommended (Schleicher
& Schuell). The nitrocellulose filter was blocked with 5%
skimmed milk in Tris-buffered saline (pH 7.8) with 0.05% Tween 20
(TBST) or phosphate-buffered saline (pH 7.4) with 0.05% Tween 20
(PBST), and incubated for 1 hour with pooled sera, diluted 1:50,
from patients with laboratory-confirmed leptospirosis. Sera were
collected from patients, identified in urban epidemics in Brazil
between 1996 and 1999, during the convalescent-phase of illness,
and were pre-absorbed with E. coli lysates prior to use to remove
antibodies to E. coli. Membranes were washed three times with TBST
or PBST, and incubated for more than 1 hour with rabbit or goat
anti-human immunoglobin antibody conjugated with alkaline
phosphatase (Sigma) in the dilution of 1:1000. Detection with NBT
(0,3 mg/ml) and BCIP (0,15 mg/ml) or development with the ECL
Western Blot Detection Reagents (Amersham) followed by exposure to
Hyperfilm (Amersham) was used to identify plaques with
antigen-antibody complexes.
[0075] Each positive plaque was stored at 4.degree. C. in 1 ml SM
(0.1 M NaCl, 8 mM MgSO.sub.4, 50 mM Tris-HCl pH 7.5; 0.01% in
gelatin, with 1-2 drops of chloroform. The lambda plaque clones
that reacted with pooled sera were subjected to two additional
stages of purification. The genomic DNA fragments inserted into
lambda bacteriophage were excised by infecting E. coli SOLR or
BM25.8 strains with the lambda clones as described by the supplier
(Stratagene and Clontech, respectively).
[0076] The sequence of the first 500-700 nucleotides of the insert
was obtained using a vector-specific primer that links adjacent to
the insert. Nucleotide sequence analysis of 131 clones identified
13 that had DNA fragment inserts, found to encode tandem repeats
approximately 90 amino acids in length. Each of the repeat
sequences were subsequently identified in Pfam 6.6
(http://pfam.wustl.edu/) to belong to the Big2 family Big2 family
of bacterial immunoglobulin-like (Big) domains.
[0077] To identify sequences that encode full-length proteins
according to the predicted amino acid sequence, the nucleotide
sequences of the clones were assembled from individual sequences
obtained by a combination of primer walking and sequencing of
nested deletions. The deletions were generated from the plasmid
clones by removal of restriction fragments extending from inside
the insert into the multicloning sites flanking the insert.
Oligonucleotides were synthesized and obtained from GIBCO BRL or
Operon. Inverse PCR (iPCR) was performed to obtain sequences
containing the remainder of the genes and flanking DNA. The UCLA
Core Sequencing Facility, the Yale/Keck Core DNA Sequencing
Facility and the University of California at Berkeley Sequencing
Facility performed the sequencing reactions.
[0078] Two L. kirschneri clones and four L. interrogans clones were
found to encode a gene which we designate bacterial
immunoglobulin-like Leptospiral protein one, bigL1. The complete
nucleotide sequence of L. kirschneri bigL1 and the predicted amino
acid sequence of the gene product is shown in SEQ ID NO: 1 and SEQ
ID NO: 2. Six L. kirschneri clones were found to encode a second
gene which we designated bigL2. The complete nucleotide sequence of
L. kirschneri bigL2 is shown in SEQ ID NO: 3. L. kirschneri bigL2
appears to be a pseudogene, an extra adenine residue occurs at
nucleotide 1011 resulting in a frameshift mutation and downstream
TAG stop codon. However, the antibody screening with pooled patient
sera was able to identify lamda clones with DNA fragments encoding
bigL2 gene products, presumably since the cloned fragments did not
have the frameshift mutation and were inserted in an orientation
that allowed expression of a product that was recognized by patient
sera. The predicted amino acid sequence of the L. kirschneri bigL2
gene product, without the frameshift mutation, is shown in SEQ ID
NO: 4. A fifth L. interrogans clone was found to encode several Big
repeats initially thought to belong to BigL1. However the upstream
DNA encoded by this fifth L. interrogans clone was found to differ
from the sequence upstream of bigL1. Sequencing the regions
flanking the bigL1 gene revealed that the fifth L. interrogans
clone corresponded to a third gene, designated bigL3, downstream of
bigL1 (FIG. 2). The complete nucleotide sequence for bigL3 was
obtained from L. kirschneri DNA and is shown in SED ID NO: 5. The
predicted amino acid sequence of the L. kirschneri bigL3 gene
product is shown in SEQ ID NO: 6.
[0079] All three bigL genes encode a signal peptide and putative
signal peptidase cleavage site largely conforming to the
spirochetal lipobox, as previously defined (Haake, D. A. 2000.
Spirochetal lipoproteins and pathogenesis. Microbiology.
146:1491-1504). Comparison of the sequences of known spirochetal
lipoproteins indicates that the spirochetal lipobox is much more
loosely defined than the E. coli lipobox. For example, while most
E. coli lipoproteins have Leu in the -3 position relative to Cys,
spirochetal lipoproteins may also have a number of other
hydrophobic amino acids in this position, including Val, Phe, and
Ile. E. coli experiments involving site-specific mutagenesis of
amino acids following cysteine indicates that acidic residues cause
sorting of lipoproteins to the cytoplasmic membrane. Sequence
analysis of leptospiral lipoproteins indicates that a similar
sorting signal is present in these bacteria. For example, LipL31 is
the only lipoprotein having an unopposed negative charge in the
first two amino acids following cysteine, and is also the only
lipoprotein sorted exclusively to the cytoplasmic membrane. Like
the outer membrane lipoproteins LipL32 and LipL41, the BigL
proteins have uncharged amino acids in the +2 and +3 positions,
indicating that they would be sorted to the outer membrane.
[0080] Following their signal peptides, all three proteins would
contain a series of tandem repeats, approximately 90-amino-acids in
length. The mature BigL1 protein would consist almost entirely of
thirteen repeats, while in contrast BigL2 and BigL3 contain twelve
repeats followed large carboxy-terminal domains. Though there is a
high degree of sequence variation among the 31 unique repeats found
in the three proteins, all of the repeats were identified by the
Pfam database as bacterial immunoglobulin-like Big protein family
with E-values as low as 4.times.e.sup.-30.
[0081] The L. interrogans and L. kirschneri versions of bigL1,
bigL2, and bigL3 were highly related, with >90% dna and amino
acid sequence identity. In both species there is a region of DNA
sequence identity involving the 5' ends of bigL1 and bigL3 (FIG.
2). The region of sequence identity begins extends from the initial
ATG start codon to position 1890 bp in both genes. The large region
of DNA sequence identity between bigL1 and bigL3 results in an
identical amino acid sequence for the first 630 amino acids
(positions 1-630) of BigL1 (SEQ ID NO: 2) and BigL3 (SEQ ID NO: 6).
This region of identity corresponds to the first six BigL domain
repeats.
EXAMPLE 2
Example 2A
Characterization of the bigL Genes and Detection of bigL DNA and
RNA
[0082] This example illustrates the distribution of multiple copies
of bigL genes among Leptospira species and methods to detect bigL
DNA and RNA in samples.
[0083] Southern Blot Analysis
[0084] Southern blot analysis was performed to identify multiple
copies of bigL genes in genomic DNA from L. interrogans strain
Fiocruz L1-130, L. kirschneri strain RM52, and L. biflexi strain
Patoc I. DNA restriction and modifying enzymes were purchased from
New England Biolabs. Genomic DNA was extracted from from 500 ml of
7-day cultures of Leptospira cells with the Blood and Cell Culture
kit (Qiagen, Valencia, Calif.). Approximately 3 mcg of DNA was
digested with 5-20 units of NsiI overnight in a final volume of 50
mcl. DNA was then purified with phenol:chloroform:isoamyl and
precipitated with 100% cold ethanol and 3M sodium acetate pH and
washed with 70% ethanol. Purified DNA was then re-digested with
5-20 units PacI overnight in a final volume of 25 mcl. The double
digested DNA was separated in a 0.8% agarose gel at 20V overnight.
The gel was then incubated twice for 30 minutes in denaturing
buffer (1.5 M NaCl, 0.5 N NaOH), and twice for 30 minutes in
neutralization buffer (1M Tris (pH 7.4) 1.5 M NaCl). Genomic DNA
was transfered onto a positively charged nylon membrane (Roche
Molecular Biochemicals, Indianapolis, Ind.) according to the method
described by Southern.
[0085] Probes were synthesized with the PCR Dig Probe Synthesis kit
(Roche, Manheim, Germany). Reactions were assembled according to
the manufacturer in a final volume of 50 mcl. Temperature cycles
for the amplification were 94.degree. C. for 5 min, 94.degree. C.
for 30 sec, 57.degree. C. for 30 s min, and 72.degree. C. for 1
min, with a final extension time of 7 min after a total of 35
cycles. Probe sequences were as follows: to amplify the bigL DNA
fragments that encodes for BigL repetitive domains, a bigL3 DNA
sequence was selected that correspond to the region that encodes
for BigL3 repetitive domains 4-6, BigL3.sub.--395
gat-ttt-aaa-gtt-aca-caa-gc and BigL3.sub.--573
aaa-ccg-gac-tac-tta-cct-tt- c-c; and to amplify bigL DNA fragments
that are specific for each of the bigL genes, sequences that encode
for C-terminal regions of the BigL gene products were selected:
BigL1.2078p, tta-cgg-cta-cag-gta-ttt-tta-cg and BigL1.2691p
att-gga-aga-ttt-cca-agt-aac-c, BigL2.5121p tat-cta-cgc-tgc-aaa-tgg
and BigL2.5865p ttg-ttg-gcg-ata-cgt-ccg, BigL3.5071p
cat-aac-tct-cct-cat-aac-a and BigL3.5548p
tat-gta-gag-ata-aga-tcc.
[0086] The UV Crosslinked membrane was subject to prehybridization
at 42.degree. C. for 1 hour in Dig Easy Hybridization solution
(Roche). Prior to hybridization, the Dig labeled probes were boiled
for 10 minutes and rapidly transferred to ice for 5 minutes. The
denatured probes were mixed with hybridization solution and
incubated overnight with the membrane at 42.degree. C. Following
hybridization, the membranes were washed twice for 5 minute at room
temperature with 2.times.SSC (NaCl, Sodium Citrate), 0.1% SDS. The
membranes were then washed twice for 30 minutes at 42.degree. C.
with 0.1 SSC, 0.1% SDS. Membranes were exposed for 1-3 minutes to
Biomax ML film (Eastman Kodak, Rochester, N.Y.) for the detection
of chemiluminescent products
[0087] FIGS. 1A and B shows the results of the Southern blots.
Probes corresponding to DNA sequences that encode BiGL repeats
hybridized to multiple DNA fragments in L. kirschneri and
interrogans (FIG. 1A). In contrast, hybridization was not
identified with digested genomic DNA from the non-pathogenic L.
biflexi. Probes based on sequences that encode for specific
C-terminal regions for each of the L. interrogans bigL gene
products hybridized to one unique fragment in digested L.
interrogans genomic DNA, therefore confirming that there are one
copy of each of the three bigL gene identified in Example 1 (FIG.
1B). These results illustrate a method of identifying specifically
pathogenic Leptospira based on detection of DNA fragments not found
in non-pathogenic Leptospira.
Example 2B
PCR Detection of bigL Gene Sequences in Leptospira Genomic DNA
[0088] This example illustrates the distribution of bigL gene in
pathogenic Leptospira. In order to detect bigL genes in other
Leptospira species, degenerate primers were designed based on an
alignment for bigL genes from L. kirschneri strain RM52 and L.
interrogans strain Fiocruz L1-130, identified in Example 1. The
sequence of the "upstream" primer, designated BigL-lup, is
5'-(GC)AAAGTTG(TC)(AG)(TC)G(TG)CTTGGCC-3' corresponding to
positions 46-65 in bigL1 and bigL3 (SEQ ID NO: 1 and 5), relative
to A of start cdon. The sequence of the "downstream" primer,
designated BigL-2dn, is
5'-(GC)(AT)ACC(AG)TC(CT)GAAAA(AG)AT(AT)CC-3' corresponding to
positions 506-487 in bigL1 and bigL3 (SEQ ID NO: 1 and 5), relative
to A of the start codon. Each primer is 20 nucleotides long. These
primers were designed to anneal to bigL2 at positions 97-116 and
590-571 relative to the A in bigL2's start codon (SEQ ID NO:
3).
[0089] PCR reactions were performed with purified genomic DNA from
high and low-passage strains of Leptospira. In FIG. 3., amplified
DNA fragments were identified in PCR reactions with genomic DNA of
strains in all four pathogenic species evaluated. Fragments had the
predicted electrophoretic mobility based on the sequences of
bigL1/bigL3 (461 bp) and bigL2 (494 bp). Amplified DNA fragments
were not identified in the two non-pathogenic Leptospira species
evaluated. Therefore this example illustrates the application of
this PCR method for identifying specifically DNA from pathogenic
Leptospira in samples.
Example 2C
Reverse Transcriptase-Polymerase Chain
[0090] Reaction (RT-PCR) Detection of Leptospira bigL RNA This
example illustrates the detection of bigL RNA in samples. L.
kirschneri strain RM52 was grown to late exponential phase, and
total RNA was extracted from 1.times.10.sup.10 leptospiral cells
using the hot-phenol method and resuspended in water following
ethanol precipitation (ref). .about.2 .mu.g of leptospiral RNA was
digested with 6 units of DNase I (Ambion) in 70 .mu.l DNase I
buffer (10 mM Tris-HCl pH 7.5, 25 mM MgCl.sub.2, 1 mM CaCl.sub.2 in
1.times.RNA secure from Ambion) for 30 min at 37. To inactivate
DNase I, 1.75 .mu.l of 25 mM EDTA was added to terminate the
reaction, and the enzyme was heat killed for 5 min at 70. RT-PCR
was performed using .about.200 ng leptospiral RNA and Omniscript RT
as described (Qiagen). The following primers were used to prime the
reverse transcriptase reaction:
1 bigL1, 5'-CGCAGAAATTTTAGAGGAACCTACAG-3' bigL2,
5'-TTTGACTCCAAGACGCAGAGGATGAT-3' bigL3,
5'-ATTTTCAAGATTTGTTCTCCAGATTT-3' bigL45,
5'-ATTACTTCTTGAACATCTGCTTGAT-3'.
[0091] The RT reactions were subjected to DNA PCR using Taq
polymerase (Qiagen) Prior to PCR, the following primers were added
to the reactions:
2 bigL1, 5'-CTGCTACGCTTGTTGACATAGAAGTA-3' bigL2,
5'-TAGAACCAACACGAAATGGCACAACA-3' bigL3,
5'-ATCCGAAGTGGCATAACTCTCCTCAT-3' bigL45,
5'-TGAAAAGAACATTACCAGCGTTGTA-3'.
[0092] Along with the primers added for reverse transcription, PCR
products of 500 bp, 479 bp, 440 bp, and 438 bp are expected. To
perform PCR, the reaction mixtures were placed in a Techne Progene
thermocycler. An initial denaturation step of 95 for 1 min was
followed by 30 cycles of denaturation at 95 for 30 sec, annealing
at 53 for 30 sec, and extension at 72 for 30 sec. A final 72
incubation for 30 sec was then performed.
[0093] The results in FIG. 4 show that RT-PCR method can detect
BigL3 transcripts and the control LipL46 transcripts. BigL1 and
BigL2 transcripts were not identified indicating that that whereas
BigL3 is expressed in Leptospira, BigL1 and BigL2 may not be.
Furthermore, these results demonstrate the application of the
RT-PCR method to identify specific BigL gene transcripts in
samples.
EXAMPLE 3
[0094] Expression and Purification of Recombinant BigL Proteins
[0095] This example illustrates the use of the DNA sequences of
bigL genes to express and purify recombinant BigL polypeptides. Two
pairs of oligonucleotides were designed for use in expressing two
regions of L. interrogans BigL3. The first region was a region
within BigL3 corresponding to the 2nd to 6th repetitive domains and
corresponded to positions 131-649 of SEQ ID NO: 6 in the L.
kirschneri BigL3DNA sequence. Oligonucleotides were designed based
upon sequence of lambda L. interrogans BigL3 clones identified in
Example 1 and their sequence are:
3 45B-1 5'-ATGGGACTCGAGATTACCGTTACACCAGCCATT-3' 45B-2
5'-ATTCCATGGTTATCCTGGAGTGAGTGTATTTGT-3'
[0096] PCR amplification with oligonucleotides 45B-1 and 45B-2 and
purified L. interrogans genomic DNA was performed to obtain DNA
fragments. These fragments were digested with XhoI and NcoI Enzymes
(New Biolabs) and then ligated into the pRSETA expression vector
(Invitrogen) (16). The cloned product was sequenced using vector
specific primers and primer walking and the sequence of the 1557 bp
product is shown in SEQ ID NO: 7. The predicted sequence of the
encoded 519 amino acid polypeptide, designated BigL3 region 1, is
shown in SEQ ID NO: 8.
[0097] A second region was selected for expression that contained
the final 200 amino acids of the C-terminal region of L.
interrogans BigL3. This region corresponded amino acid positions
1687-1886 of SEQ ID NO: 6 in L. kirschneri BigL3. The
oligonucleotides used to clone this region are:
4 BIGLCTERM1 5' aac-ctc-gag-cat-aac-tct-cct-cat-aac 3' BIGLCTERM2
5' ttc-gaa-ttc-tta-ttg-att-ctg-ttg-tct- -g 3'
[0098] PCR amplification with oligonucleotides BIGLCTERM1 and
BIGLCTERM2 and purified L. interrogans genomic DNA was performed to
obtain DNA fragments. These fragments were digested with XhoI and
EcoRI enzymes (New Biolabs) and then were ligated into the pRSETA
expression vector (Invitrogen) (16). The cloned product was
sequenced using vector specific primers and primer walking and the
nucleotide sequence of the 600 bp product is shown in SEQ ID NO: 9.
The predicted sequence of the encoded 200 amino acid polypeptide,
designated BigL3 region 2, is shown in SEQ ID NO: 10.
[0099] Recombinant proteins, rBigL regions 1 and 2, were expressed
in BL21(DE3) pLysogen (Invitrogen).
Isopropyl-.beta.-D-thiogalactopyranoside (IPTG; 2 mM final
concentration, Life Technologies) was added to log-phase cultures
of E. coli BLR(DE3)pLysS (Novagen) transformed with pRSET plasmids
encoding leptospiral DNA fragments for expression of His6-fusion
proteins. 6M guanidine hydrochloride was used to solubilize culture
pellets and His6-fusion proteins were purified by affinity
chromatography with Ni2+-nitrilotriacetic acid-agarose (Qiagen and
Pharmacia). The purity of eluted His6 fusion proteins was assessed
by gel electrophoresis and staining with Coomassie brilliant blue.
Proteins were dialyzed against PBS, 10% (v/v) glycerol, 0.025%
(w/v) sodium azide. After dialysis, the protein concentration was
determined with bicinchoninic acid (42). A Ponceau-S (Sigma Chem
Co)-stained nitrocellulose membrane after transfer of purified
BigL3 region 1 is shown in FIG. 7. The relative mobility of the
purified BigL3 was similar to the estimated molecular mass of
approximately 58 kD, which was calculated based on the predicted
amino acid sequence of the recombinant protein.
EXAMPLE 4
Example 4A
Detection of Antibodies Against Recombinant BigL Proteins
[0100] This example illustrates two among several methods that
utilize BigL polypeptides to detect antibodies in subject samples.
Furthermore, this example provides methods for a serodiagnostic
kits for identifying infection in subjects suspected of harboring
infection.
[0101] Immunoblot Detecting of Antibodies to BigL Polypeptides in
Samples from Infected Subjects
[0102] Purified recombinant BigL3 region 2 polypeptide (1 mcg/lane)
(Example 3) was subjected to sodium dodecylsulfate-polyacrylamide
12% gel eloectorphoresis (SDS-PAGE) using a discontinuous buffer
system and transferred to nitrocelulose membranes (Osmomics), as
previously described (17). The nitrocellulose filter was blocked
with TBST with 5% skimmed milk, incubated for more than 1 hour with
pooled sera from patients with labortory confirmed leptospirosis,
captured rat (Rattus norvegicus) reservoirs of Leptosprira which
had urine and kidney cultures positive for pathogenic Leptospira,
and experimental laboratory rats and rabbits, immunized with whole
L. interrogans serovar copenhageni strain Fiocruz L1-130 lysates.
As control experiments, incubations were performed with sera from
health individuals from Brazil, captured rats who had no culture or
serologic evidence for a Leptospira infection and laboratory rats
and rabbits prior to immunization. Sera were diluted 1:100 prior to
use. After washing, membranes were incubated with goat anti-human
gamma chain antibody conjugated to alkaline phosphatase (Sigma),
diluted 1:1000, for more than 1 hour. Antigen-antibody complexes
were detected by color reaction through with NBT (0,3 mg/ml) and
BCIP (0,15 mg/ml). Pooled sera from leptospirosis patients,
captured rats who were infected with pathogenic leptospires
strongly recognized purified recombinant BigL 3 region 1 protein.
However, rats immunized with whole Leptospira lysates did not
visibly bind to the BigL3 polypeptide, indicating that although
BigL3 is expressed in cultured leptospires (Example 2, FIG. 4),
there may be differential expression of the bigL3 gene. Sufficient
quantities of native BigL3 protein may not be present in vitro
whereas, during natural infection, leptospires in vivo produce
sufficient quantities of BigL3 to induce a strong immune response.
Furthermore, this example illustrates that a spectrum of animals
produce an immune response to BigL3 during infection and detection
of this immune response, and detection of antibodies to recombinant
BigL3 polypeptide can be used as a method to identify infection in
subjects.
[0103] To further illustrate the use of a detection method for
antibodies against recombinant BigL3 polypeptide, an immunoblot
evaluation was performed with individual sera of patients with
laboratory-confirmed leptospirosis, healthy individuals from Brazil
and US and patients hospitalized or evaluated in ambulatory clinics
with diagnoses other than leptospirosis. The microagglutination
test and culture isolation was used to confirm the diagnosis of
leptospirosis in patients with clinically-suspected disease (5).
The collection of sera from leptospirosis patients was during
five-year surveillance for leptospirosis in the city of Salvador,
Brazil. The collection of sera from control individuals was
obtained from pre-existing serum banks of hospitalized and clinic
patients and healthy individuals from Salvador, Brazil and through
donations from the Center for Disease Control and Prevention, USA.
A list of the sera used is shown in TABLE 1. Sera diluted 1:100
were analyzed following the method described above. The finding of
any visible colorization of the 1 mcg band of recombinant BigL3
region 1 polypeptide in the immunoblot was considered a positive
reaction.
[0104] FIG. 8 illustrates that sera from individual leptospirosis
patients react with recombinant BigL3. Table 1 summarizes the
findings that demonstrate that more than 90% of hospitalized
patients and approximately 70% of outpatients with leptospirosis
react to rBigL3 during active infection. All (100%) of the
leptospirosis patients react to rBigL3 during the
convalescent-phase of their illness. Table 2 compares
seroreactivity to rBigL3 with standard diagnostic tests. RBigL3
seroreactivity was greater during the initial phase of illness to
those observed for standard diagnostic tests. Healthy individuals
from the US and 88% of the healthy individuals from Brazil do not
react to rBigL3, demonstrating that this reaction to rBigL3 is
specific. The specificity of the reaction increases to 100% when it
is calculated based on the frequency of IgM seroreactivity among
healthy Brazilian individuals. Together, these finding illustrate
that the method has utility as a serological marker of active
infection and is the basis for a kit that can be used for diagnosis
with leptospirosis.
[0105] Table 1 also summarizes findings for rBigL3 seroreactivity
in endemic regions that have high risk for leptospirosis. 25% of
the population that resides in these regions demonstrate rBigL3 IgG
seropositivity, indicating that this reaction may be a useful
marker to identify past infection. Among patients with confirmed
leptospirosis, 56% were seroreactive agains rBigL3 during the
period two years after their infection with leptospirosis (Table
2). In the period between 2 and 4 years after infection with
leptospirosis, 18% demonstrated rBigL3 seroreactivity. Together,
these findings illustrate that a kit based on the immunoblot method
can detect a past infection with leptospirosis.
Example 4B
ELISA-based Detection of Antibodies to BigL Polypeptides in Samples
from Infected Subjects
[0106] This example illustrates that ELISA methods are useful in
detecting antibodies to BigL polypeptides and in identifying
patients with leptospirosis among those with suspected infection.
Flat-bottomed polystyrene microtiter plates (Corning) were coated
at 4.degree. C. overnight with His.sub.6-fusion rBigL3, 0.5-100
ng/well, suspended in 0.05 M sodium carbonate, pH 9.6 (16). The
plates were washed twice with distilled water and three times with
PBS, 0.05% (v/v) Tween 20 (PBST). Plates were incubated with
blocking solution (PBST/1% [w/v] bovine serum albumin) for 2 hours
at room temperature and after four washes with PBST, were stored at
-20.degree. C. until use. Wells were incubated with 50 .mu.l of
sera, diluted 50 to 200-fold in blocking solution, for 1 hour at
room temperature with agitation. After four washes with PBST, wells
were incubated with 50 .mu.l of 5,000 to 20,000-fold dilutions of
anti-human .mu. or .gamma.-chain goat antibodies conjugated to
horseradish peroxidase (Sigma) for 1 hour at room temperature with
agitation. Afterwards, plates were washed twice with PBST and three
times with PBS and incubated with 50 .mu.l/well of 0.01% (w/v)
3,3',5,5'-tetramethylbenz- idine in substrate buffer (0.03% [v/v]
hydrogen peroxide, 25 mM citric acid, 50 mM Na.sub.2HPO.sub.4, pH
5.0) for 20 minutes in the dark at room temperature. The color
reaction was stopped by adding 25 uL 2 N H.sub.2SO.sub.4 and the
absorbance at 450 nm was measured in an Emax microplate reader
(Molecular Devices, Sunnyvale, Calif.).
[0107] Initial assays were performed to determine the antigen
concentration (mcgg/well) that best discriminated between ELISA
reactions of serum samples from laboratory-confirmed leptospirosis
cases (n 4) and healthy individuals from an endemic area for
leptospirosis in Brazil (n=4). Checkerboard titrations were
performed with 50, 100 or 200-fold serum dilutions and antigen
concentrations per well of 25, 50, 100 and 200 ng. FIG. 6
illustrates that significantly increased absorbance values were
observed at all serum dilutions and rBigL3 polypeptide
concentrations for leptospirosis patients than for control
individuals.
[0108] In subsequent assays to determine sensitivity and
specificity, plates were coated with 50 ng of rBigL3. Incubations
were performed with 50 and 10,000-fold dilutions of primary sera
and secondary antibody conjugate, respectively. Individual serum
samples were tested in duplicate and the means of the two
measurements were calculated for analysis. Paired measurements that
differed by greater than 10% were retested. One positive control
serum sample which reacted with all recombinant antigens and one
negative control serum sample were included, in duplicate, on each
plate as a quality control measure. FIG. 7 illustrates that
leptospirosis patients in the acute phase of illness had
significantly increased absorbances than control individuals for
IgM and IgG seroreactivity (FIG. 7). These differences increased
when comparing absorbance values for patients in their
convalescent-phase of illness. These experiments illustrate that an
ELISA-based method for detecting antibodies against rBigL3
polypeptide is useful for identifying infection with leptospirosis
and can be used as a kit for diagnosis.
EXAMPLE 5
[0109] Induction of an Immune Response Against Leptospira in
Subjects
[0110] This example illustrates that an immune response against
BigL proteins can be induced via immunization with recombinant BigL
proteins. Purified recombinant BigL3 polypeptide derived from L.
interrogans was obtained with the method described in Example 3.
Laboratory rats (Wistar strain) were immunized with 40 mcgs of
rBigL3 in Freund's adjuvant (Sigma), and inoculated subcutaneously.
Additional immunizations were performed with 20 mcgs of rBigL3 at
weeks 3 and 6. Blood was collected 7 weeks after primary
immunization and process for serum. Immunoblots with rBigL3 (1
mcg/lane) were prepared as in Example 4. FIG. 9 illustrates the
seroreactivity of rBigL3-immunized rats. rBigL3 was an effective
immunogen inducing immunoblot rBigL3 seroreactivity with titers of
greater than 1:2500 after a total of three immunizations.
Furthermore, antibodies raised to rBigL3 polypeptide recognized
native antigens in whole Leptospira lysates (108 leptospires per
lane) (FIG. 9). A band with relative mobility at 200 kD is faintly
stained in immunoblots as are more intensely staining bands with
lower relative mobility, which may represent degradation of the 200
kD or high molecular weight BigL proteins. Seroreactivity against
these native antigens is specific since no reactions are observed
in the pre-immune sera.
[0111] Immunogenicity experiments were performed with purified
recombinant BigL polypeptides derived L. kirschneri. Purified
recombinant proteins were loaded onto a preparative 12% SDS-PAGE
gel and allowed to migrate into the separating gel by
electrophoresis. A band containing 100-200 smcg of recombinant
protein was excised from the gel, desiccated, ground to powder,
dissolved in 1 ml of water, mixed with 1 ml complete Freund's
adjuvant (Sigma), and inoculated subcutaneously and intramuscularly
in New Zealand white rabbits (Harlan Sprague Dawley) that were free
of leptospiral antibodies. Additional immunizations with similar
amounts of fusion protein in powdered acrylamide gel mixed with
incomplete Freund's adjuvant (Sigma) were administered at four and
eight weeks after primary immunization. Blood was collected from
the rabbits ten weeks after primary immunization and processed for
serum (Harlow, 1988). Immunoblots were performed as previously
described (Guerreiro et al Infect Immun 2001) with concentrations
of 108 leptospires per lane.
[0112] FIG. 10. illustrates that immunization with rBigL3 derived
from L. kirschneri induces high level antibody titers to native
BigL3 polypeptides in L. kirschneri and other pathogenic Leptospira
species such as L. interrogans. Together these findings illustrate
that immunization with rBigL polypeptides induces an immune
response against species of pathogenic spirochetes other than the
species used to design the recombinant rBigL polypeptide.
Furthermore, the antibodies produced by this method of immunization
can be used to detect pathogenic spirochetes in samples.
[0113] Finally, this example demonstrates that the presence of
native BigL polypeptides is observed in virulent low culture
passaged strains and not in avirulent attenuated high culture
passaged strains (FIG. 10). Sera from rBigL3-immunized rabbits
recognized a predicted 200 kDa corresponding to BigL3 in whole
Leptospira lysates of virulent and not avirulent attenuated
strains. This example illustrates that BigL proteins are markers
for virulence and that antibodies against BigL proteins can be used
as a method to identify virultent strains. Since BigL may be itself
a virulence factor, induction of an immune response to BigL
proteins as demonstrated in the example will be useful for
application as a vaccine.
5TABLE 1 Detection of IgG and gM antibodies against rBigL and
rLipL32 in sera from leptospirosis patients and control groups as
determined by the Western Blot method. rBigL3 seroreactivity
rLipL32 seroreactivity No. IgM IgG IgM or IgG IgM IgG IgM or IgG
Study group tested No. positive reactions (%) Hospitalized cases of
confirmed leptospirosis Acute-phase 52 37 (71) 46 (88) 48 (92) 22
(42) 21 (50) 38 (73) Convalescent-phase 52 19 (37) 52 (100) 52
(100) 21 (40) 45 (86) 46 (88) Outpatient cases of confirmed
leptospirosis Acute-phase 14 6 (42) 8 (57) 9 (64) 2 (14) 2 (14) 3
(21) Convalescent-phase 14 7 (50) 14 (100) 14 (100) 6 (42) 5 (36) 8
(57) Healthy individual control groups Non-endemic area (USA) 30 0
(0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Endemic area (Brazil) 40 0 (0) 5
(12) 5 (12) 2 (6) 0 (0) 2 (6) High risk endemic area (Brazil) 40 0
(0) 10 (25) 10 (25) 4 (10) 5 (12) 8 (20) Patient control groups
Dengue 15 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Lyme disease 15 0 (0)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) VDRL-positive 20 0 (0) 1 (5) 1 (5) 0
(0) 1 (5) 1 (5)
[0114]
6TABLE 2 Comparison of the rBigL3and rLipL32-based Western blot
with standard diagnostic tests for leptospirosis. rBigL Western
blot rLipL32 Western blot Standard diagnostic evaluation
seroreactivity seroreactivity Time period Median maximum Reciprocal
IgM or IgM or after initiation No. reciprocal MAT titer MAT titer
ELISA-IgM IgM IgG IgG IgM IgG IgG of illness tested (range) 100 No.
positive reactions (%) Acute phase (N = 52).sup.a 2-6 days 21 200
(0-1600) 12 (57) 11 (52) 12 (57) 16 (76) 17 (81) 8 (38) 8 (38) 12
(57) 7-15 days 31 400 (0-3200) 17 (55) 20 (91) 25 (81) 30 (97) 31
(100) 14 (45) 23 (74) 26 (84) Early convalescent phase (N = 52)
16-21 days 21 800 (200-12800) 21 (100) 15 (100) 7 (33) 21 (100) 21
(100) 8 (38) 18 (86) 19 (90) 21-30 days 31 1600 (0-6400) 31 (100)
21 (100) 12 (39) 31 (100) 31 (100) 13 (42) 27 (87) 27 (87) Late
convalescent phase (N = 59) 0-23 months 25 400 (0-800) 21 (84) 24
(96) 0 (0) 14 (56) 14 (56) 2 (8) 2 (8) 3 (12) 24-47 months 17 400
(100-1600) 17 (100) 7 (41) 0 (0) 3 (18) 3 (18) 2 (12) 2 (12) 3 (18)
48-78 months 17 200 (0-800) 15 (88) 5 (29) 0 (0) 3 (18) 3 (18) 2
(12) 1 (6) 3 (18) .sup.aAcute-phase serum samples were collected
upon hospital admission.
[0115] It will be apparent to those skilled in the art that various
modifications and variations can be made to the compounds and
processes of this invention. Thus, it is intended that the present
invention cover such modifications and variations, provided they
come within the scope of the appended claims and their equivalents.
Accordingly, the invention is limited only by the following
claims.
REFERENCES
[0116] 1. Levett P N. Leptospirosis. Clin Microbiol Rev.
2001;14(2):296-326.
[0117] 2. Faine S B, Adler B, Bolin C, Perolat P. Leptospira and
leptospirosis. 2nd ed Melbourne, Australia: MediSci; 1999.
[0118] 3. Farr R W. Leptospirosis. Clin Infect Dis. 1995;21(1):16;
quiz 7-8.
[0119] 4. Lomar A V, Diament D, Torres J R. Leptospirosis in Latin
America. Infect Dis Clin North Am. 2000;14(1):23-39, vii-viii.
[0120] 5. Ko A I, Galvao Reis M, Ribeiro Dourado C M, Johnson W D,
Jr., Riley L W. Urban epidemic of severe leptospirosis in Brazil.
Salvador Leptospirosis Study Group. Lancet.
1999;354(9181):820-5.
[0121] 6. Bughio N I, Lin M, Surujballi O P. Use of recombinant
flagellin protein as a tracer antigen in a fluorescence
polarization assay for diagnosis of leptospirosis. Clin Diagn Lab
Immunol. 1999;6(4):599-605.
[0122] 7. Park S H, Ahn B Y, Kim M J. Expression and immunologic
characterization of recombinant heat shock protein 58 of Leptospira
species: a major target antigen of the humoral immune response. DNA
Cell Biol. 1999;18(12):903-10.
[0123] 8. Haake D A, Walker E M, Blanco D R, Bolin C A, Miller M N,
Lovett M A. Changes in the surface of Leptospira interrogans
serovar grippotyphosa during in vitro cultivation. Infect Immun.
1991;59(3):1131-40.
[0124] 9. Haake D A, Champion C I, Martinich C, et al. Molecular
cloning and sequence analysis of the gene encoding OmpL1, a
transmembrane outer membrane protein of pathogenic Leptospira spp.
J Bacteriol. 1993;175(13):4225-34.
[0125] 10. Haake D A, Martinich C, Summers T A, et al.
Characterization of leptospiral outer membrane lipoprotein LipL36:
downregulation associated with late-log-phase growth and mammalian
infection. Infect Immun. 1998;66(4):1579-87.
[0126] 11. Haake D A, Mazel M K, McCoy A M, et al. Leptospiral
outer membrane proteins OmpL1 and LipL41 exhibit synergistic
immunoprotection. Infect Immun. 1999;67(12):6572-82.
[0127] 12. Haake D A, Chao G, Zuerner R L, et al. The leptospiral
major outer membrane protein LipL32 is a lipoprotein expressed
during mammalian infection. Infect Immun. 2000;68(4):2276-85.
[0128] 13. Shang E S, Exner M M, Summers T A, et al. The rare outer
membrane protein, OmpL1, of pathogenic Leptospira species is a
heat-modifiable porin. Infect Immun. 1995;63(8):3174-81.
[0129] 14. Shang E S, Summers T A, Haake D A. Molecular cloning and
sequence analysis of the gene encoding LipL41, a surface-exposed
lipoprotein of pathogenic Leptospira species. Infect Immun.
1996;64(6):2322-30.
[0130] 15. Yelton D B, Charon N W. Cloning of a gene required for
tryptophan biosynthesis from Leptospira biflexa serovar patoc into
Escherichia coli. Gene. 1984;28(2):147-52.
[0131] 16. Flannery B, Costa D, Carvalho F P, et al. Evaluation of
recombinant Leptospira antigen-based enzyme-linked immunosorbent
assays for the serodiagnosis of leptospirosis. Journal of Clinical
Microbiology. 2001;39(9):3303-3310.
[0132] 17. Guerreiro H, Croda J, Flannery B, et al. Leptospiral
proteins recognized during the humoral immune response to
leptospirosis in humans. Infect Immun. 2001;69(8):4958-68.
[0133]
Sequence CWU 1
1
33 1 3672 DNA Leptospira kirschneri 1 atgaagagaa cattttgtat
ttcgattctt ctttcgatgt tttttcaaag ttgtatgtct 60 tggccacttt
taaccagtct cgcgggttta gcagctggta aaaaaagtaa tgggctgccc 120
tttttccacc ttctattaag taactctgat ccagttatta caaggatcga gctcagttat
180 caaaattctt ccatcgcaaa aggtacaagt acaactctcg aagtcaccgc
aatctttgat 240 aacggaacaa atcagaatat tacggattcg acatctatcg
tttccgatgc ccaatcaatc 300 gttgacattc aaggtaacag agtcagagga
atcgcttctg gttcttccat tataaaagct 360 gaatacaacg ggatgtattc
tgaacaaaaa attacggtta caccagccac gataaactca 420 attcaagtta
cgagtttaga tgacggtata ttacctaaag gtacaaatcg tcaatttgct 480
gccatcggta tcttttcgga tggttctcat caagatattt ccaacgatcc attgatcgtt
540 tggtcttcca gtaatataga tttagttcga gtagatgatt ccggtttggc
ctcaggtatc 600 aatttaggaa cggctcatat tcgtgcatcc tttcaatcaa
aacaagcctc cgaagagata 660 actgttggtg acgctgttct ttcttctatc
caagtaactt ccaacagtcc aaatattcct 720 ctcggaaaaa aacaaaaact
cacagctact ggaatttatt cggataactc taacagggat 780 atttcctctt
ctgttatctg gaattcttct aattccacta tcgctaatat tcagaataac 840
ggaatattag aaacagctga tactggaatt gttactgttt ctgcttctag aggtaatata
900 aatggttcca taaaactaat cgtcactcct gctgccttag tttctatttc
tgtttctcct 960 acaaattctg cagtagcaaa aggtttacaa gaaaacttta
aagctacagg gatctttaca 1020 gataattcga actcagatat tacagatcaa
gttacttggg attcttctaa tccggatatt 1080 ctttccattt ccaatgcaag
tgatagccac gggttagctt ccacactcaa ccaaggaaat 1140 gttaaggtca
ccgcttccat cggtggaata caaggatcca ctgattttaa agttacacaa 1200
gaggtattaa cttccatcga agtttctcca gttttacctt caattgcaaa aggactaact
1260 cagaaattta cggcgatcgg gatttttacg gataactcca aaaaagatat
tacaaatcaa 1320 gtcacttgga attcttcttc agcaatcgca agcgtgtcta
acttagatga taataaaggt 1380 ctgggaaaag ctcacgctgt tggagacacg
actattaccg ctactttagg aaaagtttca 1440 ggtaaaactt ggtttactgt
agttcctgcg gttctcactt ctattcaaat caatcctgta 1500 aatccttctc
ttgcaaaagg gttaactcaa aaatttacgg ctactgggat ctactctgac 1560
aactctaaca aggacattac ttcctccgtt acttggttct catccgattc ttcaatcgca
1620 acaatttcaa acgccaaaaa aaatcaagga aactcttacg gagcagctac
aggagcaacg 1680 gatattaaag ccacattcgg aaaggtaagt agtccagttt
ctacgttatc cgttactgct 1740 gcaaaacttg ttgaaataca aatcacaccg
gccgctgctt ccaaagcaaa gggaatttcc 1800 gaaagattta aagcaaccgg
tatttttaca gacaactcta attccgatat tacaaatcag 1860 gtcacttgga
gttcatctaa tacagatatt cttaccgttt ccaatacaaa cgccaaacgc 1920
gggttaggtt ccactttaaa acaaggaact gttaaagtta tcgcttccat gggtggaatc
1980 gaaagttctg tagattttac cgtcacacag gctaatttga cttcgatcga
agtctctcca 2040 actcgctctt cgattgcaaa aggactaact caaaaattta
ccgctatagg tatttttacg 2100 gatcattcta agaaggatat tacagagcaa
gttacttgga agtcttcttc gaaagtatta 2160 aatatgttga atgcatccgg
tgaagaagga agaggtaagg caatttcagt cgggaaagcg 2220 accattactg
caaccttaga aaaactttcc gggaaagctg atattacagt tactcccgcg 2280
gttcttactt caattcaaat cagtcctgtg aaaccttctc ttgtaaaagg gttaacagaa
2340 aatttttctg ctacaggtat ctactctgat aattccagca aggacataac
ttcctccgtt 2400 acatggcatt cgttcaacaa ctctgttgca acgatctcga
acacgaaaaa ttaccatgga 2460 caagctcacg caaccggtac agggatagtg
ggtattaaag cgacattggg aaatgtaagc 2520 agcccagttt ccaaattatc
cgttaccgca gcagaactgg ttgagattgt gttaaatcct 2580 actttatctc
acaaggccaa gggacttact gaaaatttta aagcgaccgg cgtatttacg 2640
gacaattcga caaaagatat taccgaccag gttacttgga aatcttccaa tactgcctac
2700 gcagaaattt caaacgcaac tggaagtaaa ggggttgtta atgcactctc
gaagggaacg 2760 agtcacattt ccgctacctt aggttcaatt tcaagtgcaa
atgcgacatt ccaagttact 2820 ccagcaaaaa tagcttcgat cgaaataaca
ccaaataatt tcttcttgat caaaaaactt 2880 agttatccat ttaaagcaat
tggaatctat acggataata caaagacaga cattacaaaa 2940 caagtttcct
ggtcttcctc tgatccgaat gttgcatcga tcgataacac attttcattg 3000
gctggctcag ctaccgcaat cgatgatgga aaaacgaaca tcactgcaac gttatccgac
3060 tctatgtccg cttccactac tttgtatgtc acttctgcta cgcttgttga
catagaagta 3120 aaacctagta tcttcgttct gagtgaaggt cttacactac
aactgaccgc taccggcatc 3180 tattcggatt actctaccta tgatttgact
caggttgtaa cgtggacttc cagcgaacca 3240 tccaacattt cgatcgaaaa
tacagccggt aaaaaaggta aagtaacggc tcttgcattt 3300 ggagcttcag
aatttacggc aacctacgat tctattgaaa gtaatcgagc ttggatattt 3360
gtcaatgacg agaaatttgt aaacataacc attagttctt ctcaagtttt gacagacaag
3420 ggcttgactc aacaattcaa agcaatcgga actttcgaaa aaggtagcga
acttgacctt 3480 acggatcttg taacctggaa gtcctctgat tctaaggtag
cttctatcgg taactctaat 3540 gatgacagag gtttaataac accgctttct
gtaggttcct ctaaaatttc tgcgacttac 3600 aattctatcc atagtaactc
tattgatttt gaagtaactc cagaaatatt agcctctatt 3660 aaaacgaagc cg 3672
2 1224 PRT Leptospira kirschneri 2 Met Lys Arg Thr Phe Cys Ile Ser
Ile Leu Leu Ser Met Phe Phe Gln 1 5 10 15 Ser Cys Met Ser Trp Pro
Leu Leu Thr Ser Leu Ala Gly Leu Ala Ala 20 25 30 Gly Lys Lys Ser
Asn Gly Leu Pro Phe Phe His Leu Leu Leu Ser Asn 35 40 45 Ser Asp
Pro Val Ile Thr Arg Ile Glu Leu Ser Tyr Gln Asn Ser Ser 50 55 60
Ile Ala Lys Gly Thr Ser Thr Thr Leu Glu Val Thr Ala Ile Phe Asp 65
70 75 80 Asn Gly Thr Asn Gln Asn Ile Thr Asp Ser Thr Ser Ile Val
Ser Asp 85 90 95 Ala Gln Ser Ile Val Asp Ile Gln Gly Asn Arg Val
Arg Gly Ile Ala 100 105 110 Ser Gly Ser Ser Ile Ile Lys Ala Glu Tyr
Asn Gly Met Tyr Ser Glu 115 120 125 Gln Lys Ile Thr Val Thr Pro Ala
Thr Ile Asn Ser Ile Gln Val Thr 130 135 140 Ser Leu Asp Asp Gly Ile
Leu Pro Lys Gly Thr Asn Arg Gln Phe Ala 145 150 155 160 Ala Ile Gly
Ile Phe Ser Asp Gly Ser His Gln Asp Ile Ser Asn Asp 165 170 175 Pro
Leu Ile Val Trp Ser Ser Ser Asn Ile Asp Leu Val Arg Val Asp 180 185
190 Asp Ser Gly Leu Ala Ser Gly Ile Asn Leu Gly Thr Ala His Ile Arg
195 200 205 Ala Ser Phe Gln Ser Lys Gln Ala Ser Glu Glu Ile Thr Val
Gly Asp 210 215 220 Ala Val Leu Ser Ser Ile Gln Val Thr Ser Asn Ser
Pro Asn Ile Pro 225 230 235 240 Leu Gly Lys Lys Gln Lys Leu Thr Ala
Thr Gly Ile Tyr Ser Asp Asn 245 250 255 Ser Asn Arg Asp Ile Ser Ser
Ser Val Ile Trp Asn Ser Ser Asn Ser 260 265 270 Thr Ile Ala Asn Ile
Gln Asn Asn Gly Ile Leu Glu Thr Ala Asp Thr 275 280 285 Gly Ile Val
Thr Val Ser Ala Ser Arg Gly Asn Ile Asn Gly Ser Ile 290 295 300 Lys
Leu Ile Val Thr Pro Ala Ala Leu Val Ser Ile Ser Val Ser Pro 305 310
315 320 Thr Asn Ser Ala Val Ala Lys Gly Leu Gln Glu Asn Phe Lys Ala
Thr 325 330 335 Gly Ile Phe Thr Asp Asn Ser Asn Ser Asp Ile Thr Asp
Gln Val Thr 340 345 350 Trp Asp Ser Ser Asn Pro Asp Ile Leu Ser Ile
Ser Asn Ala Ser Asp 355 360 365 Ser His Gly Leu Ala Ser Thr Leu Asn
Gln Gly Asn Val Lys Val Thr 370 375 380 Ala Ser Ile Gly Gly Ile Gln
Gly Ser Thr Asp Phe Lys Val Thr Gln 385 390 395 400 Glu Val Leu Thr
Ser Ile Glu Val Ser Pro Val Leu Pro Ser Ile Ala 405 410 415 Lys Gly
Leu Thr Gln Lys Phe Thr Ala Ile Gly Ile Phe Thr Asp Asn 420 425 430
Ser Lys Lys Asp Ile Thr Asn Gln Val Thr Trp Asn Ser Ser Ser Ala 435
440 445 Ile Ala Ser Val Ser Asn Leu Asp Asp Asn Lys Gly Leu Gly Lys
Ala 450 455 460 His Ala Val Gly Asp Thr Thr Ile Thr Ala Thr Leu Gly
Lys Val Ser 465 470 475 480 Gly Lys Thr Trp Phe Thr Val Val Pro Ala
Val Leu Thr Ser Ile Gln 485 490 495 Ile Asn Pro Val Asn Pro Ser Leu
Ala Lys Gly Leu Thr Gln Lys Phe 500 505 510 Thr Ala Thr Gly Ile Tyr
Ser Asp Asn Ser Asn Lys Asp Ile Thr Ser 515 520 525 Ser Val Thr Trp
Phe Ser Ser Asp Ser Ser Ile Ala Thr Ile Ser Asn 530 535 540 Ala Lys
Lys Asn Gln Gly Asn Ser Tyr Gly Ala Ala Thr Gly Ala Thr 545 550 555
560 Asp Ile Lys Ala Thr Phe Gly Lys Val Ser Ser Pro Val Ser Thr Leu
565 570 575 Ser Val Thr Ala Ala Lys Leu Val Glu Ile Gln Ile Thr Pro
Ala Ala 580 585 590 Ala Ser Lys Ala Lys Gly Ile Ser Glu Arg Phe Lys
Ala Thr Gly Ile 595 600 605 Phe Thr Asp Asn Ser Asn Ser Asp Ile Thr
Asn Gln Val Thr Trp Ser 610 615 620 Ser Ser Asn Thr Asp Ile Leu Thr
Val Ser Asn Thr Asn Ala Lys Arg 625 630 635 640 Gly Leu Gly Ser Thr
Leu Lys Gln Gly Thr Val Lys Val Ile Ala Ser 645 650 655 Met Gly Gly
Ile Glu Ser Ser Val Asp Phe Thr Val Thr Gln Ala Asn 660 665 670 Leu
Thr Ser Ile Glu Val Ser Pro Thr Arg Ser Ser Ile Ala Lys Gly 675 680
685 Leu Thr Gln Lys Phe Thr Ala Ile Gly Ile Phe Thr Asp His Ser Lys
690 695 700 Lys Asp Ile Thr Glu Gln Val Thr Trp Lys Ser Ser Ser Lys
Val Leu 705 710 715 720 Asn Met Leu Asn Ala Ser Gly Glu Glu Gly Arg
Gly Lys Ala Ile Ser 725 730 735 Val Gly Lys Ala Thr Ile Thr Ala Thr
Leu Glu Lys Leu Ser Gly Lys 740 745 750 Ala Asp Ile Thr Val Thr Pro
Ala Val Leu Thr Ser Ile Gln Ile Ser 755 760 765 Pro Val Lys Pro Ser
Leu Val Lys Gly Leu Thr Glu Asn Phe Ser Ala 770 775 780 Thr Gly Ile
Tyr Ser Asp Asn Ser Ser Lys Asp Ile Thr Ser Ser Val 785 790 795 800
Thr Trp His Ser Phe Asn Asn Ser Val Ala Thr Ile Ser Asn Thr Lys 805
810 815 Asn Tyr His Gly Gln Ala His Ala Thr Gly Thr Gly Ile Val Gly
Ile 820 825 830 Lys Ala Thr Leu Gly Asn Val Ser Ser Pro Val Ser Lys
Leu Ser Val 835 840 845 Thr Ala Ala Glu Leu Val Glu Ile Val Leu Asn
Pro Thr Leu Ser His 850 855 860 Lys Ala Lys Gly Leu Thr Glu Asn Phe
Lys Ala Thr Gly Val Phe Thr 865 870 875 880 Asp Asn Ser Thr Lys Asp
Ile Thr Asp Gln Val Thr Trp Lys Ser Ser 885 890 895 Asn Thr Ala Tyr
Ala Glu Ile Ser Asn Ala Thr Gly Ser Lys Gly Val 900 905 910 Val Asn
Ala Leu Ser Lys Gly Thr Ser His Ile Ser Ala Thr Leu Gly 915 920 925
Ser Ile Ser Ser Ala Asn Ala Thr Phe Gln Val Thr Pro Ala Lys Ile 930
935 940 Ala Ser Ile Glu Ile Thr Pro Asn Asn Phe Phe Leu Ile Lys Lys
Leu 945 950 955 960 Ser Tyr Pro Phe Lys Ala Ile Gly Ile Tyr Thr Asp
Asn Thr Lys Thr 965 970 975 Asp Ile Thr Lys Gln Val Ser Trp Ser Ser
Ser Asp Pro Asn Val Ala 980 985 990 Ser Ile Asp Asn Thr Phe Ser Leu
Ala Gly Ser Ala Thr Ala Ile Asp 995 1000 1005 Asp Gly Lys Thr Asn
Ile Thr Ala Thr Leu Ser Asp Ser Met Ser Ala 1010 1015 1020 Ser Thr
Thr Leu Tyr Val Thr Ser Ala Thr Leu Val Asp Ile Glu Val 1025 1030
1035 1040 Lys Pro Ser Ile Phe Val Leu Ser Glu Gly Leu Thr Leu Gln
Leu Thr 1045 1050 1055 Ala Thr Gly Ile Tyr Ser Asp Tyr Ser Thr Tyr
Asp Leu Thr Gln Val 1060 1065 1070 Val Thr Trp Thr Ser Ser Glu Pro
Ser Asn Ile Ser Ile Glu Asn Thr 1075 1080 1085 Ala Gly Lys Lys Gly
Lys Val Thr Ala Leu Ala Phe Gly Ala Ser Glu 1090 1095 1100 Phe Thr
Ala Thr Tyr Asp Ser Ile Glu Ser Asn Arg Ala Trp Ile Phe 1105 1110
1115 1120 Val Asn Asp Glu Lys Phe Val Asn Ile Thr Ile Ser Ser Ser
Gln Val 1125 1130 1135 Leu Thr Asp Lys Gly Leu Thr Gln Gln Phe Lys
Ala Ile Gly Thr Phe 1140 1145 1150 Glu Lys Gly Ser Glu Leu Asp Leu
Thr Asp Leu Val Thr Trp Lys Ser 1155 1160 1165 Ser Asp Ser Lys Val
Ala Ser Ile Gly Asn Ser Asn Asp Asp Arg Gly 1170 1175 1180 Leu Ile
Thr Pro Leu Ser Val Gly Ser Ser Lys Ile Ser Ala Thr Tyr 1185 1190
1195 1200 Asn Ser Ile His Ser Asn Ser Ile Asp Phe Glu Val Thr Pro
Glu Ile 1205 1210 1215 Leu Ala Ser Ile Lys Thr Lys Pro 1220 3 5863
DNA Leptospira kirschneri 3 atgcctaaac atatcaacaa actcagagat
aaaaaaacgt ggccttttct tcagtttatt 60 tttattcttt ttctaacatt
cagcctattt tttttggaaa gttgcgcggc ttggccaatt 120 ttttcaggca
cacctggttt attagcaggt aaaaaaagcg gagcaaacaa ttcactttgg 180
atgctttttt taggaataga taatccgctc gaatcggagc catccgaagc agagttagat
240 cggatcgaaa tttccgtacc gaactcaaat ttagctcgag gtactacttt
acatctaaac 300 gccacagcca tctataaaga caatactcac cgagatattt
cttcggaagg atcctggtcc 360 tctacggatt cgagcattct caagctatta
acacaatctc aattcaaagg aatgaatcta 420 ggttctggaa acgttaatgt
atcctttcaa ggaaaaaacg caactacaac gttaaccgtt 480 acatccgctg
ttttgtccga tctgaccgta acttgtgtga accaaggtag tccattacct 540
gttggaatcg atcgtcaatg taaattagaa ggaatttttt cggacggtag tactcaggtt
600 ttaacttccg atccaagcgc gtcctggaac gtaacccaat cttctattgc
aggtgtaaac 660 accacaggtt tagtttccgg actttctcca ggtaacactt
ttattaccac ttcttatgga 720 agtaaaacct ccagtttgaa tgtgaccgta
agtgcggcaa cccttagctc gatctcagtg 780 actcctgcca actcaagtta
tcctcttggc aaggtccaac agtacacagc aatcggaacc 840 tacagcaatc
agtccactca agatttaaca aatcaggttt cctgggcttc tttaaatact 900
tccgttgcta cgatcgataa ttctacatcc gccaaaggta tgcttactac tcaatcaacc
960 ggttcagcaa acatcacggc aacgttaggc ggaattaccg gacagactac
tagtaaacgt 1020 cacttccgca gttcttacta gtattacgat cactcctgca
aatccaagcg tagccaatgg 1080 aaggacatta tatcttaccg ccaccggagt
tttttcggat ggtacagttt ccgacattac 1140 caaccaagta acttggtcca
gttccttaac aagtgtagct accgcagata actcaggcgg 1200 tttatccgga
agaatttccg gagtcggagt tggtagtacg aatatcaccg ccgccatcgg 1260
tggagtagat attacggttt ctttaaatgt taccaacgcc actttagaat cgattcaagt
1320 ggtttccgat tcccattcga tagctcgagg tacgtctacg tttgtacaag
cgataggagt 1380 ctactcggac ggttcttctc aaaacataag tgatcaagtt
gcctggaaca gctctaattc 1440 ttcaatatta caaatatcta atttaaatgc
agttcccaaa agagaaatac aatctccttc 1500 ttccggaggc ctaggtacag
caaggatcac cgcaacttta gaagcaatct cctcatatac 1560 cgacatctcg
gtcaatgcag caactttagt ttctatcgaa gtgtcaccca caaatccttc 1620
ggtatcttca ggacttaccg ttccttttac ggcgaccgga gtttatacgg atggaagtaa
1680 tcaaaatctg acttctcaag taacttggaa ttcctccaac acgaacagag
ctacaatcag 1740 caacgcaaac ggaactcaag gaattgcctt gggctcttct
gtcggaacta cgaacatatc 1800 agcaacgtta ggtgcggtta cttcttccgc
taccactctt acggtcacaa acgcggtttt 1860 aaattcgatc acgattactc
cgtctcttcc ttccgtagca gtaggaagaa gtctgaacct 1920 tactgcaacc
ggaacttatt ctgacggaag taaccaagat ttaactacct ccgtcgcttg 1980
gacgagtacg gattcttcca tcgtttccgt agacaacgcc tcaggtagac aggggcagac
2040 gacaggtgtt gcacaaggta acactcagat cagtgccaca ttaggcggaa
cttcttctgc 2100 tatcaatttt acggtaagtg cagcggtttt agattcaatt
caagtaactc tggaagattc 2160 tccgattgca aaaggaactt ctacaagagc
aatcgcgacg ggtgtttttt cagacggaag 2220 caatttgaat attagtgatc
aagttatttg ggatagttca caaacaaacg tgatccagct 2280 aggagtttta
gaaaccggtc ctaaaaagaa actgatgaat tctcccgcaa atggaaacag 2340
taccactgga acctcaagga tcactgcaac gttaggaggt gtgagcggat acgccgatct
2400 tacagtaatc gctccaagtt taaccagcat tcaaatcgat cctacacatc
cgagcgttgc 2460 caacggtctg actcaaaatt ttactgcaac cggagtttac
tcagatggta gcaatcagaa 2520 tctaaccgat tccgttactt gggcgtcttc
caatcctgct gttgccacga tcagcaacgc 2580 ttccggaacc aacggtaaag
ctactactct tcaaactgga tccaccaata tcagcgcgag 2640 tctgggcgcc
actacttctg atccaagtgt attaacggtt acaaacgcaa ccttaacaag 2700
tatcacgatc gctcccacct cttccttcaa catcgcaaaa ggattaaatc aagactttgt
2760 agcgaccggt tattatacag atggttcttc tagagacctg accactcaag
tcacttggaa 2820 ttcttccaat acttctaccg ctacgatcag caatgcaaac
ggaactcaag gaagaatggc 2880 cgcggtcgat actggttcta caaatatctc
cgcgtcttta ggaggaacgt atagtcagac 2940 cacaaacgta accgttacat
ctgcggttct gaattcgatc caggtttctc cagcggacat 3000 tagtgtagcc
aaaggaaaca ccaaggccta caccgcgatc ggagtatatt cagattttag 3060
cacgttagac gttacttctc aggttacctg gacttcttcc agcgtttcga tcgctacgat
3120 cagcaatgca agcggacacg aaggtttagc tacggctgta ggcacgggaa
cttccacaat 3180 taccgcaact cttggaggaa tttctaattc tacgagtttg
acggttacgg ccgccgtatt 3240 ggtttctctt tcggtaggtc ctaccaatag
ttttgtttat atgacacaaa ccaaaaattt 3300 tatggctact ggaacgtatt
ctgacggaac gatgcaggat cttacaactc aagtcacctg 3360 gacttcttcc
gatacaacct tgggaacaat cagcaacgcc ttcggaatag aaggtagggc 3420
tacaggaatt gctgccggtg ccataacgat cactgcgact ttgggaagta tcagcggaaa
3480 cacttctttg actataatct ttttagatac gatagcacct gcgatcacaa
acgtagtcgc 3540 cttaactcct actactttaa gaattacata ttccgaaaac
gtaaacgaaa cccaggcaaa 3600 aaccgcggcc aattacaaac tggctcttac
ttcttccgta accggaagtt gttcagataa 3660 cagcaacttt acttctacct
cttctgtgat tactgtttcc tcagtgagtg
gaagcggatc 3720 tgtgtttgtt ctaactctag gttcttcaca aacgtctaac
gcaccttata cgattttagt 3780 gaataaatcg ggaatacaag atctttctac
aaccccaaac aatttgggtt gtgcaaacta 3840 cggagacttc ttaggacagg
aacaaatcaa aatcgtatcc gcctcctgtg caaattccaa 3900 ttccgtgatt
ttgaatttct ctaaggctcc taaatctgga aacaatgtcg ccggttccgc 3960
agaatgtacc ggttctgcag aatgttctaa tcgttacaaa atttccggag caagcgatct
4020 tggaacaatt aacagcgtaa aggtgttaga tggaattatt tgtaacggag
caactgcaga 4080 ttccgcaaaa gtatgcgtaa ttcataattt agtacaaacc
ggagcacaat atacaatcat 4140 cactgcggat tccgtagacg gagacggatt
tgacaactca agctggggat caatccgaaa 4200 ttctttggat acagagaatc
ttcaatcttc tcccagagac agggcttcct ttttaggatg 4260 tggaacgtct
ccggtcaact ttgcagacgg accgatttcc atcgatccaa actcatccac 4320
gttcggttat ctaatcgatt ttaactctaa gatctattca ggaccaaaca attccgggaa
4380 cggagcgctt cgatttgcct atgatggaag tgttccagaa tcagttcaat
tctcctttga 4440 aaaagacaca accgttcaag acggtgacgc gactaacgta
agttcaaact cagcttcttc 4500 cagagagaat tcgatctcgg ttccgcctta
cgttacatta ggacactccg gatgtactac 4560 aaacaacgga actctttctc
taggatgtgg tccggataac gaaaacggaa gaggagtatt 4620 cgctactgga
attctttcca gcgtctccta tctatttgtt gcagctgcaa aaaccgtagc 4680
ggacggcctg ggacaatact tatttgatta tctgtattac tccgcagaca cttctactaa
4740 tacaagtttc aaatatatag atctaggatc gatcaccgga actttaaccg
ccggaacttc 4800 ttcgcttact gtactcaata atagagtgtt tgcaggtttt
gcaaagtcaa gcaacgacgg 4860 aatcggattg ttcggaggac ttaatgcacc
cgattttgga tttgtaacgt ttaactcagc 4920 ggactcagga actggatttt
gtactccagg ctccaactgc gacgcgtttg acggaaccaa 4980 aggaaaaaga
atccggatcg atttccttcc ttacttcgga ggaccgtcca ccggtttatt 5040
aggaattaat aataatgcac atccaaactg ggcgtattat atcggagtcg attccatgtt
5100 cgtatttaaa aatcgtatct atgccgcaaa cggaggatta cacgcggtag
gacataacgg 5160 ttccataata cgttctacaa ctgcagatcc aaccgcggct
tgtaccggac cggactcttg 5220 ttctaactgg gtggaaattg gacctagaac
caacacgaaa tggcacaaca gtcccacaaa 5280 caactggttc tctttagagt
taaatcaatt ttacaatctg attccgggag ataaggcgtt 5340 tgcacaattt
gccgagttca acaataacct ttatgtaact agaaccattt gtattcaaag 5400
ttctcaagcg actggaatca gaaccaatcc aggaaccgta acaggatgta cagacggaac
5460 aactacaaat cgaagggcac aactttggaa atgtgatcct acaatttcag
gaaacacgag 5520 cgaatgtgat gcagcggatt ggtcggtcgt aggcgacgac
ggaaccggaa tcacaaacat 5580 gggagattct acaaaccgaa cgatcaccat
ggtgatgaaa aacggatcct atctttacat 5640 aggatatgat aatccaaacg
gaatcagaat ttatagaacc aacgtagcca acccgggatc 5700 atcctctgcg
tcttggagtc aaatcgccgg gaacggtctc acagatgcga ctaacgttca 5760
acaaatttac tcggccgtat ccgtaccttc cggaagtatc aattatatct acgtaagcgc
5820 tggaaaaagt aacgtttctg ttcggacgta tcgtcaacaa aat 5863 4 1954
PRT Leptospira kirschneri 4 Met Pro Lys His Ile Asn Lys Leu Arg Asp
Lys Lys Thr Trp Pro Phe 1 5 10 15 Leu Gln Phe Ile Phe Ile Leu Phe
Leu Thr Phe Ser Leu Phe Phe Leu 20 25 30 Glu Ser Cys Ala Ala Trp
Pro Ile Phe Ser Gly Thr Pro Gly Leu Leu 35 40 45 Ala Gly Lys Lys
Ser Gly Ala Asn Asn Ser Leu Trp Met Leu Phe Leu 50 55 60 Gly Ile
Asp Asn Pro Leu Glu Ser Glu Pro Ser Glu Ala Glu Leu Asp 65 70 75 80
Arg Ile Glu Ile Ser Val Pro Asn Ser Asn Leu Ala Arg Gly Thr Thr 85
90 95 Leu His Leu Asn Ala Thr Ala Ile Tyr Lys Asp Asn Thr His Arg
Asp 100 105 110 Ile Ser Ser Glu Gly Ser Trp Ser Ser Thr Asp Ser Ser
Ile Leu Lys 115 120 125 Leu Leu Thr Gln Ser Gln Phe Lys Gly Met Asn
Leu Gly Ser Gly Asn 130 135 140 Val Asn Val Ser Phe Gln Gly Lys Asn
Ala Thr Thr Thr Leu Thr Val 145 150 155 160 Thr Ser Ala Val Leu Ser
Asp Leu Thr Val Thr Cys Val Asn Gln Gly 165 170 175 Ser Pro Leu Pro
Val Gly Ile Asp Arg Gln Cys Lys Leu Glu Gly Ile 180 185 190 Phe Ser
Asp Gly Ser Thr Gln Val Leu Thr Ser Asp Pro Ser Ala Ser 195 200 205
Trp Asn Val Thr Gln Ser Ser Ile Ala Gly Val Asn Thr Thr Gly Leu 210
215 220 Val Ser Gly Leu Ser Pro Gly Asn Thr Phe Ile Thr Thr Ser Tyr
Gly 225 230 235 240 Ser Lys Thr Ser Ser Leu Asn Val Thr Val Ser Ala
Ala Thr Leu Ser 245 250 255 Ser Ile Ser Val Thr Pro Ala Asn Ser Ser
Tyr Pro Leu Gly Lys Val 260 265 270 Gln Gln Tyr Thr Ala Ile Gly Thr
Tyr Ser Asn Gln Ser Thr Gln Asp 275 280 285 Leu Thr Asn Gln Val Ser
Trp Ala Ser Leu Asn Thr Ser Val Ala Thr 290 295 300 Ile Asp Asn Ser
Thr Ser Ala Lys Gly Met Leu Thr Thr Gln Ser Thr 305 310 315 320 Gly
Ser Ala Asn Ile Thr Ala Thr Leu Gly Gly Ile Thr Gly Gln Thr 325 330
335 Thr Val Asn Val Thr Ser Ala Val Leu Thr Ser Ile Thr Ile Thr Pro
340 345 350 Ala Asn Pro Ser Val Ala Asn Gly Arg Thr Leu Tyr Leu Thr
Ala Thr 355 360 365 Gly Val Phe Ser Asp Gly Thr Val Ser Asp Ile Thr
Asn Gln Val Thr 370 375 380 Trp Ser Ser Ser Leu Thr Ser Val Ala Thr
Ala Asp Asn Ser Gly Gly 385 390 395 400 Leu Ser Gly Arg Ile Ser Gly
Val Gly Val Gly Ser Thr Asn Ile Thr 405 410 415 Ala Ala Ile Gly Gly
Val Asp Ile Thr Val Ser Leu Asn Val Thr Asn 420 425 430 Ala Thr Leu
Glu Ser Ile Gln Val Val Ser Asp Ser His Ser Ile Ala 435 440 445 Arg
Gly Thr Ser Thr Phe Val Gln Ala Ile Gly Val Tyr Ser Asp Gly 450 455
460 Ser Ser Gln Asn Ile Ser Asp Gln Val Ala Trp Asn Ser Ser Asn Ser
465 470 475 480 Ser Ile Leu Gln Ile Ser Asn Leu Asn Ala Val Pro Lys
Arg Glu Ile 485 490 495 Gln Ser Pro Ser Ser Gly Gly Leu Gly Thr Ala
Arg Ile Thr Ala Thr 500 505 510 Leu Glu Ala Ile Ser Ser Tyr Thr Asp
Ile Ser Val Asn Ala Ala Thr 515 520 525 Leu Val Ser Ile Glu Val Ser
Pro Thr Asn Pro Ser Val Ser Ser Gly 530 535 540 Leu Thr Val Pro Phe
Thr Ala Thr Gly Val Tyr Thr Asp Gly Ser Asn 545 550 555 560 Gln Asn
Leu Thr Ser Gln Val Thr Trp Asn Ser Ser Asn Thr Asn Arg 565 570 575
Ala Thr Ile Ser Asn Ala Asn Gly Thr Gln Gly Ile Ala Leu Gly Ser 580
585 590 Ser Val Gly Thr Thr Asn Ile Ser Ala Thr Leu Gly Ala Val Thr
Ser 595 600 605 Ser Ala Thr Thr Leu Thr Val Thr Asn Ala Val Leu Asn
Ser Ile Thr 610 615 620 Ile Thr Pro Ser Leu Pro Ser Val Ala Val Gly
Arg Ser Leu Asn Leu 625 630 635 640 Thr Ala Thr Gly Thr Tyr Ser Asp
Gly Ser Asn Gln Asp Leu Thr Thr 645 650 655 Ser Val Ala Trp Thr Ser
Thr Asp Ser Ser Ile Val Ser Val Asp Asn 660 665 670 Ala Ser Gly Arg
Gln Gly Gln Thr Thr Gly Val Ala Gln Gly Asn Thr 675 680 685 Gln Ile
Ser Ala Thr Leu Gly Gly Thr Ser Ser Ala Ile Asn Phe Thr 690 695 700
Val Ser Ala Ala Val Leu Asp Ser Ile Gln Val Thr Leu Glu Asp Ser 705
710 715 720 Pro Ile Ala Lys Gly Thr Ser Thr Arg Ala Ile Ala Thr Gly
Val Phe 725 730 735 Ser Asp Gly Ser Asn Leu Asn Ile Ser Asp Gln Val
Ile Trp Asp Ser 740 745 750 Ser Gln Thr Asn Val Ile Gln Leu Gly Val
Leu Glu Thr Gly Pro Lys 755 760 765 Lys Lys Leu Met Asn Ser Pro Ala
Asn Gly Asn Ser Thr Thr Gly Thr 770 775 780 Ser Arg Ile Thr Ala Thr
Leu Gly Gly Val Ser Gly Tyr Ala Asp Leu 785 790 795 800 Thr Val Ile
Ala Pro Ser Leu Thr Ser Ile Gln Ile Asp Pro Thr His 805 810 815 Pro
Ser Val Ala Asn Gly Leu Thr Gln Asn Phe Thr Ala Thr Gly Val 820 825
830 Tyr Ser Asp Gly Ser Asn Gln Asn Leu Thr Asp Ser Val Thr Trp Ala
835 840 845 Ser Ser Asn Pro Ala Val Ala Thr Ile Ser Asn Ala Ser Gly
Thr Asn 850 855 860 Gly Lys Ala Thr Thr Leu Gln Thr Gly Ser Thr Asn
Ile Ser Ala Ser 865 870 875 880 Leu Gly Ala Thr Thr Ser Asp Pro Ser
Val Leu Thr Val Thr Asn Ala 885 890 895 Thr Leu Thr Ser Ile Thr Ile
Ala Pro Thr Ser Ser Phe Asn Ile Ala 900 905 910 Lys Gly Leu Asn Gln
Asp Phe Val Ala Thr Gly Tyr Tyr Thr Asp Gly 915 920 925 Ser Ser Arg
Asp Leu Thr Thr Gln Val Thr Trp Asn Ser Ser Asn Thr 930 935 940 Ser
Thr Ala Thr Ile Ser Asn Ala Asn Gly Thr Gln Gly Arg Met Ala 945 950
955 960 Ala Val Asp Thr Gly Ser Thr Asn Ile Ser Ala Ser Leu Gly Gly
Thr 965 970 975 Tyr Ser Gln Thr Thr Asn Val Thr Val Thr Ser Ala Val
Leu Asn Ser 980 985 990 Ile Gln Val Ser Pro Ala Asp Ile Ser Val Ala
Lys Gly Asn Thr Lys 995 1000 1005 Ala Tyr Thr Ala Ile Gly Val Tyr
Ser Asp Phe Ser Thr Leu Asp Val 1010 1015 1020 Thr Ser Gln Val Thr
Trp Thr Ser Ser Ser Val Ser Ile Ala Thr Ile 1025 1030 1035 1040 Ser
Asn Ala Ser Gly His Glu Gly Leu Ala Thr Ala Val Gly Thr Gly 1045
1050 1055 Thr Ser Thr Ile Thr Ala Thr Leu Gly Gly Ile Ser Asn Ser
Thr Ser 1060 1065 1070 Leu Thr Val Thr Ala Ala Val Leu Val Ser Leu
Ser Val Gly Pro Thr 1075 1080 1085 Asn Ser Phe Val Tyr Met Thr Gln
Thr Lys Asn Phe Met Ala Thr Gly 1090 1095 1100 Thr Tyr Ser Asp Gly
Thr Met Gln Asp Leu Thr Thr Gln Val Thr Trp 1105 1110 1115 1120 Thr
Ser Ser Asp Thr Thr Leu Gly Thr Ile Ser Asn Ala Phe Gly Ile 1125
1130 1135 Glu Gly Arg Ala Thr Gly Ile Ala Ala Gly Ala Ile Thr Ile
Thr Ala 1140 1145 1150 Thr Leu Gly Ser Ile Ser Gly Asn Thr Ser Leu
Thr Ile Ile Phe Leu 1155 1160 1165 Asp Thr Ile Ala Pro Ala Ile Thr
Asn Val Val Ala Leu Thr Pro Thr 1170 1175 1180 Thr Leu Arg Ile Thr
Tyr Ser Glu Asn Val Asn Glu Thr Gln Ala Lys 1185 1190 1195 1200 Thr
Ala Ala Asn Tyr Lys Leu Ala Leu Thr Ser Ser Val Thr Gly Ser 1205
1210 1215 Cys Ser Asp Asn Ser Asn Phe Thr Ser Thr Ser Ser Val Ile
Thr Val 1220 1225 1230 Ser Ser Val Ser Gly Ser Gly Ser Val Phe Val
Leu Thr Leu Gly Ser 1235 1240 1245 Ser Gln Thr Ser Asn Ala Pro Tyr
Thr Ile Leu Val Asn Lys Ser Gly 1250 1255 1260 Ile Gln Asp Leu Ser
Thr Thr Pro Asn Asn Leu Gly Cys Ala Asn Tyr 1265 1270 1275 1280 Gly
Asp Phe Leu Gly Gln Glu Gln Ile Lys Ile Val Ser Ala Ser Cys 1285
1290 1295 Ala Asn Ser Asn Ser Val Ile Leu Asn Phe Ser Lys Ala Pro
Lys Ser 1300 1305 1310 Gly Asn Asn Val Ala Gly Ser Ala Glu Cys Thr
Gly Ser Ala Glu Cys 1315 1320 1325 Ser Asn Arg Tyr Lys Ile Ser Gly
Ala Ser Asp Leu Gly Thr Ile Asn 1330 1335 1340 Ser Val Lys Val Leu
Asp Gly Ile Ile Cys Asn Gly Ala Thr Ala Asp 1345 1350 1355 1360 Ser
Ala Lys Val Cys Val Ile His Asn Leu Val Gln Thr Gly Ala Gln 1365
1370 1375 Tyr Thr Ile Ile Thr Ala Asp Ser Val Asp Gly Asp Gly Phe
Asp Asn 1380 1385 1390 Ser Ser Trp Gly Ser Ile Arg Asn Ser Leu Asp
Thr Glu Asn Leu Gln 1395 1400 1405 Ser Ser Pro Arg Asp Arg Ala Ser
Phe Leu Gly Cys Gly Thr Ser Pro 1410 1415 1420 Val Asn Phe Ala Asp
Gly Pro Ile Ser Ile Asp Pro Asn Ser Ser Thr 1425 1430 1435 1440 Phe
Gly Tyr Leu Ile Asp Phe Asn Ser Lys Ile Tyr Ser Gly Pro Asn 1445
1450 1455 Asn Ser Gly Asn Gly Ala Leu Arg Phe Ala Tyr Asp Gly Ser
Val Pro 1460 1465 1470 Glu Ser Val Gln Phe Ser Phe Glu Lys Asp Thr
Thr Val Gln Asp Gly 1475 1480 1485 Asp Ala Thr Asn Val Ser Ser Asn
Ser Ala Ser Ser Arg Glu Asn Ser 1490 1495 1500 Ile Ser Val Pro Pro
Tyr Val Thr Leu Gly His Ser Gly Cys Thr Thr 1505 1510 1515 1520 Asn
Asn Gly Thr Leu Ser Leu Gly Cys Gly Pro Asp Asn Glu Asn Gly 1525
1530 1535 Arg Gly Val Phe Ala Thr Gly Ile Leu Ser Ser Val Ser Tyr
Leu Phe 1540 1545 1550 Val Ala Ala Ala Lys Thr Val Ala Asp Gly Leu
Gly Gln Tyr Leu Phe 1555 1560 1565 Asp Tyr Leu Tyr Tyr Ser Ala Asp
Thr Ser Thr Asn Thr Ser Phe Lys 1570 1575 1580 Tyr Ile Asp Leu Gly
Ser Ile Thr Gly Thr Leu Thr Ala Gly Thr Ser 1585 1590 1595 1600 Ser
Leu Thr Val Leu Asn Asn Arg Val Phe Ala Gly Phe Ala Lys Ser 1605
1610 1615 Ser Asn Asp Gly Ile Gly Leu Phe Gly Gly Leu Asn Ala Pro
Asp Phe 1620 1625 1630 Gly Phe Val Thr Phe Asn Ser Ala Asp Ser Gly
Thr Gly Phe Cys Thr 1635 1640 1645 Pro Gly Ser Asn Cys Asp Ala Phe
Asp Gly Thr Lys Gly Lys Arg Ile 1650 1655 1660 Arg Ile Asp Phe Leu
Pro Tyr Phe Gly Gly Pro Ser Thr Gly Leu Leu 1665 1670 1675 1680 Gly
Ile Asn Asn Asn Ala His Pro Asn Trp Ala Tyr Tyr Ile Gly Val 1685
1690 1695 Asp Ser Met Phe Val Phe Lys Asn Arg Ile Tyr Ala Ala Asn
Gly Gly 1700 1705 1710 Leu His Ala Val Gly His Asn Gly Ser Ile Ile
Arg Ser Thr Thr Ala 1715 1720 1725 Asp Pro Thr Ala Ala Cys Thr Gly
Pro Asp Ser Cys Ser Asn Trp Val 1730 1735 1740 Glu Ile Gly Pro Arg
Thr Asn Thr Lys Trp His Asn Ser Pro Thr Asn 1745 1750 1755 1760 Asn
Trp Phe Ser Leu Glu Leu Asn Gln Phe Tyr Asn Leu Ile Pro Gly 1765
1770 1775 Asp Lys Ala Phe Ala Gln Phe Ala Glu Phe Asn Asn Asn Leu
Tyr Val 1780 1785 1790 Thr Arg Thr Ile Cys Ile Gln Ser Ser Gln Ala
Thr Gly Ile Arg Thr 1795 1800 1805 Asn Pro Gly Thr Val Thr Gly Cys
Thr Asp Gly Thr Thr Thr Asn Arg 1810 1815 1820 Arg Ala Gln Leu Trp
Lys Cys Asp Pro Thr Ile Ser Gly Asn Thr Ser 1825 1830 1835 1840 Glu
Cys Asp Ala Ala Asp Trp Ser Val Val Gly Asp Asp Gly Thr Gly 1845
1850 1855 Ile Thr Asn Met Gly Asp Ser Thr Asn Arg Thr Ile Thr Met
Val Met 1860 1865 1870 Lys Asn Gly Ser Tyr Leu Tyr Ile Gly Tyr Asp
Asn Pro Asn Gly Ile 1875 1880 1885 Arg Ile Tyr Arg Thr Asn Val Ala
Asn Pro Gly Ser Ser Ser Ala Ser 1890 1895 1900 Trp Ser Gln Ile Ala
Gly Asn Gly Leu Thr Asp Ala Thr Asn Val Gln 1905 1910 1915 1920 Gln
Ile Tyr Ser Ala Val Ser Val Pro Ser Gly Ser Ile Asn Tyr Ile 1925
1930 1935 Tyr Val Ser Ala Gly Lys Ser Asn Val Ser Val Arg Thr Tyr
Arg Gln 1940 1945 1950 Gln Asn 5 5658 DNA Leptospira kirschneri 5
atgaagagaa cattttgtat ttcgattctt ctttcgatgt tttttcaaag ttgtatgtct
60 tggccacttt taaccagtct cgcgggttta gcagctggta aaaaaagtaa
tgggctgccc 120 tttttccacc ttctattaag taactctgat ccagttatta
caaggatcga gctcagttat 180 caaaattctt ccatcgcaaa aggtacaagt
acaactctcg aagtcaccgc aatctttgat 240 aacggaacaa atcagaatat
tacggattcg acatctatcg tttccgatgc ccaatcaatc 300 gttgacattc
aaggtaacag agtcagagga atcgcttctg gttcttccat tataaaagct 360
gaatacaacg ggatgtattc tgaacaaaaa attacggtta caccagccac gataaactca
420 attcaagtta cgagtttaga tgacggtata ttacctaaag gtacaaatcg
tcaatttgct 480 gccatcggta tcttttcgga tggttctcat caagatattt
ccaacgatcc attgatcgtt 540 tggtcttcca gtaatataga tttagttcga
gtagatgatt ccggtttggc ctcaggtatc 600 aatttaggaa cggctcatat
tcgtgcatcc tttcaatcaa aacaagcctc cgaagagata 660 actgttggtg
acgctgttct ttcttctatc caagtaactt ccaacagtcc aaatattcct 720
ctcggaaaaa aacaaaaact cacagctact ggaatttatt cggataactc taacagggat
780 atttcctctt ctgttatctg gaattcttct aattccacta tcgctaatat
tcagaataac 840 ggaatattag aaacagctga tactggaatt gttactgttt
ctgcttctag aggtaatata 900 aatggttcca taaaactaat cgtcactcct
gctgccttag tttctatttc tgtttctcct 960 acaaattctg cagtagcaaa
aggtttacaa gaaaacttta aagctacagg gatctttaca 1020 gataattcga
actcagatat tacagatcaa gttacttggg attcttctaa tccggatatt 1080
ctttccattt ccaatgcaag tgatagccac gggttagctt ccacactcaa ccaaggaaat
1140 gttaaggtca ccgcttccat cggtggaata caaggatcca ctgattttaa
agttacacaa 1200 gaggtattaa cttccatcga agtttctcca gttttacctt
caattgcaaa aggactaact 1260 cagaaattta cggcgatcgg gatttttacg
gataactcca aaaaagatat tacaaatcaa 1320 gtcacttgga attcttcttc
agcaatcgca agcgtgtcta acttagatga taataaaggt 1380 ctgggaaaag
ctcacgctgt tggagacacg actattaccg ctactttagg aaaagtttca 1440
ggtaaaactt ggtttactgt agttcctgcg gttctcactt ctattcaaat caatcctgta
1500 aatccttctc ttgcaaaagg gttaactcaa aaatttacgg ctactgggat
ctactctgac 1560 aactctaaca aggacattac ttcctccgtt acttggttct
catccgattc ttcaatcgca 1620 acaatttcaa acgccaaaaa aaatcaagga
aactcttacg gagcagctac aggagcaacg 1680 gatattaaag ccacattcgg
aaaggtaagt agtccagttt ctacgttatc cgttactgct 1740 gcaaaacttg
ttgaaataca aatcacaccg gccgctgctt ccaaagcaaa gggaatttcc 1800
gaaagattta aagcaaccgg tatttttaca gacaactcta attccgatat tacaaatcag
1860 gtcacttgga gttcatctaa tacagatatt gctgaaatta caaataccag
aggaagcaaa 1920 ggtattacaa atacactcac tcccggatcg agtgaaatat
ccgccgctct cggttcaatc 1980 aaaagttcta aagtaatatt gaaggtaact
ccggcacaat tgatttccat tgcagtaaca 2040 cctacaaatc catcagttgc
aaaaggtcta atacgacaat ttaaagccac cggaacatat 2100 acggatcatt
ccgtacaaga cgtgactgcc ctagctacct ggtcttcttc caatcccaga 2160
aaagcaatgg ttaacaacgt tacaggttcg gttacaacag tggctaccgg aaatacaaat
2220 attaaagcaa cgatagactc catatccgga tcttccgttt tgaatgtcac
tcctgcactt 2280 cttacttcta tcgagataac accgacgatt aactctatca
ctcacggtct tacaaaacaa 2340 tttaaagcga ctggtatctt ttcagataaa
tctactcaaa atttgactca gcttgtaact 2400 tggatttctt ccgatccctc
caagatcaag atcgaaaata actccggtat agcaacagct 2460 tctgcattag
gaagttcgaa tattacggcc atctacaaat ttgtccaaag ttccccaatt 2520
ccgatcacag tcactgactt aaaactgaaa agtataacta tcagtccttc ctcaagttca
2580 atagccaaag gattgaccca acaatttaaa gcgatcggaa cttttataga
tggttctgaa 2640 caagaaatta cgaatcttgt gacctggtat tcctccaaat
ccgatattgt tcctatcaat 2700 aattctgcgg gtaaaaaagg tttagcgacc
gcactctcaa taggttcctc caacatctcc 2760 gcaatttaca attctataag
cagtaataaa ataaatttta atgtaagcgc cgccacgtta 2820 gattccatta
aaatcaatcc agtcaacaat aacatcgcca agggacttac ccaacaatat 2880
actgcgcttg gcgtttattc agactccacc attcaggaca tcagcgattt agttacatgg
2940 tccagttcca attctgactc gatcagcatc tccaattcga ccggaaccaa
gggaaaagcg 3000 accgctttac agattggaaa gagcaaaatt accgcgactt
acaattccat ttcgaaaaac 3060 ataaatctaa ctgtcagcgc agcaactctc
tcttcgattt ttatatctcc taccaataca 3120 aatataaaca ccaccgtatc
aaaacaattc tttgcaatgg gaacgtattc ggacggaacc 3180 aaaacggatt
taacttcttc ggttacatgg tccagttcga atcaagctca agcaaaggtg 3240
agtaacgcat ctgaaacgaa aggattggtt acagggatta cttctggaaa tcctataatc
3300 acagcgacct acggctcagt gtcgggaaat acaattctca cagtaaacaa
aaccgacacg 3360 atagctccga cggttcaatc ggtagtttct ttatcaccta
ctaccatcca agttgtatat 3420 tcagaatcca taaacaatca ggaagccctt
gatttatcca attacaaaat aattaatagt 3480 tccaattttt acggacattg
ttcggataat acggacttca attccaattc tcaaaccgca 3540 gatttttctc
ttagtagtat caaaggaagt aaaaatactt ttacgattac actttcacat 3600
tcacaaatct taaacaaatc atacacactt gtagtcaaca aacaaggaat tcacgatctt
3660 tcttccattc caaattcctt aagttgtcca aataactctg attttatagg
aaaagaacaa 3720 ctcaaactta caagtgcagt ttgtaattcc ttaaaccaag
tgatcgtttc tttttccaaa 3780 cctttatatt ctggaaagga agtaacaaaa
tccgtggaat gttcaaatcc gtcccaatgt 3840 gaatccagat ataaatttgc
aggtgtgtct tcattgggaa gtattacgag cgttagaatt 3900 ttagatggaa
aagtatgcgg tggagcaccg gcagactcct cgaaaatatg tttaacacac 3960
tcccttcttc aatcaggtgg tcaatatacg atcatcgccg caaatgattt gaacggagac
4020 ggctttgaca acaaatcctg gggagcaatt cgagattcat tcgatcaaga
aaacctacaa 4080 ccttctccga aagatagaat caactttata ggttgtggaa
attcccctct caactttatg 4140 gatggcccga tcgtgtcaga tccttttgga
gacggttccg atttcggctc tcttgtagat 4200 tacaacaatc aaatctatct
aggaccgaat gtaaaaggaa accaagcagc tcgattcaat 4260 tacgacggaa
cttttccgga atctattttc ttttctttta cccaagataa aaatgccact 4320
aaccgtgctt cttcaagaga tggaggaatt ccggttccga attacgttac gatcggtcat
4380 accggttgta ctctcaatag tgcagacatc actactggat gtggtccaga
taacgaagat 4440 ggacgtgggg tttttgccac cggatcatta gacaaaaaat
ctcatatttt tatagcaggt 4500 tcaaaaccaa ggagattcaa ctatctctat
tattcctcag ataccgatac aaaccttaat 4560 tttaaatata tcagtatggg
aaaaattact ggattggcga ctgcaggaac ttcatctatc 4620 gcagttctag
acgatcggat ccatgtaggt tttgcaaaaa aaaatcaaaa tctaaacgca 4680
cctgatttcg gtaaaatcac ctttaataca tccgagcaca atcgatgtgc aattgtaaac
4740 aactgtgaag cctctgacgg ataccgcggt aatcgtttta gaatcgatag
aatgccttac 4800 tttggcggcg gctccgtgga tgcagtcaat tataaaactc
ataaatctga taattcctcg 4860 atcaactggg gttattatgt gggaatagat
tctctattcg tttttaaaga aaaactttac 4920 gccgcaaacg gaggatttcc
aaattcatta cataatggaa gtataataca ctctaccagt 4980 gcaaatccta
gtccttgtga aggaatcaat cgttgttcca gttggaaaga cacagcacct 5040
agatccaatc cgaagtggca taactctcct cataccaatt ggttttcact ggagcttaca
5100 aagtatcgag atttaattcc ggcggataaa gcattctctc aattcgcaga
atttaacgga 5160 agattgtatg taacaagaac gatctgtgta acgaaagaag
atcactccgg actcagacaa 5220 agtttacaaa ctttgaaagg ttgtacagac
ggaagttata caaatcgaag acctcaactt 5280 tggaaatgtg atccgactct
aaccggcgat acaacaacct gcgaagcaaa agattggtct 5340 ttagtaggag
ataatggaac cgggtttacg aatttcggag acgattccaa tcacagtatg 5400
acgatggtag ttgcaagtgg atcttatctc tacgtaggtt ttgacaacga aaacggaatt
5460 caaatctgga gaacaaatct tgaaaatcct ggaagttcat cacacgactg
ggagcctata 5520 ggaataggcg gattaagaga cgttaccaat cgtcaaattt
attcggctat atccggaatg 5580 aattttggtg taaatttcgt atatataagc
gtaggaaata aagatcaacc ggttaaaatt 5640 tacagacaac agaaccaa 5658 6
1886 PRT Leptospira kirschneri 6 Met Lys Arg Thr Phe Cys Ile Ser
Ile Leu Leu Ser Met Phe Phe Gln 1 5 10 15 Ser Cys Met Ser Trp Pro
Leu Leu Thr Ser Leu Ala Gly Leu Ala Ala 20 25 30 Gly Lys Lys Ser
Asn Gly Leu Pro Phe Phe His Leu Leu Leu Ser Asn 35 40 45 Ser Asp
Pro Val Ile Thr Arg Ile Glu Leu Ser Tyr Gln Asn Ser Ser 50 55 60
Ile Ala Lys Gly Thr Ser Thr Thr Leu Glu Val Thr Ala Ile Phe Asp 65
70 75 80 Asn Gly Thr Asn Gln Asn Ile Thr Asp Ser Thr Ser Ile Val
Ser Asp 85 90 95 Ala Gln Ser Ile Val Asp Ile Gln Gly Asn Arg Val
Arg Gly Ile Ala 100 105 110 Ser Gly Ser Ser Ile Ile Lys Ala Glu Tyr
Asn Gly Met Tyr Ser Glu 115 120 125 Gln Lys Ile Thr Val Thr Pro Ala
Thr Ile Asn Ser Ile Gln Val Thr 130 135 140 Ser Leu Asp Asp Gly Ile
Leu Pro Lys Gly Thr Asn Arg Gln Phe Ala 145 150 155 160 Ala Ile Gly
Ile Phe Ser Asp Gly Ser His Gln Asp Ile Ser Asn Asp 165 170 175 Pro
Leu Ile Val Trp Ser Ser Ser Asn Ile Asp Leu Val Arg Val Asp 180 185
190 Asp Ser Gly Leu Ala Ser Gly Ile Asn Leu Gly Thr Ala His Ile Arg
195 200 205 Ala Ser Phe Gln Ser Lys Gln Ala Ser Glu Glu Ile Thr Val
Gly Asp 210 215 220 Ala Val Leu Ser Ser Ile Gln Val Thr Ser Asn Ser
Pro Asn Ile Pro 225 230 235 240 Leu Gly Lys Lys Gln Lys Leu Thr Ala
Thr Gly Ile Tyr Ser Asp Asn 245 250 255 Ser Asn Arg Asp Ile Ser Ser
Ser Val Ile Trp Asn Ser Ser Asn Ser 260 265 270 Thr Ile Ala Asn Ile
Gln Asn Asn Gly Ile Leu Glu Thr Ala Asp Thr 275 280 285 Gly Ile Val
Thr Val Ser Ala Ser Arg Gly Asn Ile Asn Gly Ser Ile 290 295 300 Lys
Leu Ile Val Thr Pro Ala Ala Leu Val Ser Ile Ser Val Ser Pro 305 310
315 320 Thr Asn Ser Ala Val Ala Lys Gly Leu Gln Glu Asn Phe Lys Ala
Thr 325 330 335 Gly Ile Phe Thr Asp Asn Ser Asn Ser Asp Ile Thr Asp
Gln Val Thr 340 345 350 Trp Asp Ser Ser Asn Pro Asp Ile Leu Ser Ile
Ser Asn Ala Ser Asp 355 360 365 Ser His Gly Leu Ala Ser Thr Leu Asn
Gln Gly Asn Val Lys Val Thr 370 375 380 Ala Ser Ile Gly Gly Ile Gln
Gly Ser Thr Asp Phe Lys Val Thr Gln 385 390 395 400 Glu Val Leu Thr
Ser Ile Glu Val Ser Pro Val Leu Pro Ser Ile Ala 405 410 415 Lys Gly
Leu Thr Gln Lys Phe Thr Ala Ile Gly Ile Phe Thr Asp Asn 420 425 430
Ser Lys Lys Asp Ile Thr Asn Gln Val Thr Trp Asn Ser Ser Ser Ala 435
440 445 Ile Ala Ser Val Ser Asn Leu Asp Asp Asn Lys Gly Leu Gly Lys
Ala 450 455 460 His Ala Val Gly Asp Thr Thr Ile Thr Ala Thr Leu Gly
Lys Val Ser 465 470 475 480 Gly Lys Thr Trp Phe Thr Val Val Pro Ala
Val Leu Thr Ser Ile Gln 485 490 495 Ile Asn Pro Val Asn Pro Ser Leu
Ala Lys Gly Leu Thr Gln Lys Phe 500 505 510 Thr Ala Thr Gly Ile Tyr
Ser Asp Asn Ser Asn Lys Asp Ile Thr Ser 515 520 525 Ser Val Thr Trp
Phe Ser Ser Asp Ser Ser Ile Ala Thr Ile Ser Asn 530 535 540 Ala Lys
Lys Asn Gln Gly Asn Ser Tyr Gly Ala Ala Thr Gly Ala Thr 545 550 555
560 Asp Ile Lys Ala Thr Phe Gly Lys Val Ser Ser Pro Val Ser Thr Leu
565 570 575 Ser Val Thr Ala Ala Lys Leu Val Glu Ile Gln Ile Thr Pro
Ala Ala 580 585 590 Ala Ser Lys Ala Lys Gly Ile Ser Glu Arg Phe Lys
Ala Thr Gly Ile 595 600 605 Phe Thr Asp Asn Ser Asn Ser Asp Ile Thr
Asn Gln Val Thr Trp Ser 610 615 620 Ser Ser Asn Thr Asp Ile Ala Glu
Ile Thr Asn Thr Arg Gly Ser Lys 625 630 635 640 Gly Ile Thr Asn Thr
Leu Thr Pro Gly Ser Ser Glu Ile Ser Ala Ala 645 650 655 Leu Gly Ser
Ile Lys Ser Ser Lys Val Ile Leu Lys Val Thr Pro Ala 660 665 670 Gln
Leu Ile Ser Ile Ala Val Thr Pro Thr Asn Pro Ser Val Ala Lys 675 680
685 Gly Leu Ile Arg Gln Phe Lys Ala Thr Gly Thr Tyr Thr Asp His Ser
690 695 700 Val Gln Asp Val Thr Ala Leu Ala Thr Trp Ser Ser Ser Asn
Pro Arg 705 710 715 720 Lys Ala Met Val Asn Asn Val Thr Gly Ser Val
Thr Thr Val Ala Thr 725 730 735 Gly Asn Thr Asn Ile Lys Ala Thr Ile
Asp Ser Ile Ser Gly Ser Ser 740 745 750 Val Leu Asn Val Thr Pro Ala
Leu Leu Thr Ser Ile Glu Ile Thr Pro 755 760 765 Thr Ile Asn Ser Ile
Thr His Gly Leu Thr Lys Gln Phe Lys Ala Thr 770 775 780 Gly Ile Phe
Ser Asp Lys Ser Thr Gln Asn Leu Thr Gln Leu Val Thr 785 790 795 800
Trp Ile Ser Ser Asp Pro Ser Lys Ile Lys Ile Glu Asn Asn Ser Gly 805
810 815 Ile Ala Thr Ala Ser Ala Leu Gly Ser Ser Asn Ile Thr Ala Ile
Tyr 820 825 830 Lys Phe Val Gln Ser Ser Pro Ile Pro Ile Thr Val Thr
Asp Leu Lys 835 840 845 Leu Lys Ser Ile Thr Ile Ser Pro Ser Ser Ser
Ser Ile Ala Lys Gly 850 855 860 Leu Thr Gln Gln Phe Lys Ala Ile Gly
Thr Phe Ile Asp Gly Ser Glu 865 870 875 880 Gln Glu Ile Thr Asn Leu
Val Thr Trp Tyr Ser Ser Lys Ser Asp Ile 885 890 895 Val Pro Ile Asn
Asn Ser Ala Gly Lys Lys Gly Leu Ala Thr Ala Leu 900 905 910 Ser Ile
Gly Ser Ser Asn Ile Ser Ala Ile Tyr Asn Ser Ile Ser Ser 915 920 925
Asn Lys Ile Asn Phe Asn Val Ser Ala Ala Thr Leu Asp Ser Ile Lys 930
935 940 Ile Asn Pro Val Asn Asn Asn Ile Ala Lys Gly Leu Thr Gln Gln
Tyr 945 950 955 960 Thr Ala Leu Gly Val Tyr Ser Asp Ser Thr Ile Gln
Asp Ile Ser Asp 965 970 975 Leu Val Thr Trp Ser Ser Ser Asn Ser Asp
Ser Ile Ser Ile Ser Asn 980 985 990 Ser Thr Gly Thr Lys Gly Lys Ala
Thr Ala Leu Gln Ile Gly Lys Ser 995 1000 1005 Lys Ile Thr Ala Thr
Tyr Asn Ser Ile Ser Lys Asn Ile Asn Leu Thr 1010 1015 1020 Val Ser
Ala Ala Thr Leu Ser Ser Ile Phe Ile Ser Pro Thr Asn Thr 1025 1030
1035 1040 Asn Ile Asn Thr Thr Val Ser Lys Gln Phe Phe Ala Met Gly
Thr Tyr 1045 1050 1055 Ser Asp Gly Thr Lys Thr Asp Leu Thr Ser Ser
Val Thr Trp Ser Ser 1060 1065 1070 Ser Asn Gln Ala Gln Ala Lys Val
Ser Asn Ala Ser Glu Thr Lys Gly 1075 1080 1085 Leu Val Thr Gly Ile
Thr Ser Gly Asn Pro Ile Ile Thr Ala Thr Tyr 1090 1095 1100 Gly Ser
Val Ser Gly Asn Thr Ile Leu Thr Val Asn Lys Thr Asp Thr 1105 1110
1115 1120 Ile Ala Pro Thr Val Gln Ser Val Val Ser Leu Ser Pro Thr
Thr Ile 1125 1130 1135 Gln Val Val Tyr Ser Glu Ser Ile Asn Asn Gln
Glu Ala Leu Asp Leu 1140 1145 1150 Ser Asn Tyr Lys Ile Ile Asn Ser
Ser Asn Phe Tyr Gly His Cys Ser 1155 1160 1165 Asp Asn Thr Asp Phe
Asn Ser Asn Ser Gln Thr Ala Asp Phe Ser Leu 1170 1175 1180 Ser Ser
Ile Lys Gly Ser Lys Asn Thr Phe Thr Ile Thr Leu Ser His 1185 1190
1195 1200 Ser Gln Ile Leu Asn Lys Ser Tyr Thr Leu Val Val Asn Lys
Gln Gly 1205 1210 1215 Ile His Asp Leu Ser Ser Ile Pro Asn Ser Leu
Ser Cys Pro Asn Asn 1220 1225 1230 Ser Asp Phe Ile Gly Lys Glu Gln
Leu Lys Leu Thr Ser Ala Val Cys 1235 1240 1245 Asn Ser Leu Asn Gln
Val Ile Val Ser Phe Ser Lys Pro Leu Tyr Ser 1250 1255 1260 Gly Lys
Glu Val Thr Lys Ser Val Glu Cys Ser Asn Pro Ser Gln Cys 1265 1270
1275 1280 Glu Ser Arg Tyr Lys Phe Ala Gly Val Ser Ser Leu Gly Ser
Ile Thr 1285 1290 1295 Ser Val Arg Ile Leu Asp Gly Lys Val Cys Gly
Gly Ala Pro Ala Asp 1300 1305 1310 Ser Ser Lys Ile Cys Leu Thr His
Ser Leu Leu Gln Ser Gly Gly Gln 1315 1320 1325 Tyr Thr Ile Ile Ala
Ala Asn Asp Leu Asn Gly Asp Gly Phe Asp Asn 1330 1335 1340 Lys Ser
Trp Gly Ala Ile Arg Asp Ser Phe Asp Gln Glu Asn Leu Gln 1345 1350
1355 1360 Pro Ser Pro Lys Asp Arg Ile Asn Phe Ile Gly Cys Gly Asn
Ser Pro 1365 1370 1375 Leu Asn Phe Met Asp Gly Pro Ile Val Ser Asp
Pro Phe Gly Asp Gly 1380 1385 1390 Ser Asp Phe Gly Ser Leu Val Asp
Tyr Asn Asn Gln Ile Tyr Leu Gly 1395 1400 1405 Pro Asn Val Lys Gly
Asn Gln Ala Ala Arg Phe Asn Tyr Asp Gly Thr 1410 1415 1420 Phe Pro
Glu Ser Ile Phe Phe Ser Phe Thr Gln Asp Lys Asn Ala Thr 1425 1430
1435 1440 Asn Arg Ala Ser Ser Arg Asp Gly Gly Ile Pro Val Pro Asn
Tyr Val 1445 1450 1455 Thr Ile Gly His Thr Gly Cys Thr Leu Asn Ser
Ala Asp Ile Thr Thr 1460 1465 1470 Gly Cys Gly Pro Asp Asn Glu Asp
Gly Arg Gly Val Phe Ala Thr Gly 1475 1480 1485 Ser Leu Asp Lys Lys
Ser His Ile Phe Ile Ala Gly Ser Lys Pro Arg 1490 1495 1500 Arg Phe
Asn Tyr Leu Tyr Tyr Ser Ser Asp Thr Asp Thr Asn Leu Asn 1505 1510
1515 1520 Phe Lys Tyr Ile Ser Met Gly Lys Ile Thr Gly Leu Ala Thr
Ala Gly 1525 1530 1535 Thr Ser Ser Ile Ala Val Leu Asp Asp Arg Ile
His Val Gly Phe Ala 1540 1545 1550 Lys Lys Asn Gln Asn Leu Asn Ala
Pro Asp Phe Gly Lys Ile Thr Phe 1555 1560 1565 Asn Thr Ser Glu His
Asn Arg Cys Ala Ile Val Asn Asn Cys Glu Ala 1570 1575 1580 Ser Asp
Gly Tyr Arg Gly Asn Arg Phe Arg Ile Asp Arg Met Pro Tyr 1585 1590
1595 1600 Phe Gly Gly Gly Ser Val Asp Ala Val Asn Tyr Lys Thr His
Lys Ser 1605 1610 1615 Asp Asn Ser Ser Ile Asn Trp Gly Tyr Tyr Val
Gly Ile Asp
Ser Leu 1620 1625 1630 Phe Val Phe Lys Glu Lys Leu Tyr Ala Ala Asn
Gly Gly Phe Pro Asn 1635 1640 1645 Ser Leu His Asn Gly Ser Ile Ile
His Ser Thr Ser Ala Asn Pro Ser 1650 1655 1660 Pro Cys Glu Gly Ile
Asn Arg Cys Ser Ser Trp Lys Asp Thr Ala Pro 1665 1670 1675 1680 Arg
Ser Asn Pro Lys Trp His Asn Ser Pro His Thr Asn Trp Phe Ser 1685
1690 1695 Leu Glu Leu Thr Lys Tyr Arg Asp Leu Ile Pro Ala Asp Lys
Ala Phe 1700 1705 1710 Ser Gln Phe Ala Glu Phe Asn Gly Arg Leu Tyr
Val Thr Arg Thr Ile 1715 1720 1725 Cys Val Thr Lys Glu Asp His Ser
Gly Leu Arg Gln Ser Leu Gln Thr 1730 1735 1740 Leu Lys Gly Cys Thr
Asp Gly Ser Tyr Thr Asn Arg Arg Pro Gln Leu 1745 1750 1755 1760 Trp
Lys Cys Asp Pro Thr Leu Thr Gly Asp Thr Thr Thr Cys Glu Ala 1765
1770 1775 Lys Asp Trp Ser Leu Val Gly Asp Asn Gly Thr Gly Phe Thr
Asn Phe 1780 1785 1790 Gly Asp Asp Ser Asn His Ser Met Thr Met Val
Val Ala Ser Gly Ser 1795 1800 1805 Tyr Leu Tyr Val Gly Phe Asp Asn
Glu Asn Gly Ile Gln Ile Trp Arg 1810 1815 1820 Thr Asn Leu Glu Asn
Pro Gly Ser Ser Ser His Asp Trp Glu Pro Ile 1825 1830 1835 1840 Gly
Ile Gly Gly Leu Arg Asp Val Thr Asn Arg Gln Ile Tyr Ser Ala 1845
1850 1855 Ile Ser Gly Met Asn Phe Gly Val Asn Phe Val Tyr Ile Ser
Val Gly 1860 1865 1870 Asn Lys Asp Gln Pro Val Lys Ile Tyr Arg Gln
Gln Asn Gln 1875 1880 1885 7 1557 DNA Leptospira interrogans 7
attaccgtta caccagccat tcttaactca attcaagtta cgagtttaga gtcaggtata
60 ctacctaaag gtactaatcg tcaattctca gccatcggta tcttttcgga
tggttctcat 120 caggatattt ccaacgaacc actgatcgtt tggtcttcca
gtaatcctga tttggttcga 180 gtagatgatt cagggttggc atcagggatc
aatttaggaa cagctcatat tcgtgcatcc 240 tttcaatcaa aacaaggggc
tgaagaaatg accgttggag atgctgttct ctctcaaatc 300 caagtaactt
caaacgatct gaatattcct ctcggaaaaa aacaaaaact aacagctacg 360
ggaatctatt cggataactc taacagggat atttcctctt ctgttatttg gaattcttct
420 aattccacta tcgctaatat tcaaaacaac ggaatattag aaacagctga
tactggtatt 480 gtcactgttt ctgcttctag cgagaatata atcggatccg
taaaactaat cgttactcca 540 gcagccttag tttctatttc tgtttctccg
acaaattcta cagttgcaaa aggtttacaa 600 gaaaacttta aagctacagg
gatctttaca gataattcaa actcggatat taccgaccaa 660 gttacttggg
attcttctaa taccgatatt ctctcaattt ccaatgcaag tgatagccac 720
ggattagctt ccacactcaa ccaagggaat gttaaagtca ctgcttccat cggtggaata
780 caaggatcca ctgattttaa agttacacaa gctgcattga cttccatcga
agtctctcca 840 actcgcactt ccattgcaaa aggactaact caaaagttta
ctgcgatcgg gatttttacg 900 gataactcta agaaggatat tacggatcaa
gtcacttgga attcttcttc agcaatcgta 960 agcgtgtcta acttagacaa
caataaaggt ctgggaaaaa ccaactcagt tggaaacacg 1020 actattaccg
caaccttagg aaaagtttca ggtaacactt ggtttactgt agttcctgcg 1080
gttctcactt ctattcaaat caatcctgta aatccttctc ttgcaaaagg gttaactcaa
1140 aaatttacgg ctactgggat ctactctgac aactctaaca aggacattac
ttccgctgtt 1200 acgtggttct catccgattc ttcaatcgcg acgatttcaa
acgcccaaaa aaatcaagga 1260 aacgcttacg gagcagctac aggagcaacg
gatattaaag ccacattcgg aaaggtaagt 1320 agtccggttt ctacgttatc
tgttacagct gcaaagcttg ttgaaatcca aatcacaccg 1380 gctgctgctt
ccaaagcaaa gggactcaca gaaagattca aggctactgg tatctttacg 1440
gataactcaa attccgatat tacaaatcaa gttacctgga attcctctaa tacggatatt
1500 gctgaaatta aaaataccag tggaagtaaa ggtattacaa atacactcac tccagga
1557 8 519 PRT Leptospira interrogans 8 Ile Thr Val Thr Pro Ala Ile
Leu Asn Ser Ile Gln Val Thr Ser Leu 1 5 10 15 Glu Ser Gly Ile Leu
Pro Lys Gly Thr Asn Arg Gln Phe Ser Ala Ile 20 25 30 Gly Ile Phe
Ser Asp Gly Ser His Gln Asp Ile Ser Asn Glu Pro Leu 35 40 45 Ile
Val Trp Ser Ser Ser Asn Pro Asp Leu Val Arg Val Asp Asp Ser 50 55
60 Gly Leu Ala Ser Gly Ile Asn Leu Gly Thr Ala His Ile Arg Ala Ser
65 70 75 80 Phe Gln Ser Lys Gln Gly Ala Glu Glu Met Thr Val Gly Asp
Ala Val 85 90 95 Leu Ser Gln Ile Gln Val Thr Ser Asn Asp Leu Asn
Ile Pro Leu Gly 100 105 110 Lys Lys Gln Lys Leu Thr Ala Thr Gly Ile
Tyr Ser Asp Asn Ser Asn 115 120 125 Arg Asp Ile Ser Ser Ser Val Ile
Trp Asn Ser Ser Asn Ser Thr Ile 130 135 140 Ala Asn Ile Gln Asn Asn
Gly Ile Leu Glu Thr Ala Asp Thr Gly Ile 145 150 155 160 Val Thr Val
Ser Ala Ser Ser Glu Asn Ile Ile Gly Ser Val Lys Leu 165 170 175 Ile
Val Thr Pro Ala Ala Leu Val Ser Ile Ser Val Ser Pro Thr Asn 180 185
190 Ser Thr Val Ala Lys Gly Leu Gln Glu Asn Phe Lys Ala Thr Gly Ile
195 200 205 Phe Thr Asp Asn Ser Asn Ser Asp Ile Thr Asp Gln Val Thr
Trp Asp 210 215 220 Ser Ser Asn Thr Asp Ile Leu Ser Ile Ser Asn Ala
Ser Asp Ser His 225 230 235 240 Gly Leu Ala Ser Thr Leu Asn Gln Gly
Asn Val Lys Val Thr Ala Ser 245 250 255 Ile Gly Gly Ile Gln Gly Ser
Thr Asp Phe Lys Val Thr Gln Ala Ala 260 265 270 Leu Thr Ser Ile Glu
Val Ser Pro Thr Arg Thr Ser Ile Ala Lys Gly 275 280 285 Leu Thr Gln
Lys Phe Thr Ala Ile Gly Ile Phe Thr Asp Asn Ser Lys 290 295 300 Lys
Asp Ile Thr Asp Gln Val Thr Trp Asn Ser Ser Ser Ala Ile Val 305 310
315 320 Ser Val Ser Asn Leu Asp Asn Asn Lys Gly Leu Gly Lys Thr Asn
Ser 325 330 335 Val Gly Asn Thr Thr Ile Thr Ala Thr Leu Gly Lys Val
Ser Gly Asn 340 345 350 Thr Trp Phe Thr Val Val Pro Ala Val Leu Thr
Ser Ile Gln Ile Asn 355 360 365 Pro Val Asn Pro Ser Leu Ala Lys Gly
Leu Thr Gln Lys Phe Thr Ala 370 375 380 Thr Gly Ile Tyr Ser Asp Asn
Ser Asn Lys Asp Ile Thr Ser Ala Val 385 390 395 400 Thr Trp Phe Ser
Ser Asp Ser Ser Ile Ala Thr Ile Ser Asn Ala Gln 405 410 415 Lys Asn
Gln Gly Asn Ala Tyr Gly Ala Ala Thr Gly Ala Thr Asp Ile 420 425 430
Lys Ala Thr Phe Gly Lys Val Ser Ser Pro Val Ser Thr Leu Ser Val 435
440 445 Thr Ala Ala Lys Leu Val Glu Ile Gln Ile Thr Pro Ala Ala Ala
Ser 450 455 460 Lys Ala Lys Gly Leu Thr Glu Arg Phe Lys Ala Thr Gly
Ile Phe Thr 465 470 475 480 Asp Asn Ser Asn Ser Asp Ile Thr Asn Gln
Val Thr Trp Asn Ser Ser 485 490 495 Asn Thr Asp Ile Ala Glu Ile Lys
Asn Thr Ser Gly Ser Lys Gly Ile 500 505 510 Thr Asn Thr Leu Thr Pro
Gly 515 9 600 DNA Leptospira interrogans 9 cataactctc ctcataacaa
ttggttttca ctggagctta caaagtatcg gaatttaatt 60 ccggcggata
aagcattctc tcaattcgca gaatttaacg gaagattgta tgtaacaaga 120
acgatctgcg taacgaaaga agatcactcc ggactcagac aaagtttaca aactgtggaa
180 ggttgtacgg acggaagtta tacaaatcga agaccccaac tttggaaatg
tgatccgact 240 ctaaccggcg atacaacaac ctgcgaagca gaagattggt
ctttagtagg agataacgga 300 accggattta caaactttgg agacaattcc
aatcacagta tgacgatgat ggttgcaagt 360 ggatcttatc tctacatagg
ttttgataac gaaaacggaa ttcaaatctg gagaacaaat 420 cttgaaaatc
ctggaagttc atcacacaac tgggaaccta taggaatagg cggattaaga 480
gacgttacca atcgtcaaat ttattcggct atatccggaa tgaattttgg tgtaaatttc
540 gtatatataa gcgtaggaaa caaaaataaa ccggtcaaaa tttacagaca
acagaatcaa 600 10 200 PRT Leptospira interrogans 10 His Asn Ser Pro
His Asn Asn Trp Phe Ser Leu Glu Leu Thr Lys Tyr 1 5 10 15 Arg Asn
Leu Ile Pro Ala Asp Lys Ala Phe Ser Gln Phe Ala Glu Phe 20 25 30
Asn Gly Arg Leu Tyr Val Thr Arg Thr Ile Cys Val Thr Lys Glu Asp 35
40 45 His Ser Gly Leu Arg Gln Ser Leu Gln Thr Val Glu Gly Cys Thr
Asp 50 55 60 Gly Ser Tyr Thr Asn Arg Arg Pro Gln Leu Trp Lys Cys
Asp Pro Thr 65 70 75 80 Leu Thr Gly Asp Thr Thr Thr Cys Glu Ala Glu
Asp Trp Ser Leu Val 85 90 95 Gly Asp Asn Gly Thr Gly Phe Thr Asn
Phe Gly Asp Asn Ser Asn His 100 105 110 Ser Met Thr Met Met Val Ala
Ser Gly Ser Tyr Leu Tyr Ile Gly Phe 115 120 125 Asp Asn Glu Asn Gly
Ile Gln Ile Trp Arg Thr Asn Leu Glu Asn Pro 130 135 140 Gly Ser Ser
Ser His Asn Trp Glu Pro Ile Gly Ile Gly Gly Leu Arg 145 150 155 160
Asp Val Thr Asn Arg Gln Ile Tyr Ser Ala Ile Ser Gly Met Asn Phe 165
170 175 Gly Val Asn Phe Val Tyr Ile Ser Val Gly Asn Lys Asn Lys Pro
Val 180 185 190 Lys Ile Tyr Arg Gln Gln Asn Gln 195 200 11 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
probe 11 gattttaaag ttacacaagc 20 12 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic probe 12 aaaccggact
acttaccttt cc 22 13 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic probe 13 ttacggctac aggtattttt acg 23
14 22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic probe 14 attggaagat ttccaagtaa cc 22 15 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic probe 15
tatctacgct gcaaatgg 18 16 18 DNA Artificial Sequence Description of
Artificial Sequence Synthetic probe 16 ttgttggcga tacgtccg 18 17 19
DNA Artificial Sequence Description of Artificial Sequence
Synthetic probe 17 cataactctc ctcataaca 19 18 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic probe 18
tatgtagaga taagatcc 18 19 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 19 saaagttgyr ygkcttggcc 20 20 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 20
swaccrtcyg aaaaratwcc 20 21 26 DNA Artificial Sequence Description
of Artificial Sequence Primer 21 cgcagaaatt ttagaggaac ctacag 26 22
26 DNA Artificial Sequence Description of Artificial Sequence
Primer 22 tttgactcca agacgcagag gatgat 26 23 26 DNA Artificial
Sequence Description of Artificial Sequence Primer 23 attttcaaga
tttgttctcc agattt 26 24 25 DNA Artificial Sequence Description of
Artificial Sequence Primer 24 attacttctt gaacatctgc ttgat 25 25 26
DNA Artificial Sequence Description of Artificial Sequence Primer
25 ctgctacgct tgttgacata gaagta 26 26 26 DNA Artificial Sequence
Description of Artificial Sequence Primer 26 tagaaccaac acgaaatggc
acaaca 26 27 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 27 atccgaagtg gcataactct cctcat 26 28 25 DNA
Artificial Sequence Description of Artificial Sequence Primer 28
tgaaaagaac attaccagcg ttgta 25 29 33 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 29
atgggactcg agattaccgt tacaccagcc att 33 30 33 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 30 attccatggt tatcctggag tgagtgtatt tgt 33 31 27
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 31 aacctcgagc ataactctcc tcataac 27 32 28
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 32 ttcgaattct tattgattct gttgtctg 28 33 6
PRT Artificial Sequence Description of Artificial Sequence 6x His
tag 33 His His His His His His 1 5
* * * * *
References