US20040058323A1

US20040058323A1 - Proteins with repetitive bacterial-Ig-like (big) domains present in leptospira species

Info

Publication number: US20040058323A1
Application number: US10/147,299
Authority: US
Inventors: Albert Ko; Mitermayer Reis; Julio Croda; Isadora Siqueira; James Matsunaga; Lee Riley; Michele Barocchi; Tracy Young
Original assignee: Ko Albert I.; Reis Mitermayer Galvao; Croda Julio Henrique; Siqueira Isadora Cristina; James Matsunaga; Riley Lee W.; Barocchi Michele A.; Young Tracy Ann
Current assignee: Fundacao Oswaldo Cruz
Priority date: 2002-05-17
Filing date: 2002-05-17
Publication date: 2004-03-25
Also published as: US8124110B2; US20120121639A1; US20180186867A1; US8216594B2; US20130230903A1; JP2006516083A; ES2295464T3; US20160222069A1; US8802835B2; ATE374943T1; US20120270301A1; AR039035A1; US20150329599A1; US9133250B2; US20110311570A1; US8021673B2; US20140308734A1; US20100183656A1; US7718183B2; BR0215719A

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.

Description

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

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).

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%.

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).

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.

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.

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.

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.

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).

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

FIGS. 1A and B show a Southern blot analysis of bigL gene sequences in Leptospira. Genomic DNA (3 mcg/lane) from [0021] 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^th-6^threpetitive 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).
FIG. 2 shows a schematic diagram of the genomic organization of the region encoding the BigL1 and BigL3 proteins in [0022] 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.
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 [0023] 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).
FIG. 4 shows amplified products from RT-PCR of RNA extracts of [0024] 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.
FIG. 5 shows the immunoblot reactivity of pooled sera from patients and animal reservoirs infected with pathogenic Leptospira and laboratory animals immunized with whole [0025] 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).
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 ([0026] OD 450 nm) and standard deviations are represented in the graphs.
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 ([0027] OD 450 nm) values.
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 [0028] region 1 of BigL3 (2^ndto 6^thBig 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.
FIG. 9 shows the immunoblot reactivity of rat anti-rBigL3 antisera to rBigL3 and native angigen from [0029] L. interrogans lysates. Immunoblots were prepared with purified rBigL3 (1 mg/lane; lanes 3, 5, 7, 9) and whole antigen preparations (10⁸leptospira 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
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[0030] ⁸leptospira 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

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. [0031]
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 [0032] E. coli, Yersinia and Bordetella, which have virulence functions such as host cell adhesion.
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. [0033]
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). [0034]
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). [0035]
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. [0036]
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. [0037]
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. [0038]
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. [0039]
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. [0040]
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. [0041]
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. [0042]
[0043] Step 1—The screening the positive clones consisted basically of the following steps:
(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. [0044]
(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. [0045]
(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. [0046]
[0047] Step 2—Subcloning expression and purification of the protein
Drawing of two oligonucleotides with base in sequences of two proteins BigL [0048]
Amplification by PCR of the initial BigL portion encoding for part of the repetitive region, from those oligonucleotides [0049]
Sequencing of the product of the amplification [0050]
Subcloning of the region-encoding by the product sequenced [0051]
Expression of the recombinant protein. [0052]
Purification of the recombinant protein. [0053]
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. [0054]
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. [0055]
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. [0056]
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. [0057]
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. [0058]
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. [0059]
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. [0060]
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. [0061]
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. [0062]
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: [0063]
1—detecting one of polypeptides, BigL1, BigL2 and BigL3, or their functionally equivalent sequences; [0064]
2—detecting nucleic acid encoding for BigL1, BigL2 and BigL3 or their functionally equivalent sequences; [0065]
3—detecting antibodies for such polypeptides, BigL1, BigL2 and BigL3, or their functionally equivalent sequences. [0066]
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. [0067]
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. [0068]
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. [0069]
The present invention will be now described with reference to the Examples, which are should not be considered as limitative of the present invention. [0070]

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° 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 10⁷by intraperitoneal inoculation.
[0072] Escherichia coli XL1-Blue MRF′mcrA)183ΔmcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′proAB lacI^qZΔM15 Tn10 (Tetr)] (Stratagene) and E. coli PLK-F′ (endA1 gyrA96 hsdR17 lac recA1 relA1 supE44 thi-1 [F′ lacIqZΔM15]) were used as the host strains for infection with the λZap II (Stratagene) and λTrip1Ex (Clontech) vectors, respectively. E. coli SOLR (e14⁻ [mcrA], A[mcrCB-hsdSMR-mrr]171 sbcC recB recJ umuC::Tn5[Kan^r] uvrC lac gyrA96 relA1 thi-1 endA1 λ^r, [F′ proAB lacI^qZΔM15], Su-[non-suppressing]] and E. coli BM25.8 (supE44 thi Δlac-proAB [F′ traD36 proAB⁺ lacI^qZΔM15] λimm434(kan^r) P1 (cam^r) hsdR (r^K12−m^K12−)) were used for in vivo excision of the pBluescript and pTrip1Ex phagemids, respectively. BLR(DE3)pLysS [F⁻ ompT hsdS_B(r_B-m_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 μg/ml ampicillin, 100 μg/ml carbenicillin, or 25 μg/ml chloramphenicol where appropriate. Antibiotics were purchased from Sigma.

Example 1B

Isolation and Characterization of bigL Genes

This example illustrates the identification and isolation of the bigL genes. Genomic DNA was prepared from virulent, low-passage [0073] 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.
For screening of genomic library, approximately 103 pfu approximately were plated on a lawn of [0074] 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.
Each positive plaque was stored at 4° C. in 1 ml SM (0.1 M NaCl, 8 mM MgSO[0075] 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).
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. [0076]
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. [0077]
Two [0078] 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.
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 [0079] 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.
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×e[0080] ⁻³⁰.
The [0081] 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

This example illustrates the distribution of multiple copies of bigL genes among Leptospira species and methods to detect bigL DNA and RNA in samples. [0082]
Southern Blot Analysis [0083]
Southern blot analysis was performed to identify multiple copies of bigL genes in genomic DNA from [0084] 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.
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° C. for 5 min, 94° C. for 30 sec, 57° C. for 30 s min, and 72° 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[0085] _—395 gat-ttt-aaa-gtt-aca-caa-gc and BigL3_—573 aaa-ccg-gac-tac-tta-cct-ttc-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.
The UV Crosslinked membrane was subject to prehybridization at 42° 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° C. Following hybridization, the membranes were washed twice for 5 minute at room temperature with 2×SSC (NaCl, Sodium Citrate), 0.1% SDS. The membranes were then washed twice for 30 minutes at 42° 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 [0086]
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 [0087] 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

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 [0088] 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 códon. 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).
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. [0089]

Example 2C

Reverse Transcriptase-Polymerase Chain

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 1×10¹⁰leptospiral cells using the hot-phenol method and resuspended in water following ethanol precipitation (ref). ˜2 μg of leptospiral RNA was digested with 6 units of DNase I (Ambion) in 70 μl DNase I buffer (10 mM Tris-HCl pH 7.5, 25 mM MgCl₂, 1 mM CaCl₂in 1×RNA secure from Ambion) for 30 min at 37. To inactivate DNase I, 1.75 μl 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 ˜200 ng leptospiral RNA and Omniscript RT as described (Qiagen). The following primers were used to prime the reverse transcriptase reaction:


	bigL1,	5′-CGCAGAAATTTTAGAGGAACCTACAG-3′

	bigL2,	5′-TTTGACTCCAAGACGCAGAGGATGAT-3′

	bigL3,	5′-ATTTTCAAGATTTGTTCTCCAGATTT-3′

	bigL45,	5′-ATTACTTCTTGAACATCTGCTTGAT-3′.

The RT reactions were subjected to DNA PCR using Taq polymerase (Qiagen) Prior to PCR, the following primers were added to the reactions:


	bigL1,	5′-CTGCTACGCTTGTTGACATAGAAGTA-3′

	bigL2,	5′-TAGAACCAACACGAAATGGCACAACA-3′

	bigL3,	5′-ATCCGAAGTGGCATAACTCTCCTCAT-3′

	bigL45,	5′-TGAAAAGAACATTACCAGCGTTGTA-3′.

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. [0092]
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. [0093]

EXAMPLE 3

Expression and Purification of Recombinant BigL Proteins [0094]
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 [0095] 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:

45B-1 5′-ATGGGACTCGAGATTACCGTTACACCAGCCATT-3′

45B-2 5′-ATTCCATGGTTATCCTGGAGTGAGTGTATTTGT-3′
PCR amplification with oligonucleotides 45B-1 and 45B-2 and purified [0096] 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.
A second region was selected for expression that contained the final 200 amino acids of the C-terminal region of [0097] 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:

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′
PCR amplification with oligonucleotides BIGLCTERM1 and BIGLCTERM2 and purified [0098] 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.
Recombinant proteins, [0099] rBigL regions 1 and 2, were expressed in BL21(DE3) pLysogen (Invitrogen). Isopropyl-β-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

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. [0100]
Immunoblot Detecting of Antibodies to BigL Polypeptides in Samples from Infected Subjects [0101]
Purified [0102] 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.
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 [0103] recombinant BigL3 region 1 polypeptide in the immunoblot was considered a positive reaction.
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. [0104]
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. [0105]

Example 4B

ELISA-based Detection of Antibodies to BigL Polypeptides in Samples from Infected Subjects

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° C. overnight with His[0106] 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° C. until use. Wells were incubated with 50 μl 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 μl of 5,000 to 20,000-fold dilutions of anti-human μ or γ-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 μl/well of 0.01% (w/v) 3,3′,5,5′-tetramethylbenzidine in substrate buffer (0.03% [v/v] hydrogen peroxide, 25 mM citric acid, 50 mM Na₂HPO₄, pH 5.0) for 20 minutes in the dark at room temperature. The color reaction was stopped by adding 25 uL 2 N H₂SO₄and the absorbance at 450 nm was measured in an Emax microplate reader (Molecular Devices, Sunnyvale, Calif.).
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. [0107]
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. [0108]

EXAMPLE 5

Induction of an Immune Response Against Leptospira in Subjects [0109]
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 [0110] 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.
Immunogenicity experiments were performed with purified recombinant BigL polypeptides derived [0111] 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.
FIG. 10. illustrates that immunization with rBigL3 derived from [0112] 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.

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.

TABLE 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)

TABLE 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

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)^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)

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. [0115]

REFERENCES

1. Levett P N. Leptospirosis. [0116] Clin Microbiol Rev. 2001;14(2):296-326.
2. Faine S B, Adler B, Bolin C, Perolat P. [0117] Leptospira and leptospirosis. 2nd ed Melbourne, Australia: MediSci; 1999.
3. Farr R W. Leptospirosis. [0118] Clin Infect Dis. 1995;21(1):16; quiz 7-8.
4. Lomar A V, Diament D, Torres J R. Leptospirosis in Latin America. [0119] Infect Dis Clin North Am. 2000;14(1):23-39, vii-viii.
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. [0120] Lancet. 1999;354(9181):820-5.
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. [0121] Clin Diagn Lab Immunol. 1999;6(4):599-605.
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. [0122] DNA Cell Biol. 1999;18(12):903-10.
8. Haake D A, Walker E M, Blanco D R, Bolin C A, Miller M N, Lovett M A. Changes in the surface of [0123] Leptospira interrogans serovar grippotyphosa during in vitro cultivation. Infect Immun. 1991;59(3):1131-40.
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. [0124] J Bacteriol. 1993;175(13):4225-34.
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. [0125] Infect Immun. 1998;66(4):1579-87.
11. Haake D A, Mazel M K, McCoy A M, et al. Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection. [0126] Infect Immun. 1999;67(12):6572-82.
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. [0127] Infect Immun. 2000;68(4):2276-85.
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. [0128] Infect Immun. 1995;63(8):3174-81.
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. [0129] Infect Immun. 1996;64(6):2322-30.
15. Yelton D B, Charon N W. Cloning of a gene required for tryptophan biosynthesis from [0130] Leptospira biflexa serovar patoc into Escherichia coli. Gene. 1984;28(2):147-52.
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. [0131] Journal of Clinical Microbiology. 2001;39(9):3303-3310.
17. Guerreiro H, Croda J, Flannery B, et al. Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans. [0132] Infect Immun. 2001;69(8):4958-68.
[0133]
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

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.