Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

Abstract

Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

INTRODUCTION

Leptospires are spirochete bacteria classified into nonpathogenic, intermediate, or pathogenic species and inhabit soil and freshwater reservoirs, predominately in tropical regions (1). Infection with pathogenic Leptospira spp. can result in a large range of clinical manifestations, including fever, renal failure, jaundice, hemorrhage, meningitis, and death. Leptospirosis, the severe manifestation of disease, has an annual global incidence of over 1 million human cases and a case fatality rate of from 5% to 20% (2, 3). Pathogenic Leptospira spp. can colonize the renal tubules of a wide variety of wild and domesticated mammals. Rats are asymptomatic carriers and serve as the main reservoir of pathogenic Leptospira spp. The bacteria are shed in the urine of infected animals and persist in freshwater (4, 5), providing an opportunity for the bacteria to infect a new host. Leptospires are unable to cross the skin barrier; they can, however, access the underlying tissues via cuts/wounds in the skin. After gaining access to underlying tissue, leptospires rapidly disseminate to the interstitial space in the kidneys and liver (6, 7). While generally extracellular, studies have demonstrated transient intracellular localization in macrophages (8–10).

Leptospires resemble Gram-negative bacteria, in that they contain an inner membrane, a periplasmic space with peptidoglycan, and an outer membrane where the lipopolysaccharide (LPS) is anchored. The bacteria are highly motile, and previous studies have demonstrated that both full motility (11, 12) and an intact LPS (13) are required for successful colonization of the host. Unlike the majority of other pathogenic Gram-negative bacteria, Leptospira genomes lack the genes encoding delivery systems, such as type III and IV secretion systems, and classical virulence proteins, such as toxins and effectors (14).

Protein secretion systems have not been experimentally identified in Leptospira (15). Genes orthologous to those encoding secretion systems are present in Leptospira genomes, including a type I secretion system which consists of an inner membrane ATP binding cassette protein, a periplasmic adaptor, and a TolC channel-forming outer membrane protein (16). While relatively simple, the type I secretion system is responsible for the export of a variety of proteins with different functions in bacteria (17). Leptospira genomes also contain genes orthologous to those encoding type II secretion proteins. The type II secretion system consists of various proteins that reside in the inner membrane and a pilus-like structure that can polymerize to drive proteins to the extracellular space through an outer membrane pore (for a review, see reference 18). To date, other bacterial secretion systems have also been characterized (nonflagellar type III and type IV, V, VI, and VII secretion systems), but Leptospira genomes do not contain orthologous genes that encode proteins which would assemble into these systems. Consequently, protein export to the extracellular space is likely mediated by type I and II secretion systems or through some other yet to be discovered mechanism(s) in Leptospira.

The extracellular proteome of cells is more accurately termed the exoproteome, which is defined to consist of the proteins in extracellular proximity to a biological system arising from secretion, other transport mechanisms, and/or cell lysis (19). Proteins delivered to the extracellular space likely serve essential functions for the pathogenic and saprophytic lifestyles of Leptospira spp. Previous studies focusing on extracellular proteins identified Leptospira interrogans proteins in culture supernatants (20), while another study used a bioinformatics-based approach to identify potential outer membrane and extracellular proteins (21). It has also been demonstrated that culture supernatants of L. interrogans contain proteases which can interfere with the host complement defense against Leptospira spp. (22), that an extracellular enolase interacts with host plasminogen (23), and that an extracellular collagenase can degrade host collagen (24). Combined, these studies implicated extracellular proteins in the leptospiral infection process.

Here, we used label-free quantitative proteomics to analyze and characterize the L. interrogans exoproteome. We identified proteins transported to the extracellular space and categorized these proteins functionally to gain further insight into L. interrogans biology, encompassing the pathogenic and saprophytic existence of these bacteria. These experiments guided our subsequent in vivo experiments, which provided significant insight into the extent of L. interrogans pathogenicity.

MATERIALS AND METHODS

Bacterial strains and culturing.

Leptospira interrogans serovar Manilae strain L495 was culture maintained in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (25, 26) at 30°C. L. interrogans transposon mutagenesis has been described previously (27–29), and the L495 transposon mutants used in this study were obtained from an in-house-maintained library of mutants. The mutant strains were culture maintained as described above for the parent strain. To perform proteomic analysis on culture supernatants, EMJH medium was constituted with the following modifications. Albumin was omitted from the recipe, and Tween 80 and glycerol were added to 0.01% (vol/vol). A separate batch of modified EMJH medium was made to contain 120 mM NaCl. Prior to shifting to modified EMJH medium, L. interrogans was cultured in EMJH medium at 30°C to a density of 10⁹ bacteria per ml, and the bacteria were subsequently pelleted via centrifugation at 3,200 × g for 15 min, using a swinging-bucket rotor. The pelleted bacteria were washed 3 times with modified EMJH medium using 20 ml of medium for each washing step. The bacteria were then resuspended in modified EMJH medium and enumerated via dark-field microscopy. The bacteria were subsequently diluted in modified EMJH medium to a concentration of 10⁸ bacteria per ml in a total volume of 100 ml for each condition (for incubation at 30°C and 37°C and in modified EMJH medium with 120 mM NaCl at 30°C) in two biological replicates for each condition. After 18 h of incubation, the bacteria were enumerated via dark-field microscopy to validate bacterial viability. Technical limitations prevented the growth of bacteria at 37°C in modified EMJH medium containing 120 mM NaCl, as bacterial viability was affected under these conditions.

Leptospira culture supernatants.

The leptospires were centrifuged at 3,200 × g for 10 min, and the pelleted bacteria were separated from the culture supernatants by siphoning of the supernatants. The pellets were stored at −20°C, and the culture supernatants were transferred to Vivaspin 20 1,000,000-molecular-weight-cutoff (MWCO) polyethersulfone (PES) ultrafiltration devices (Sartorius Stedim Biotech, Goettingen, Germany). The latter step ensured the removal of any remaining whole L. interrogans cells from the culture supernatants. Subsequent centrifugation was performed according to the manufacturer's instructions, and all manipulations were carried out at room temperature. The flowthrough was then concentrated using Amicon Ultra centrifugal filters (Ultracel 3,000 MWCO; Merck Millipore Ltd., Cork, Ireland) and subsequently with low-volume Amicon Ultra 0.5-ml 3,000-MWCO filters (Merck Millipore Ltd.). Each 100-ml culture supernatant was concentrated down to a final volume of 20 μl.

To obtain protein from the pelleted whole Leptospira cells, the bacterial pellets were resuspended in the respective modified EMJH medium to a final volume of 200 μl and sonicated to lyse the bacteria. The protein concentration was measured via UV spectrometry at 280 nm. Samples were diluted 3:1 in Laemmli (4×) sample loading buffer, and equal concentrations of protein were used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Equal protein loading across all gel lanes was confirmed via densitometry.

Recombinant protein and antisera.

Recombinant LIC12209 (encoding a Leptospira beta-propeller protein of 52 kDa [LβP52]) was produced as previously described (30) with the following modifications. The predicted lβP52 gene was amplified from L. interrogans serovar Manilae strain L495 genomic DNA by PCR with the primer pair LIC12209F (5′-GGGGCTAGCATGAGAAAATTTTACATT-3′) and LIC12209R (5′-CCCGCGGCCGCTTATCTATATTTTACA-3′). The underlined nucleotides indicate restriction sites (NheI and NotI in primers LIC12209F and LIC12209R, respectively). The PCR product was cloned into the TOPO vector (Invitrogen Life Technologies, Saint Aubin, France) and transformed into Escherichia coli DH5α cells (Invitrogen Life Technologies). The amplicon was released from the vector by digestion with NheI and NotI and recloned into the pET-28(a) expression plasmid. The pET-28(a) vector allows expression of a recombinant protein with an N-terminal 6× His (polyhistidine) tag. The cloned sequence was confirmed by DNA sequencing (Eurofins, Courtaboeuf, France).

Protein expression was achieved in E. coli strain BL21(DE3). E. coli BL21(DE3) containing the recombinant plasmid was cultured at 37°C under kanamycin selection to an optical density (at 600 nm) of 0.6 to 0.8. Recombinant protein synthesis was induced by the addition of 1 mM (final concentration) isopropyl-β-d-1-thiogalactopyranoside (IPTG). After 3 h, the cells were harvested by centrifugation at 4,000 × g for 30 min and the bacterial pellets were resuspended in 5 ml buffer A (100 mM NaH₂PO₄, 10 mM Tris-HCl, pH 8.0). The bacterial cell pellets were lysed via sonication, and recombinant protein, in the form of inclusion bodies, was then separated by centrifugation at 17,000 × g for 20 min and solubilized in 5 ml of buffer B (100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 8.0). The protein was purified by metal-chelating chromatography in a fast-flow column with 0.4 ml of nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen, Courtaboeuf, France) using the manufacturer's protocol. All fractions were analyzed by 12% SDS-PAGE.

Rabbit antisera generated against the flagellar protein FlaA2 (31), chaperone protein GroEL (32), and Ig-like repeat domain protein 1 LigA (32) were generously provided by David Haake. Rabbit antiserum against recombinant LβP52 (rLβP52) protein was generated as follows. Recombinant protein LβP52 was dialyzed against buffer 1 (100 mM NaH₂PO₄, 10 mM Tris-HCl, 5 M urea, pH 8) for 48 h, followed by a second dialysis step against buffer 2 (100 mM NaH₂PO₄, 10 mM Tris-HCl, 2 M urea, pH 8) for 48 h, and 50 μg of protein was submitted for antiserum production in rabbits by Covalab R&D in Biotechnology (France).

To obtain L. interrogans-positive sera, Hartley male guinea pigs (Charles River Laboratories) were obtained at 6 weeks of age and weighed between 450 and 500 g. Guinea pigs (n = 6) were anesthetized via intramuscular injection of 40 mg ketamine and 4 mg xylazine (per kg of body weight), and blood (∼1 ml) was collected via cardiac heart puncture and placed into 10-ml Venosafe plastic tubes (Terumo, Guyancourt, France). Animals were then maintained under normal care conditions for 7 days and then injected intraperitoneally with 10⁵ L. interrogans strain L495 spirochetes in 1 ml albumin-free EMJH medium. Blood samples were collected from infected animals by terminal cardiac puncture of anesthetized animals at 14 days postchallenge. Serum was collected by incubation of blood samples in Venosafe plastic tubes (Terumo) at room temperature for 30 min, followed by centrifugation at 1,500 × g for 10 min. The supernatant was collected for use in immunoblot experiments. The protocols for the animal experiments conformed to the guidelines of the Animal Care and Use Committees of the Institut Pasteur (Paris, France).

Protein immunoblotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblotting were performed as previously described (33, 34), with the following modifications. The SDS-PAGE and immunoblot experiments were performed using 10 μg of total protein from whole-cell lysates and culture supernatants. Antiserum to FlaA2, GroEL, LigA, and LβP52 was used at 1:2,000, 1:8,000, 1:750, and 1:1,000, respectively. Guinea pig Leptospira-positive and preimmune sera were used at 1:100, and goat polyclonal secondary antibody to guinea pig IgG-Fc (horseradish peroxidase [HRP]) (Abcam, Paris, France) and IgM-Fc (HRP) (Acris, Montluçon, France) were used at a dilution of 1:20,000.

Protein treatment preceding mass spectrometry.

For mass spectrometry experiments, 10 μg of protein was allowed to migrate 10 mm into a 4 to 12% gradient TGX gel (Bio-Rad, Marnes-la-Coquette, France), without the addition of any loading dyes. Samples were excised, diced, and digested with trypsin as previously described (35). Briefly, gel pieces were washed multiple times in 25 mM ammonium bicarbonate in 50% acetonitrile, reduced with 10 mM dithiothreitol (DTT) at 37°C for 60 min, and then alkylated using 55 mM iodoacetamide for 60 min. Gel pieces were dried in a SpeedVac apparatus (Thermo Savant, Hemel Hempstead, United Kingdom), and then 200 ng of trypsin (Promega, Southampton, United Kingdom) in 25 mM ammonium bicarbonate was added to each gel piece and the gel pieces were digested overnight at 37°C. Peptides were extracted by sonicating the gel pieces in 50% acetonitrile containing 0.1% formic acid for 30 min. After vacuum concentration, the tryptic peptides were concentrated on an in-house-manufactured C₁₈ desalting tip prior to mass spectrometry analysis (36).

Mass spectrometry.

Protein samples were analyzed on an Ultimate 3000 RSLCnano high-performance liquid chromatography (HPLC) system (Dionex, Camberley, United Kingdom), run in direct injection mode, coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Electron, Hemel Hempstead, United Kingdom). Samples were resolved on a 25-cm by 75-μm (inner diameter) picotip analytical column (New Objective, Woburn, MA, USA) which was packed in-house with ProntoSIL 120-3 C₁₈ Ace-EPS-phase, 3-μm-diameter beads (Bischoff Chromatography, Germany). The system was operated at a flow rate of 300 nl min⁻¹, and a 120-min gradient was used to separate the peptides. The mass spectrometer was operated in the Top 10 data-dependent acquisition mode. Precursor scans were performed in the Orbitrap column at a resolving power of 70,000, from which the 10 most intense precursor ions were selected by the quadrupole and fragmented by higher-energy collisional dissociation (HCD) at a normalized collision energy of 28%. The quadrupole isolation window was set at 3 m/z. Charge state +1 ions and undetermined charge state ions were rejected from selection for fragmentation. Dynamic exclusion was enabled for 40 s. Data were converted from RAW to MGF files using the ProteoWizard program (36).

Mass spectrometry data analysis.

Data were searched against the L. interrogans serovar Copenhageni strain Fiocruz L1-130 proteome using the Andromeda algorithm (37) through MaxQuant software (version 1.5.2.8) (38). Parameters can be found in Table S3 in the supplemental material. Contaminants, reverse decoy proteins, and proteins identified only by a modification site were removed; majority protein identifiers were used as protein identifiers. Raw protein abundances were used for further analysis. Missing values were replaced with the minimum for each sample. The resulting protein intensities were log₂ transformed and quantile normalized (39), using the method implemented in the normalizeBetweenArrays function of the limma package (40, 41). An empirical Bayes moderated t statistic, as implemented in the R limma package (40), was applied to identify proteins with a significant difference in abundance. Temperature and osmotic variables were used as blocking factors, to account for any additional variance in the data. The Benjamini and Hochberg multiple-testing correction was applied to control the false discovery rate (42).

Data analyses downstream of identification and quantification.

To be deemed an exoprotein and/or a protein potentially localized to the extracellular space via active transport, proteins must have displayed an enriched abundance in supernatants compared to the respective abundance in whole cells with an adjusted P value of <0.05. To compare exoprotein quantities in culture supernatants (culture supernatants at 37°C versus 30°C and culture supernatants in NaCl versus culture supernatants at 30°C), protein abundances were compared to assess relative abundance (see Table S1 in the supplemental material).

Assignment of proteins to COGs.

The protein products of Leptospira genomes were automatically classified into clusters of orthologous groups (COGs) by use of the MicroScope platform (43). These data were used to sort the detected exoproteins into COGs, and the resulting frequencies were compared to those predicted genome-wide. Statistical analysis was performed by assuming a binomial distribution, where assignment of exoproteins into a COG would be considered a success and the absence of assignment would be considered a failure. The percentage of coding sequences classified into a COG (as calculated in MicroScope genome-wide) was used as the probability of observing a success, and the total number of detected exoproteins (n = 325) was used as the sampling size to generate COG-specific binomial probability distributions. A P value of <0.01 was used as a cutoff for significance.

Mutant strains, growth rates, and infection experiments.

The LIC10465 (ligA, encoding Ig-like repeat domain protein 1) mutant was obtained in L. interrogans serovar Manilae strain L495 by cloning an internal fragment of ligA lacking the 5′ and 3′ ends of the open reading frame in a conjugative suicide vector carrying a spectinomycin resistance cassette. We obtained integration of the plasmid via a single-crossover event, which generated two copies of the targeted gene, one with a deletion at the 5′ end of the gene and the other with a deletion at the 3′ end, thereby rendering it inactive, as confirmed by immunoblotting and immunofluorescence assay (data not shown). Other mutants were obtained by transposon mutagenesis in the parental strain (L. interrogans serovar Manilae strain L495), and insertion sites were initially identified via semirandom PCR (27–29). The insertion sites for exoprotein mutants were validated via PCR, and the primers, insertion sites, and transposon mutants are listed in Table S4 in the supplemental material.

Exoprotein LIC13006 (lenC, encoding an endostatin-like protein), LIC12208 (encoding a lipoprotein with beta-propeller repeats), LIC13060 (lipL36, encoding a lipoprotein), LIC10373 (encoding a lipoprotein with beta-propeller repeats), LIC10898 (lipL48, encoding an exoprotein with the highest absolute abundance in the present study), LIC11977 (encoding a cyclic nucleotide binding protein), LIC11852 (encoding an O-acetylhomoserine [thiol] lyase), LIC10713 (lruB, encoding a lipoprotein), and LIC10465 (ligA) mutants and L. interrogans serovar Manilae strain L495 were compared for in vitro growth rates in EMJH medium at 30°C. Bacterial growth was measured on a daily basis by measuring the optical densities at 420 nm via spectroscopy. Exoprotein mutants that demonstrated growth rates similar to those of L. interrogans serovar Manilae strain L495 were used in subsequent virulence measurement experiments in Mongolian gerbils (Janvier). To measure virulence, groups of 4 gerbils were injected intraperitoneally with 10⁴ bacteria per animal. Animals were administered L. interrogans serovar Manilae strain L495 or the LIC13006 (lenC), LIC12208, LIC13060 (lipL36), LIC10373, LIC10898 (lipL48), LIC10465 (ligA), or LIC11977 mutant. Animals were monitored on a daily basis for 23 days and sacrificed when moribund. All strains were isolated by culture of samples collected from the animals postmortem, and genotypes were confirmed via PCR. The protocols for the animal experiments conformed to the guidelines of the Animal Care and Use Committees of the Institut Pasteur (Paris, France).

RESULTS

Validation of methodology and overview of WCPs and exoproteins.

The method utilized in the present study for the separation of culture supernatants from whole bacteria has not been previously described, and validation that culture supernatants were free of nonextracellular proteins was required. To survey the proteins found in culture supernatants, whole-cell proteins (WCPs) and culture supernatant proteins (CSPs) from each culture condition were subjected to SDS-PAGE and immunoblot analyses (Fig. 1). The Coomassie-stained SDS-polyacrylamide gel revealed a visual difference in protein banding between WCPs and CSPs (Fig. 1A). Additionally, the majority and/or the most abundant CSPs migrated between 25 and 90 kDa, whereas bands as large as 260 kDa were observed for WCPs (Fig. 1A). There was no visual difference in protein banding in response to temperature or osmotic shifts in WCPs or CSPs (Fig. 1A).

FIG 1.

Differential protein localization and expression in leptospiral culture supernatants. L. interrogans was cultured at 30°C or 37°C or in medium containing 120 mM NaCl. Proteins from whole cells and culture supernatants were subsequently used in immunoblot experiments with the indicated antisera. (A) A representative Coomassie-stained SDS-polyacrylamide gel demonstrating disparate protein compositions when proteins from whole-cell lysates were compared to those from culture supernatants. LD, molecular mass marker. (B) Protein immunoblot demonstrating the localization of flagellar protein FlaA2 in whole cells but not in the supernatant. (C) Protein immunoblot suggesting increased expression of the chaperone protein GroEL at 37°C. (D) Protein immunoblot indicating expression of immunoglobulin protein LigA in CSPs and increased expression in the CSPs from L. interrogans exposed to 120 mM NaCl. (E) Protein immunoblot confirming expression of the LβP52 protein in culture supernatants and increased expression in CSPs from L. interrogans exposed to 120 mM NaCl.

Leptospires are flagellated bacteria, and the flagellum is anchored into the inner membrane of the bacteria (31). Antiserum generated against the 27.2-kDa periplasmic flagellar protein (FlaA2) revealed reactivity with proteins migrating between 25 and 35 kDa in WCPs but no reactivity with CSPs in protein immunoblots (Fig. 1B). Antiserum generated against the chaperone protein GroEL was used in immunoblot experiments (Fig. 1C) to validate the bacterial response to a temperature shift from 30°C to 37°C (44). The WCPs from Leptospira exposed to 37°C displayed a slight increase in GroEL reactivity (Fig. 1C). Detectable levels of GroEL were observed in all CSP samples, albeit at levels significantly lower than those in WCP samples (Fig. 1C). Using reasoning similar to that described above, WCPs and CSPs were subjected to immunoblot analysis using antiserum cross-reactive with L. interrogans Ig-like repeat domain protein 1 (LigA with a molecular mass of 128 kDa) (Fig. 1D) to assess the bacterial response to an osmotic shift (45, 46). The LigA protein was detected in CSP samples, with pronounced reactivity being observed in CSP samples from L. interrogans exposed to 120 mM NaCl (Fig. 1D). Immunoblot analysis of WCPs and CSPs also confirmed detection of the LβP52 protein in CSP samples only, with elevated expression being observed in samples exposed to 120 mM NaCl (Fig. 1E).

The immunoblot experiments described above demonstrated that the CSP preparations were prepared and the temperature and osmotic shift experiments were performed in a manner acceptable for subsequent quantitative global proteome analyses using mass spectrometry. Thus, WCPs and CSPs (in replicate) were subjected to liquid chromatography-tandem mass spectrometry on a Q Exactive Orbitrap mass spectrometer for the identification and relative quantification of proteins, using label-free quantification. These analyses resulted in the detection of 1,073 to 1,293 proteins in WCP preparations and 540 to 712 proteins in CSP preparations (Table 1; see also Table S1 in the supplemental material). The range of proteins detected was culture condition dependent. Other notable differences between the samples included the detection of relatively large proteins (300 kDa) in WCPs which were absent in CSPs and detection of the FlaA2 and LIC10371 proteins in WCPs and CSPs, respectively, but not vice versa (Table 1). The LIC10371 protein consists of multiple repeating beta-propeller domains which we found to also be present in 4 other exoproteins and displayed a high abundance in all culture supernatants. Both the FlaA2 and LIC10371 proteins served as controls for demonstrating the appropriate separation of CSPs from WCPs. Label-free protein quantification revealed a dynamic range of protein quantities approaching 5 orders of magnitude (Table 1; see also Table S1 in the supplemental material).

TABLE 1.

Overview of proteomic results

Leptospira fraction and growth condition	No. of unique proteins detecteda	Mol wt (Da)		Protein intensity (highest/lowest)	Avgb
		Smallest protein	Largest protein		FlaA2c	LIC10371d
Whole cells in pellet
30°C	1,073	∼11,000	∼300,000	1.5E10/1.0E6	5.5E8	Not detected
37°C	1,141	∼11,000	∼300,000	1.9E10/3.1E5	2.8E8	Not detected
NaCl	1,293	∼9,000	∼300,000	3.1E10/3.1E5	1.1E8	Not detected
Culture supernatant
30°C	712	∼9,000	∼270,000	1.2E10/2.3E5	Not detected	3.2E8
37°C	553	∼10,000	∼224,000	2.2E10/1.6E5	Not detected	2.5E8
NaCl	540	∼10,000	∼219,000	1.7E10/1.0E5	Not detected	4.2E8

^aFor combined experimental replicates.

^bAverage between experiments.

^cPeriplasmic localization.

^dExtracellular localization.

Identification of exported proteins via relative protein quantities.

The protein abundances in CSPs relative to those in WCPs enabled assignment of exoproteins and those potentially localized to the extracellular space via active transport, and this approach identified 325 exoproteins (see Table S2 in the supplemental material). The relative abundance of these exoproteins in supernatants compared to that in whole cells ranged from a 1.5-fold higher abundance in supernatants than in whole cells to detection only in supernatants (Table 2; see also Table S1 in the supplemental material).

TABLE 2.

The 20 most abundant exoproteins in the culture supernatant of L. interrogans

EMBL/GenBank/DDBJ CDS accession no.	Locus tag	Protein	COGa	WCPb	CSPc	CSP/WCP	RQd
AAS69512.1	LIC10898	LipL48	U	1.2E+09	1.3E+10	10.8	1.0
AAS70653.1	LIC12082	Cysteine synthase	E	2.5E+09	1.1E+10	4.6	0.9
AAS71860.1	LIC13318	Fatty acid synthase subunit beta	I	6.5E+08	3.8E+09	5.8	0.3
AAS72009.1	LIC13470	Ferredoxin-NADP reductase (EC 1.18.1.2)	P	3.6E+08	2.3E+09	6.4	0.2
AAS72270.1	LIC20249	Aconitate hydratase	C	8.8E+08	2.2E+09	2.5	0.2
AAS70370.1	LIC11781	Malate dehydrogenase	C	6.0E+08	2.1E+09	3.4	0.2
AAS69960.1	LIC11359	MaoC	I	3.2E+08	2.0E+09	6.2	0.2
AAS71788.1	LIC13244	Isocitrate dehydrogenase	C	8.3E+08	1.9E+09	2.4	0.1
AAS68881.1	LIC10253	Alcohol dehydrogenase	C	3.0E+08	1.9E+09	6.3	0.1
AAS71429.1	LIC12876	Elongation factor G	J	1.2E+09	1.8E+09	1.5	0.1
AAS68844.1	LIC10216	Phosphoenolpyruvate carboxykinase	C	4.2E+08	1.8E+09	4.3	0.1
AAS70553.1	LIC11977	Cyclic nucleotide binding protein	R	5.3E+08	1.8E+09	3.3	0.1
AAS71933.1	LIC13393	Ketol-acid reductoisomerase	E/H	8.2E+08	1.6E+09	1.9	0.1
AAS69801.1	LIC11194	Putative citrate lyase		9.8E+08	1.5E+09	1.5	0.1
AAS68639.1	LIC10002	DNA polymerase III, beta subunit	L	3.6E+08	1.4E+09	4.0	0.1
AAS70661.1	LIC12090	Glyceraldehyde-3-phosphate dehydrogenase	G	8.2E+08	1.3E+09	1.6	0.1
AAS69456.1	LIC10842	4-Hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7)	M/E	1.6E+08	1.1E+09	6.8	0.1
AAS69086.1	LIC10465	LigA		1.7E+08	1.1E+09	6.4	0.1
Q72NJ3.1	LIC12841	ll-Diaminopimelate aminotransferase (EC 2.6.1.83)	E	1.4E+08	1.0E+09	7.0	0.1
AAS70438.1	LIC11852	O-Acetylhomoserine (thiol) lyase	E	2.4E+08	9.8E+08	4.1	0.1

^aCOG, clusters of orthologous groups. See the Fig. 2 legend for descriptions of the categories.

^bWCP, average protein intensity for proteins from whole bacteria.

^cCSP, average protein intensity for proteins in supernatants.

^dRQ, protein quantities in supernatants relative to the quantity of LipL48.

To further evaluate the assignment of exoproteins, the corresponding primary sequences were used in bioinformatic analyses to predict N-terminal signal peptides using the Phobius program (47) and nonclassical protein export using the SecretomeP (version 2.0) server (48) (see Table S2 in the supplemental material). Of the 325 proteins deemed to be exported, 89 were predicted to contain an N-terminal signal peptide and 23 were predicted to be exported via a nonclassical pathway (defined as pathways that export proteins lacking classical signal peptides) (see Table S2 in the supplemental material).

The exoproteins identified in Table S2 in the supplemental material were then used in basic local alignment searches to identify the numbers of proteins unique to pathogenic Leptospira (Table 3) and those conserved in the nonpathogenic species Leptospira biflexa (see Table S2 in the supplemental material). These analyses revealed the majority (274 out of 325, 84.3%) of exoproteins to be conserved in L. biflexa.

TABLE 3.

Exoproteins unique to pathogenic Leptospira

Locus taga	Protein namea	Gene	COGb	N-terminal signal peptidec	Nonclassical secretiond	Virulencee
LIC10061	Uncharacterized protein			Yes		Not tested
LIC10241	Uncharacterized protein		D			Not tested
LIC10371	Putative lipoprotein			Yes		Not tested
LIC10373	Putative lipoprotein			Yes		0/4
LIC10465	Ig-like repeat domain protein 1	ligA		Yes		0/4
LIC10520	Uncharacterized protein			Yes		Not tested
LIC10548	Uracil-DNA glycosylase		L			Not tested
LIC10637	Ribosomal protein serine acetyltransferase		J			Not tested
LIC10657	Sphingomyelinase C	sphH		Yes		Not tested
LIC10711	Cytoplasmic membrane protein			Yes		Not tested
LIC10713	Putative lipoprotein		P	Yes		Not tested
LIC10774	Putative lipoprotein			Yes		Not tested
LIC10793	Surface antigen	orfC	S	Yes		Not tested
LIC10927	Putative lipoprotein			Yes		Not tested
LIC10987	Uncharacterized protein					Not tested
LIC10988	Cytoplasmic membrane protein		E	Yes		Not tested
LIC11009	Uncharacterized protein			Yes		Not tested
LIC11046	Uncharacterized protein					Not tested
LIC11096	Uncharacterized protein		S			Not tested
LIC11207	Putative lipoprotein			Yes		Not tested
LIC11334	Uncharacterized protein		S	Yes		Not tested
LIC11687	Endonuclease			Yes		Not tested
LIC11860	3-Hydroxyisobutyrate dehydrogenase	mmsB	I	Yes		Not tested
LIC11996	Uncharacterized protein			Yes		Not tested
LIC12032	Catalase (EC 1.11.1.6)	katE	P		Yes	Avirulentf
LIC12048	Uncharacterized protein			Yes		Not tested
LIC12084	Uncharacterized protein					Not tested
LIC12139	UDP-N-acetylglucosamine 2-epimerase	rffE	M			Not tested
LIC12158	Putative hydroxyacid aldolase protein		G			Not tested
LIC12166	Alcohol dehydrogenase		C			Not tested
LIC12193	Uncharacterized protein					Not tested
LIC12208	Putative lipoprotein			Yes		0/4
LIC12209	Putative lipoprotein (LβP52)			Yes		Not tested
LIC12232	Thymidylate synthase	thyX	F			Not tested
LIC12323	Type III beta-ketoacyl synthase-like protein		I		Yes	Not tested
LIC12331	Uncharacterized protein		G	Yes		Not tested
LIC12341	Uncharacterized protein			Yes		Not tested
LIC12353	Uncharacterized protein			Yes		Not tested
LIC12410	Uncharacterized protein			Yes		Not tested
LIC12581	Uncharacterized protein		S		Yes	Not tested
LIC12736	Putative lipoprotein			Yes		Not tested
LIC12739	MaoC family protein		I	Yes		Not tested
LIC13060	LipL36	lipL36		Yes		0/4
LIC13066	Putative lipoprotein			Yes		Not tested
LIC13261	Uncharacterized protein		R			Not tested
LIC13354	Uncharacterized protein				Yes	Not tested
LIC13361	Uncharacterized protein		L	Yes		Not tested
LIC13428	Uncharacterized protein					Not tested
LIC20053	Uncharacterized protein					Not tested
LIC20077	Polysaccharide deacetylase		G		Yes	Not tested
LIC20196	Uncharacterized protein			Yes		Not tested

^aProteins with putative orthologous alignments between L. interrogans and other pathogenic Leptospira species are defined as proteins satisfying a BLASTp alignment threshold of a minimum of 40% sequence identity on 80% of the length of the protein.

^bCOG, clusters of orthologous groups. Clusters of orthologous groups were retrieved from the MicroScope platform.

^cN-terminal signal peptide predictions were predicted using Phobius.

^dNonclassical secretion predictions were performed using SecretomeP (version 2.0).

^eVirulence is given as gerbil survival, in which the data indicate the number of gerbils that survived/total number of gerbils tested. Groups of 4 gerbils were inoculated with 10⁴ bacteria. The animals were monitored for 23 days.

^fSee reference 34.

Overrepresentation of exoproteins in COGs relating to energy production and metabolism.

Exoproteins from Table S2 in the supplemental material were assigned to clusters of orthologous groups (COGs) on the basis of the automatic classification of Leptospira genomes in the MicroScope platform (43) (Fig. 2A). Compared to the genome-wide expected frequencies, exoproteins in the COGs cell motility (COG N), signal transduction mechanisms (COG T), replication recombination and repair (COG L), general function (COG R), function unknown (COG S), and unclassified (COG −) were underrepresented (Fig. 2B). In contrast, there was a 2- to 4-fold overrepresentation of exoproteins classified in the COGs energy production and conversion (COG C), amino acid transport and metabolism (COG E), nucleotide transport and metabolism (COG F), carbohydrate transport and metabolism (COG G), and lipid transport and metabolism (COG I) (Fig. 2B). Additionally, of the 20 most abundant exoproteins, 13 were categorized in at least one of the overrepresented categories (Table 2).

FIG 2.

Classification of L. interrogans exoproteins suggests that most proteins are involved in metabolic processes. (A) Exoproteins were assigned to COGs using GenoScope (43) guidelines. The values indicate the number of exoproteins classified into each category. (B) The frequencies of exoproteins in each COG were compared to those expected genome-wide. Statistical analyses were performed by assuming a binomial distribution using a P-value cutoff of <0.01. *, a significant difference between the observed number of exoproteins and the genome-wide expected probabilities for the given COG in a sample of 325 proteins. The COG categories (and the predicted percentages of L. interrogans genes in the respective COGs) are as follows: D, cell cycle control, cell division, and chromosome partitioning (0.9%); M, cell wall/membrane/envelope biogenesis (5.3%); N, cell motility (2.4%); O, posttranslational modification, protein turnover, and chaperones (3.2%); T, signal transduction mechanisms (5.9%); U, intracellular trafficking, secretion, and vesicular transport (1.7%); V, defense mechanisms (1.6%); W, extracellular structures; Z, cytoskeleton (0.06%); B, chromatin structure and dynamics (0.04%); J, translation, ribosomal structure, and biogenesis (3.5%); K, transcription (3.1%); L, replication, recombination, and repair (4.6%); C, energy production and conversion (3.2%); E, amino acid transport and metabolism (7.0%); F, nucleotide transport and metabolism (1.6%); G, carbohydrate transport and metabolism (3.6%); H, coenzyme transport and metabolism (2.8%); I, lipid transport and metabolism (2.9%); P, inorganic ion transport and metabolism (4.5%); Q, secondary metabolite biosynthesis, transport, and catabolism (1.8%); R, general function prediction only (11.0%); S, function unknown (5.0%); −, unclassified (39.0%).

Altered exoprotein abundance in response to temperature and osmotic shifts.

The optimal temperature for the in vitro growth of Leptospira is 30°C. Comparison of the protein abundance in CSPs from Leptospira shifted to 37°C or to modified medium containing 120 mM NaCl (∼300 mosmol/liter⁻¹), which mimics physiological conditions in human plasma, to that in CSPs from Leptospira at 30°C suggested altered expression of exoproteins in response to temperature (91 in total) and 120 mM NaCl (140 in total) (see Table S1 in the supplemental material). Protein quantities were validated via immunoblot experiments (Fig. 1D and E) for the LigA protein (which was present at levels 6-fold higher in response to 120 mM NaCl via determination of relative protein intensity) and LβP52 protein (which was present at levels 20-fold higher in response to 120 mM NaCl via determination of relative protein intensity), confirming the increased expression of these proteins in response to 120 mM NaCl.

Exoproteins with potential moonlighting functions.

Moonlighting proteins are a class of proteins in which a single polypeptide chain performs more than one biochemical function (49). Classification of exoproteins into COGs revealed the overrepresentation of proteins involved in nutrient uptake and metabolism, with the latter comprising numerous proteins involved in the glycolytic pathway (Fig. 2; see also Table S2 in the supplemental material). Enzymes in the glycolytic pathway have been implicated as having moonlighting properties in other bacteria (50–53) and in Leptospira (23). To assess potential moonlighting properties for Leptospira exoproteins, proteins that have been experimentally characterized to display moonlighting properties in other microorganisms were collected from the MoonProt database (49). The primary sequences of these proteins were then used in basic local alignment searches against the L. interrogans proteome. The search results were compared with the proteomic data in Tables S1 and S2 in the supplemental material to identify orthologous proteins in the proteomic data. This approach identified in the supernatant 32 proteins that could be classified as moonlighting proteins (Table 4). A total of 45 proteins orthologous to moonlighting proteins were identified in L. interrogans; 15 of these were exoproteins, and 17 others were detected in supernatants (to be deemed exoproteins, proteins had to display a statistically significantly higher abundance in supernatants).

TABLE 4.

Potential and confirmed moonlighting proteins in Leptospira culture supernatants

EMBL/GenBank/DDBJ CDS accession no.	Locus tag	Gene/annotation	Moonlighting function(s) in other organismsa
AAS71252.1b	LIC12694	Glutamate synthase (NADPH) alpha chain precursor	Binds plasminogen, fibronectin, laminin, and collagen I
AAS69825.1b	LIC11219	Peroxiredoxin	Molecular chaperones
AAS71651.1	LIC13105	Glucose-6-phosphate isomerase	Binds laminin and collagen I
AAS72117.1	LIC20088	Pyrophosphate-fructose-6-phosphate 1-phosphotransferase	Binds invertase and plasminogen
AAS69569.1	LIC10958	Alcohol dehydrogenase	Binds plasminogen, fibronectin, laminin, and collagen II
AAS70661.1	LIC12090	Glyceraldehyde-3-phosphate dehydrogenase	NAD ribosylating activity; binds mucin, Caco-2 cells, invertase, fibronectin, laminin, type I collagen, plasminogen, uPAR/CD87 receptor, and transferrin-binding protein
AAS69802.1	LIC11195	Ornithine carbamoyltransferase	Binds fibronectin
AAS70607.1	LIC12032	Catalase	Binds plasminogen
AAS70662.1	LIC12091	Phosphoglycerate kinase	Binds plasminogen
AAS72270.1	LIC20249	Aconitate hydratase	Iron-responsive protein, binds iron-responsive elements
AAS68899.1b	LIC10272	Translation elongation factor G	Binds mucin
AAS69466.1	LIC10852	Uridylate kinase	Transcriptional regulator
AAS70653.1	LIC12082	Cysteine synthase	Transcriptional regulator
AAS71909.1	LIC13367	Sulfite reductase	Transcriptional regulator
AAS69936.1b	LIC11335	GroEL	Binds glycosphingolipids, mucins, epithelial cells, and DNA; a toxin
AAS69145.1b	LIC10524	Heat shock protein 70 (DnaK)	Binds plasminogen and invertase
AAS71428.1b	LIC12875	Elongation factor Tu (Tuf)	Binds human cells, mucins, fibronectin, factor H, and plasminogen
AAS70536.1b	LIC11954	Enolase	Binds plasminogen, laminin, fibronectin, and mucin
AAS70976.1b	LIC12407	Glutamine synthetase protein (GlnA)	Binds plasminogen, fibronectin, laminin, collagen I, and transcription factor TnrA
AAS68791.1b	LIC10162	Fumarate hydratase (aspartate ammonia-lyase)	Binds plasminogen
AAS71913.1b	LIC13371	Sulfate adenylyltransferase subunit 2 (transcription elongation factor)	Binds plasminogen
AAS70665.1b	LIC12094	Triosephosphate isomerase	Binds plasminogen
AAS72270.1	LIC20249	Aconitate hydratase	Binds untranslated regions of mRNA
AAS71933.1	LIC13393	Ketol-acid reductoisomerase (EC 1.1.1.86)	Maintains mitochondrial DNA stability
AAS69525.1b	LIC10913	Transketolase alpha subunit protein	Transcriptional regulator
AAS70287.1b	LIC11698	Tyrosyl-tRNA synthetase	Promotes folding of group 1 introns
AAS70990.1	LIC12422	Aspartate aminotransferase A	Transcription regulation
AAS69466.1	LIC10852	Uridylate kinase (UK) (EC 2.7.4.22)	Required for microautophagy
AAS69380.1b	LIC10763	Alanyl-tRNA synthetase	Transcription regulator
AAS69367.1b	LIC10750	50S ribosomal protein L1	Translational repressor
AAS71870.1b	LIC13328	Isocitrate dehydrogenase	Binds mRNA
AAS68702.1b	LIC10065	dCTP deaminase	Hydrolysis of the triphosphate moiety

^aMoonlighting functions were obtained from MoonProt (49).

^bProteins were detected in supernatants but displayed an adjusted P value of >0.05 when comparing protein abundances between supernatants and whole-cell pellets. A P value of >0.05 is indicative of a protein abundance in culture supernatants equal to or less than that in whole cells.

Exoproteins are immunogenic and not essential for Leptospira virulence.

To begin to characterize exoproteins in the context of pathogenesis, WCPs and CSPs were used in immunoblot experiments with L. interrogans-positive guinea pig sera to assess the antibody response to exoproteins, which would also be suggestive of exoprotein expression during the infection process (Fig. 3). These analyses revealed that IgG and IgM in L. interrogans-positive guinea pig sera was reactive against L. interrogans exoproteins (Fig. 3A and B) and no reactivity of the control preinfection sera (Fig. 3C and D). Exoprotein reactivity with IgG in L. interrogans-positive guinea pig sera was significantly less prominent than that of WCPs, and a similar trend was observed with IgM reactivity (Fig. 3A and B). Comparison of the reactivity of IgG and IgM in L. interrogans-positive guinea pig sera against exoproteins was distinguishable, in that the proteins displaying reactivity with IgG did not display reactivity with IgM and vice versa (Fig. 3A and B). Furthermore, CSPs from 120 mM NaCl exposure led to altered IgG and IgM reactivity against exoproteins compared to that of CSPs from the 30°C and 37°C exposures (Fig. 3A and B). Specifically, reduced IgG and IgM reactivity was observed for protein bands migrating between 70 and 100 kDa (Fig. 3A and B), and increased IgG reactivity was observed for two proteins migrating at 35 kDa and 15 kDa (Fig. 3A).

FIG 3.

L. interrogans-positive sera display IgM and IgG reactivity to leptospiral exoproteins. Guinea pig sera obtained prior to and after L. interrogans infection were used in protein immunoblot experiments to test immunoglobulin reactivity with whole L. interrogans lysates and culture supernatants. (A) Protein immunoblot comparing IgG reactivity with whole-cell proteins and exoproteins when using L. interrogans-positive sera. (B) Protein immunoblot comparing IgM reactivity to whole-cell proteins and exoproteins when using L. interrogans-positive sera. (C) Protein immunoblot demonstrating a lack of IgG reactivity to whole-cell and extracellular proteins when using preinfection sera. (D) Protein immunoblot demonstrating a lack of IgM reactivity to whole-cell and extracellular proteins when using preinfection sera.

To further assess the necessity of exoproteins for L. interrogans viability in vitro and within the host, select L. interrogans mutants in which an exoprotein-encoding gene had been inactivated were tested for in vitro growth rates and for disease manifestation in gerbils. We used for further characterization nine mutants in which we inactivated genes encoding exoproteins that previous studies suggested are involved in the infection process (LigA [54] and LruB [55–57]) or that were detected in high abundance in supernatants in the present study. In vitro growth rates identified two genes, lruB (LIC10713) and the gene for O-acetylhomoserine (thiol) lyase (LIC11852), that, when inactivated, resulted in significantly reduced in vitro growth of L. interrogans (Table 5). The other tested L. interrogans mutants (with mutations in exoprotein-encoding genes) did not display an in vitro growth defect (Table 5) and were subsequently used to challenge gerbils via intraperitoneal injection (Table 5). Gerbils challenged with these mutants displayed mortality rates similar to those challenged with the wild-type parent strain (Table 5).

TABLE 5.

Inactivation of select exoprotein-encoding genes has no effect on L. interrogans virulence in the animal infection model

Inactivated locus	Gene	Relative protein intensity in CSPs vs WCPs	Presence of an orthologue in L. biflexaa	In vitro growthb	Virulencec
LIC10713	lruB	Present in CSPs only	No	Limited	Not tested
LIC11852	O-Acetylhomoserine (thiol) lyase	4.7	Yes	Limited	Not tested
LIC13006	lenC	Present in CSPs only	Yes	Same as wtd	0/4
LIC12208	Putative lipoprotein (beta-propeller)	Present in CSPs only	No	Same as wt	0/4
LIC13060	lipL36	4.3	No	Same as wt	0/4
LIC10373	Putative lipoprotein (beta-propeller)	490	No	Same as wt	1/4
LIC10898	lipL48	10.8	Yes	Same as wt	0/4
LIC11977	Cyclic nucleotide binding protein	3.4	Yes	Same as wt	0/4
LIC10465	ligA	6.4	No	Same as wt	0/4

^aProteins with orthologous alignments between L. interrogans and L. biflexa are defined as proteins satisfying a BLASTp alignment threshold of a minimum of 40% sequence identity over 80% of the length of the protein.

^bGrowth of wild-type and mutant strains in liquid EMJH medium incubated at 30°C. Growth was monitored by measuring the optical density at 420 nm on a daily basis.

^cVirulence is given as gerbil survival, in which the data indicate the number of gerbils that survived/total number of gerbils tested. Groups of 4 gerbils were inoculated with 10⁴ bacteria. The animals were monitored for 23 days.

^dwt, wild type.

DISCUSSION

Global characterization of L. interrogans exoproteins has revealed the majority of exoproteins to be involved in metabolic and energy generation functions, which are likely essential for survival in the diverse environments encountered by these bacteria. It has been suggested that pathogenic L. interrogans strains evolved from nonpathogenic strains (58), and the former likely retained the majority of these exoprotein-encoding genes from the predecessors. Classification of exoproteins into COGs indicated that proteins with unknown functions were underrepresented, suggesting involvement of these proteins in yet to be characterized biological processes occurring within the bacteria. In contrast, the COGs amino acid, carbohydrate, and lipid uptake and metabolism were overrepresented, indicative of exoprotein involvement in nutrient acquisition and metabolism.

In addition to their metabolic activities, 32 proteins detected in culture supernatants displayed orthology to moonlighting proteins in other microorganisms. The moonlighting properties of two proteins detected in supernatants have already been demonstrated in Leptospira (23, 59). One of these proteins, phosphopyruvate hydratase (Eno/LIC11954), has been characterized as an enolase and displays plasminogen binding activity (23). The other protein, elongation factor Tu (Tuf/LIC12875), has been detected on the surface of Leptospira and displays plasminogen and factor H binding (59). A catalase (KatE/LIC12032), another potential moonlighting protein, has previously been characterized to be required for Leptospira oxidative stress resistance and virulence (34), but the plasminogen binding capacity of this protein (as demonstrated for Candida albicans catalase [60]) remains to be elucidated in L. interrogans. In accordance with a potential role in host-pathogen interactions, 5 putative L. interrogans moonlighting proteins have been shown to be immunoreactive (61), suggestive of their expression during the infection process. An exoprotein (not detected in the present study) directly associated with pathogenesis has been characterized as a collagenase required for tissue invasiveness and virulence in animals (24), while another protein (Lsa32/LIC11089), detected in culture supernatants in the present study, has been characterized and demonstrates laminin and plasminogen binding capacity (62). A known Leptospira virulence factor, high-temperature protein G (HtpG) (63), was detected at a high abundance in all culture supernatants, suggesting that the extracellular presence of this protein is a factor contributing to disease pathogenesis in animals through either unidentified moonlighting properties or the host inflammatory response to this protein. Taken together, these observations make a compelling case for exoprotein-mediated host-pathogen interactions and disease pathogenesis.

While exoprotein function can be associated with disease pathogenesis, the L. interrogans mutants tested in this study whose exoprotein-encoding genes were disrupted caused disease manifestations in animals similar to those caused by the parent strain. It should be highlighted that one of the inactivated genes encoded LipL48 (LIC10898), which was the most abundant exoprotein and was detected at levels 11-fold higher in culture supernatants than in whole bacteria. Similarly, two other inactivated genes, LIC11977, encoding a cyclic nucleotide binding protein, and ligA, encoding Ig-like repeat domain protein 1, were also detected at a high abundance in supernatants. It has previously been demonstrated that LigA confers binding of Leptospira spp. to host cells and fibronectin (54), implicative of a role for this protein in the infection process. It should also be noted that immunization of hamsters using LigA antigen provides protection against challenge with L. interrogans (64), indicating that LigA is an effective antigen for immune clearance of Leptospira. Other notable proteins included the endostatin-like protein LenC, which has been shown to bind fibronectin (65) and was unique to supernatants, and an L. interrogans antigen, LipL36. Genes annotated as lipoproteins were inactivated in two other L. interrogans mutants. Our curiosity for these genes (LIC12208 and LIC10373) stemmed from the observation that these exoproteins, among 3 others, contained multiple repeating beta-propeller domains and were unique to culture supernatants. In contrast to the lack of an impact on disease pathogenesis, inactivation of two exoprotein-encoding genes, annotated as a lipoprotein (containing an imelysin peptidase domain; lruB, or LIC10713) and an O-acetylhomoserine (thiol) lyase (LIC11852), rendered the bacteria with in vitro growth defects. The LruB (imelysine-like) protein is categorized in the COG class P (inorganic ion transport and metabolism), and structural analysis of imelysine-like proteins implicates a role in iron uptake (66). The other vital exoprotein, O-acetylhomoserine (thiol) lyase, is in COG class E (amino acid transport and metabolism) and is likely involved in methionine and cysteine regulation (67). It follows that of the 325 exoproteins, 50 were classified in COG class E, further implicating exoprotein function in heterotrophic processes.

The lack of involvement in disease pathogenesis by the majority of exoproteins is in agreement with the findings of previous studies on L. interrogans virulence genes which have demonstrated that the majority of genes are dispensable for virulence (27, 68). Moreover, with the exception of a catalase gene (katE) (34) and a collagenase gene (24), the genes that have been demonstrated to be essential for virulence, such as HtpG (63), Loa22 (69), and those involved in motility (11, 12), LPS biosynthesis (13), heme metabolism (70), and adhesion (71, 72), have orthologues in the nonpathogenic species L. biflexa. In combination with the finding that the majority of L. interrogans exoproteins have orthologues in L. biflexa, this evidence further supports the theory that L. interrogans evolved from L. biflexa. How L. interrogans became successful at establishing infection and L. biflexa did not remains to be clarified. The present study identified 51 exoproteins that were unique to L. interrogans, and the functioning of these proteins, in addition to those conserved in L. biflexa, may confer the pathogenic traits observed for L. interrogans.

The methodology used in the present study identified 325 exoproteins (the L. interrogans genome encodes ∼4,000 putative proteins), which is nearly 5-fold higher than the 66 proteins reported in a previous study (20). We propose two explanations for the observed difference in the number of proteins detected: first, protein precipitation, which was performed in the previous study and which can result in protein loss due to inefficient precipitation and resolubilization, was omitted in the present study. Second, the present study identified proteins under three different culture conditions, which would increase the number of proteins detected. Comparing protein quantities in whole cells versus those in the extracellular space under different culturing conditions had the advantage of identifying proteins unique to the extracellular space. It follows that at least a subset of the proteins in culture supernatants would be detected as a result of bacterial lysis or release from the outer membrane due to experimental manipulation, and relative protein quantification allows the satisfactory identification of putative actively transported proteins. Quantitative proteomics also provided information on the relative abundance of proteins in the extracellular space. In line with a role in heterotrophic processes, of the top 20 most abundant proteins in culture supernatants, the majority were involved in metabolic processes.

Bioinformatic analyses of the primary sequences of the 325 exoproteins suggested that Leptospira predominantly export proteins via a mechanism yet to be characterized. Of the 325 identified exoproteins, 89 (∼27%) contained an N-terminal signal sequence and 23 (∼7%) were predicted to be exported through a nonclassical protein export system. How the other 213 (∼66%) proteins are exported by L. interrogans remains to be determined. It should be emphasized that these proteins are likely exported and were not detected as a result of experimental manipulation. Evidence supporting this claim was the lack of flagellar proteins and the relatively low abundance of proteins highly expressed (in the bacteria) in the supernatants, such as LipL32 and GroEL.

The present study provides significant insight into exoprotein function in the context of the lifestyle of L. interrogans both inside and outside the host. A survey of the L. interrogans exoproteins under laboratory growth conditions has revealed that the majority of exoproteins are dedicated to heterotrophic processes but that the same proteins may also contribute to the pathogenic life cycle of the bacteria through the plasticity or moonlighting functions. These results lead us to propose a shift in how we view disease manifestation resulting from an infection with the extracellular pathogen L. interrogans. The numerous exoproteins functioning in nutrient uptake within the host and their potential moonlighting activities participating in binding to host components could lead to the disruption of normal biological processes in the host. Finally, any resulting deleterious effects on the host would likely be further compounded by the host inflammatory response to these proteins. Thus, future research should focus on how these proteins function within the host to acquire nutrients, on how they interact with host components, and on the ensuing immune response of the host to these proteins.