Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti‐leptospiral strategies.     

Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA

Bacterial specific primers were used to amplify 23S rRNA genes from a representative strain from each of the 23 serogroups of the pathogenic Leptospira interrogans and 8 strains from 6 serogroups of the non-pathogenic Leptospira biflexa. Only regions of extreme variability, which had been identified on the basis of homology-based search of all the 23S rRNA sequences available in GenBank database, were sequenced from the amplified products. PCR primers that had the potential to distinguish L. interrogans from L. biflexa species were designed from the derived sequences and a sensitive PCR protocol developed. The PCR method enabled the differentiation of the 59 strains of the 23 serogroups of L. interrogans from the 8 strains of 6 serogroups of L. biflexa. Further investigation by 16S rDNA sequencing of two strains of L. interrogans, which gave unexpected PCR results, provided evidence that they had been misclassified and hence we propose to reassign them to L. biflexa.     

Contribution of Complement System pathways to the killing of Leptospira spp.

The Complement System (CS) plays an important role in the immune response against leptospirosis and can be activated by the Alternative and Lectin Pathways (Innate Immunity) and by the Classical Pathway (Acquired Immunity). Here we analyzed a broad range of nonpathogenic and pathogenic Leptospira strains considering their interaction with each CS pathway. We determined bacterial survival rate and CS protein deposition in the presence of purified proteins, specific component depleted sera and NHS treated with the chelating agents EDTA (inhibits all three activation pathways) or EGTA (inhibits the Classical and Lectin Pathways). We suggest that the Lectin and the Alternative Pathways have an important role to eliminate saprophytic leptospires since i) approximately 50% survival of both saprophytic strains was observed in the presence of MBL-deficient serum; ii) approximately 50% survival of Leptospira biflexa Patoc I was observed in the presence of NHS – EGTA and iii) C1q-depleted serum caused significant bacterial lysis. In all serovars investigated the deposition of C5–C9 proteins on saprophytic Leptospira strains was more pronounced when compared to pathogenic species confirming previous studies in the literature. No difference on C3 deposition was observed between nonpathogenic and pathogenic strains. In conclusion, Leptospira strains interact to different degrees with CS proteins, especially those necessary to form MAC, indicating that some strains and specific ligands could favor the binding of certain CS proteins.     

Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes

Leptospirosis, a widespread zoonosis, is a re-emerging infectious disease caused by pathogenic Leptospira species. In Taiwan, Leptospira santarosai serovar Shermani is the most frequently isolated serovar, causing both renal and systemic infections. This study aimed to generate a L. santarosai serovar Shermani genome sequence and categorize its hypothetical genes, particularly those associated with virulence. The genome sequence consists of 3,936,333 nucleotides and 4033 predicted genes. Additionally, 2244 coding sequences could be placed into clusters of orthologous groups and the number of genes involving cell wall/membrane/envelope biogenesis and defense mechanisms was higher than that of other Leptospira spp. Comparative genetic analysis based on BLASTX data revealed that about 73% and 68.8% of all coding sequences have matches to pathogenic L. interrogans and L. borgpetersenii, respectively, and about 57.6% to saprophyte L. biflexa. Among the hypothetical proteins, 421 have a transmembrane region, 172 have a signal peptide and 17 possess a lipoprotein signature. According to PFAM prediction, 32 hypothetical proteins have properties of toxins and surface proteins mediated bacterial attachment, suggesting they may have roles associated with virulence. The availability of the genome sequence of L. santarosai serovar Shermani and the bioinformatics re-annotation of leptospiral hypothetical proteins will facilitate further functional genomic studies to elucidate the pathogenesis of leptospirosis and develop leptospiral vaccines.     

Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira

S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.     

Identification of Cell-Binding Adhesins of Leptospira interrogans

Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.     

Whole Genome Analysis of Leptospira licerasiae Provides Insight into Leptospiral Evolution and Pathogenicity

The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010^T and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness and its unique antigenic characteristics.     

Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

Leptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n = 11), transport (n = 6), membrane structure (n = 5), stress response (n = 2), DNA damage repair (n = 1), and other processes (n = 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenic L. interrogans.     

A widely conserved bacterial cytoskeletal component influences unique helical shape and motility of the spirochete Leptospira biflexa

Leptospires and other members of the evolutionarily ancient phylum of Spirochaetes are bacteria often characterized by long, highly motile spiral‐ or wave‐shaped cells. Morphology and motility are critical factors in spirochete physiology, contributing to the ability of these bacteria to successfully colonize diverse environments. However, the mechanisms conferring the helical structure of Leptospira spp. have yet to be fully elucidated. We have identified five Leptospira biflexa bactofilin proteins, a recently characterized protein family with cytoskeletal properties. These five bactofilins are conserved in all species of the Leptospiraceae, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility and a decreased ability to tolerate cell wall stressors. The change in the helical spacing, combined with the motility and cell wall integrity defects, showcases the intimate relationship and coevolution between shape and motility in these spirochetes.     

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.

Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo³⁵S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major ³⁵S-labeled proteins. The major incorporation of ³⁵S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major ³⁵S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the ³⁵S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

     

Occurrence of 3-O-Methylmannose in the Polysaccharide of Leptospira biflexa Urawa

3-O-methylmannose was identified by gas-liquid chromatography-mass spectrometry in the acid hydrolysate of the polysaccharide of Leptospira biflexa Urawa.     

Structural and Functional Characterization of an Orphan ATP-Binding Cassette ATPase Involved in Manganese Utilization and Tolerance in Leptospira spp.

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn²⁺, we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn²⁺, suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn²⁺ toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg²⁺-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/β subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.     

Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira

S10-spc-alpha is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-alpha locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-alpha locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-alpha locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.     

Isolation of a Seawater Tolerant Leptospira spp. from a Southern Right Whale (Eubalaena australis)

Leptospirosis is the most widespread zoonotic disease in the world. It is caused by pathogenic spirochetes of the genus Leptospira spp. and is maintained in nature through chronic renal infection of carrier animals. Rodents and other small mammals are the main reservoirs. Information on leptospirosis in marine mammals is scarce; however, cases of leptospirosis have been documented in pinniped populations from the Pacific coast of North America from southern California to British Columbia. We report the isolation of a Leptospira spp. strain, here named Manara, from a kidney sample obtained from a Southern Right Whale (Eubalaena australis) calf, which stranded dead in Playa Manara, Península Valdés, Argentina. This strain showed motility and morphology typical of the genus Leptospira spp. under dark-field microscopy; and grew in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and Fletcher medium after 90 days of incubation at 28°C. Considering the source of this bacterium, we tested its ability to grow in Fletcher medium diluted with seawater at different percentages (1%, 3%, 5%, 7% and 10% v/v). Bacterial growth was detected 48 h after inoculation of Fletcher medium supplemented with 5% sea water, demonstrating the halophilic nature of the strain Manara. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the pathogenic species of the genus Leptospira spp., with sequence similarities within the range 97–100%, and closely related to L. interrogans. Two different PCR protocols targeting genus-specific pathogenic genes (G1-G2, B64I-B64II and LigB) gave positive results, which indicates that the strain Manara is likely pathogenic. Further studies are needed to confirm this possibility as well as determine its serogroup. These results could modify our understanding of the epidemiology of this zoonosis. Until now, the resistance and ability to grow in seawater for long periods of time had been proven for the strain Muggia of L. biflexa, a saprophytic species. To the best of our knowledge, this is the first isolation of a Leptospira sp. from cetaceans. Our phenotypic data indicate that strain Manara represents a novel species of the genus Leptospira, for which the name Leptospira brihuegai sp. nov. is proposed.     

Isolation,characterization and analysis of the agglutinative activity of a lectin from <Emphasis Type="Italic">Crotalaria spectabilis</Emphasis>

Lectins are proteins with ability to recognize specific carbohydrates. These are present in virtually all organisms and have increasing applications in biotechnology. Here, our aim was to purify lectins from seeds of Crotalaria spectabilis Roth and determine their agglutinative ability. In this study, 45 g of seeds were milled, their proteins were precipitated by acetone or ammonium sulfate and purified by exclusion and ion-exchange chromatography. An isolated lectin was submitted to tests for hemagglutination and inhibition of hemagglutinating activity by carbohydrates as well as tests for its response to chelating and reducing agents. Our results show that the apparent molecular weight (as determined by SDS-PAGE) of the lectin is 30 kDa, and the tests for inhibition of erythrocytes’ agglutinative activity by sugars were positive for d-galactose and N-acetyl-d-galactosamine. Data obtained with the chelating agent EDTA demonstrated the presence of divalent cations in the protein structure. However, the reducing agent 2-mercaptoethanol was unable to inhibit the protein’s bioactivity. The lectin agglutinated the blood groups A, B, AB and O, as well as bacterial lineages from the species Leptospira interrogans and Leptospira biflexa, indicating a prospective application in the diagnosis and treatment of leptospirosis.     

Leptospira species promote a pro‐inflammatory phenotype in human neutrophils

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. Neutrophils are key cells against bacterial pathogens but can also contribute to tissue damage. Because the information regarding the role of human neutrophils in leptospirosis is scant, we comparatively analysed the human neutrophil's response to saprophytic Leptospira biflexa serovar Patoc (Patoc) and the pathogenic Leptospira interrogans serovar Copenhageni (LIC). Both species triggered neutrophil responses involved in migration, including the upregulation of CD11b expression, adhesion to collagen, and the release of IL‐8. In addition, both species increased levels of pro‐inflammatory IL‐1β and IL‐6 associated with the inflammasome and NFκB pathway activation and delayed neutrophil apoptosis. LIC was observed on the neutrophil surface and not phagocytized. In contrast, Patoc generated intracellular ROS associated with its uptake. Neutrophils express the TYRO3, AXL, and MER receptor protein tyrosine kinases (TAM), but only LIC selectively increased the level of AXL. TLR2 but not TLR4‐blocking antibodies abrogated the IL‐8 secretion triggered by both Leptospira species. In summary, we demonstrate that Leptospira species trigger a robust neutrophil activation and pro‐inflammatory response. These findings may be useful to find new diagnostic markers and therapeutic strategies against leptospirosis.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

Analysis of differential proteomes in pathogenic and non‐pathogenic Leptospira: Potential pathogenic and virulence factors

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel‐based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non‐pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non‐pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.     

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

Background

Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.

Methodology/Principal Findings

For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.

Conclusions/Significance

The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.