Proteomic profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures.

Leptospirosis is a global zoonotic disease affecting humans, domestic, and wild animals. Leptospira are typically shed in the urine of reservoir hosts which persist in suitable environments where incidental host transmission occurs after direct contact with infected urine or contaminated environments. Interestingly, serologically identical L. borgpetersenii serovar Hardjo strains JB197 and HB203 show divergent disease severity in the hamster model; JB197 causes severe acute infection while HB203 causes persistent chronic infection. Historically, serovar Hardjo was limited to culture at 29 °C, but utilization of HAN media allows propagation from host tissues at 37 °C. Here, the proteome of strains JB197 and HB203 were characterized after culture from experimentally challenged hamsters at 29 °C and 37 °C. Comparative analyses of JB197 and HB203 samples cultured at 29 °C yielded 425 significantly differentially expressed (DE) proteins, while strains at 37 °C yielded 613 DE proteins including prominent outer membrane proteins and known virulence factors. In agreement, membrane protein GO terms were identified by STRING network analyses along with numerous metabolic KEGG pathways consistent with condition differences. Within strain, JB197 cultured at 29 °C vs 37 °C identified 529 DE proteins, while HB203 identified 524 DE proteins. Investigating differential protein profiles provide insights into strain specific behaviors with implications for better understanding host-pathogen interactions, disease transmission, and response to environmental conditions which can contribute to vaccine development, diagnostic improvement, and ultimately leptospirosis control. SIGNIFICANCE: Leptospirosis is a devastating zoonotic disease affecting humans, wild and domestic animals around the globe. Different species and serovars of Leptospira can affect various animal host species differently; for instance, a serovar that is asymptomatic in the rat may cause severe disease in a dog or human. These differences in host response are not only found at the species and serovar level for Leptospira, but also at the strain level. A prime example comes from strains JB197 and HB203, both species L. borgpetersenii, both serovar Hardjo. Interestingly, JB197 causes a severe acute infection in the hamster while HB203 causes an asymptomatic chronic infection. Understanding these unique relationships between pathogen and host species is important, especially in the context of prevention technologies such as vaccine design, where the strain of Leptospira used as a bacterin might have different efficiencies in different hosts. In this study, proteomic profiles of strains JB197 and HB203 were analyzed, and results revealed diverse protein expression profiles of outer membrane proteins, as well as proteins functioning in motility and growth.

Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic Leptospira.

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with Leptospira borgpetersenii serogroup Ballum (n = 48) and/or Leptospira kirschneri serogroup Icterohaemorrhagiae (n = 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them: L. borgpetersenii serogroup Ballum in LR1, LR5, and LR37, and L. kirschneri serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of Leptospira concurrently. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.IMPORTANCEPathogenic Leptospira, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of Leptospira may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic Leptospira from the same host. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.

Evaluation of Leptospira interrogans knockdown mutants for LipL32, LipL41, LipL21, and OmpL1 proteins.

Introduction: Leptospirosis is a worldwide zoonosis caused by pathogenic and virulent species of the genus Leptospira, whose pathophysiology and virulence factors remain widely unexplored. Recently, the application of CRISPR interference (CRISPRi) has allowed the specific and rapid gene silencing of major leptospiral proteins, favoring the elucidation of their role in bacterial basic biology, host-pathogen interaction and virulence. Episomally expressed dead Cas9 from the Streptococcus pyogenes CRISPR/Cas system (dCas9) and single-guide RNA recognize and block transcription of the target gene by base pairing, dictated by the sequence contained in the 5' 20-nt sequence of the sgRNA. Methods: In this work, we tailored plasmids for silencing the major proteins of L. interrogans serovar Copenhageni strain Fiocruz L1-130, namely LipL32, LipL41, LipL21 and OmpL1. Double- and triple-gene silencing by in tandem sgRNA cassettes were also achieved, despite plasmid instability. Results: OmpL1 silencing resulted in a lethal phenotype, in both L. interrogans and saprophyte L. biflexa, suggesting its essential role in leptospiral biology. Mutants were confirmed and evaluated regarding interaction with host molecules, including extracellular matrix (ECM) and plasma components, and despite the dominant abundance of the studied proteins in the leptospiral membrane, protein silencing mostly resulted in unaltered interactions, either because they intrinsically display low affinity to the molecules assayed or by a compensation mechanism, where other proteins could be upregulated to fill the niche left by protein silencing, a feature previously described for the LipL32 mutant. Evaluation of the mutants in the hamster model confirms the augmented virulence of the LipL32 mutant, as hinted previously. The essential role of LipL21 in acute disease was demonstrated, since the LipL21 knockdown mutants were avirulent in the animal model, and even though mutants could still colonize the kidneys, they were found in markedly lower numbers in the animals' liver. Taking advantage of higher bacterial burden in LipL32 mutant-infected organs, protein silencing was demonstrated in vivo directly in leptospires present in organ homogenates. Discussion: CRISPRi is now a well-established, attractive genetic tool that can be applied for exploring leptospiral virulence factors, leading to the rational for designing more effective subunit or even chimeric recombinant vaccines.

LIC12254 Is a Leptospiral Protein That Interacts with Integrins via the RGD Motif.

Pathogenic leptospires can bind to receptors on mammalian cells such as cadherins and integrins. Leptospira effectively adheres to cells, overcomes host barriers and spreads into the bloodstream, reaching internal target organs such as the lungs, liver and kidneys. Several microorganisms produce proteins that act as ligands of integrins through the RGD motif. Here, we characterized a leptospiral RGD-containing protein encoded by the gene lic12254. In silico analysis of pathogenic, intermediate and saprophytic species showed that LIC12254 is highly conserved among pathogenic species, and is unique in presenting the RGD motif. The LIC12254-coding sequence is greatly expressed in the virulent Leptospira interrogans L1-130 strain compared with the culture-attenuated L. interrogans M20 strain. We also showed that the recombinant protein rLIC12254 binds to αVß8 and α8 human integrins most likely via the RGD motif. These interactions are dose-dependent and saturable, a typical property of receptor-ligand interactions. The binding of the recombinant protein lacking this motif-rLIC12254 ΔRAA-to αVß8 was almost totally abolished, while that with the α8 human integrin was decreased by 65%. Taken together, these results suggest that this putative outer membrane protein interacts with integrins via the RGD domain and may play a key role in leptospirosis pathogenesis.

Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity.

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/ß subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for ß-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin ß-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

Proteomic dataset comparing strains of Leptospira borgpetersenii serovar Hardjo cultured at different temperatures.

Leptospirosis is a global zoonotic bacterial disease which is a threat for humans and most mammals. Bacterin vaccines for leptospirosis are available however they are severely limited in cross protection between serogroups. Leptospira typically colonize the kidneys of reservoir hosts where they are subsequently shed in the urine and persist in the environment and can thus be indirectly or directly transmitted to incidental hosts. Leptospira borgpetersenii serovar Hardjo is the primary cause of leptospirosis in cattle which can result in abortion, unhealthy calves, and rebreed problems. This dataset comprises proteomic profiles of four strains of L. borgpetersenii serovar Hardjo propagated at the routinely utilized culture temperature of 29 °C, and a newly achieved culture temperature of 37 °C, which more closely emulates the temperature of an infected host. The strains analyzed include JB197 (established strain that causes Hardjo atypical acute disease in the hamster model of leptospirosis), HB203 (established strain, causes typical chronic disease in hamsters), as well as TC129 and TC273 (recently isolated strains from the central United States). Differential expression profiles were detected not only between strains but also within strains between culture temperatures. Mass spectrometry data are available via ProteomeXchange with identifier PXD032831.

A New Recombinant Multiepitope Chimeric Protein of Leptospira interrogans Is a Promising Marker for the Serodiagnosis of Leptospirosis.

The zoonotic disease leptospirosis is caused by pathogenic species of the genus Leptospira and was recently included in the list of Neglected Diseases by the World Health Organization. Leptospirosis burden is estimated to have over a million human cases and cause 60 thousand deaths annually, in addition to its economic impact and veterinary concern. The microscopic agglutination test (MAT), recommended by the World Health Organization, exhibits reduced sensitivity at the beginning of the disease, in addition to being technically difficult. New recombinant antigens are being pursued for rapid and specific serodiagnostic tests, especially in the initial phase of the disease, and chimeric multiepitope proteins are a strategy with a great potential to be implemented in serology. Based on previous subproteomic results, we designed a synthetic construct comprising 10 conserved leptospiral surface antigens, and the recombinant protein was purified and evaluated regarding its diagnostic potential. The protein termed rChi2 was recognized by antibodies in serum from patients both at the onset (MAT-) and in the convalescent (MAT+) phase in 75 and 82% of responders, respectively. In addition, rChi2 immunization in hamsters elicited a strong humoral response, and anti-rChi2 antibodies recognized several immobilized intact Leptospira species, validating its potential as an early, broad, and cross-reactive diagnostic test.

A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems.

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

Some like it hot, some like it cold; proteome comparison of Leptospira borgpetersenii serovar Hardjo strains propagated at different temperatures.

Leptospirosis is a global zoonotic disease affecting humans and livestock species. Bacterin vaccines lack cross protection between serogroups, and include multiple serovars propagated at 29 °C. Recent work demonstrated substantial variation in the transcriptome of identical species and serovars of Leptospira. Here, substantial differences in protein abundance profiles were identified in Leptospira borgpetersenii serovar Hardjo; strain HB203, which was isolated in the 1980s, compared to newer strains TC129 and TC273 isolated in 2016, and whether they were propagated at the routine temperature of 29 °C, compared to 37 °C which more closely emulates host infection. While 388 and 385 significantly differentially expressed (DE) proteins (FDR of 0.01) were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 29 °C respectively, only 66 and 4 DE proteins were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 37 °C respectively. Within each strain comparing temperatures, HB203 had 524 significantly DE proteins, TC129 had 347 DE proteins, and TC273 had 569 DE proteins. Data are available via ProteomeXchange with identifier PXD032831. Results highlight significant differential protein expression among identical serovars of L. borgpetersenii suggesting that bacterin vaccine design can benefit from consideration of strains employed and effects of temperature on growth. SIGNIFICANCE: Leptospirosis is a zoonotic disease caused by spirochete bacteria of the genus Leptospira. While leptospirosis affects over one million people per year, symptoms range vastly in severity from completely asymptomatic, to flu-like, to multi-organ failure and death in severe cases. Incidental hosts become infected after encountering pathogens directly from contact with another host, including domestic or wildlife animals, or indirectly from contaminated environments. Though animal vaccines exist, they lack cross protection across serogroups, and instead rely on inclusion of multiple carefully selected serovars from laboratory strains prepared at ~29 °C. Recent interest in gene expression at the Leptospira strain level, along with a newly achieved culture temperature of 37 °C (which more closely resembles host body temperature), led us to investigate the proteomic profiles of an older, established challenge strain HB203 in comparison to TC129 and TC273, two strains isolated in 2016 from abattoir cattle in the central United States. Herein, we identify substantial proteomic differences not only between strains of the same species and serovar, but notably between growth temperatures, collectively suggesting that bacterin vaccine composition may benefit from investigating strain selection and the temperature employed for growth of the bacteria used in bacterin preparation.

Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico, USA.

BACKGROUND: Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology. METHODOLOGY/PRINCIPAL FINDINGS: We collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup. CONCLUSIONS/SIGNIFICANCE: Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.

A Review on Host-Leptospira Interactions: What We Know and Future Expectations.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is considered a neglected infectious disease of human and veterinary concern. Our group has been investigating proteins annotated as hypothetical, predicted to be located on the leptospiral surface. Because of their location, these proteins may have the ability to interact with various host components, which could allow establishment of the infection. These proteins act as adherence factors by binding to host receptor molecules, such as the extracellular matrix (ECM) components laminin and glycosaminoglycans to help bacterial colonization. Leptospira also interacts with the host fibrinolytic system, which has been demonstrated to be a powerful tool for invasion mechanisms. The interaction with fibrinogen and thrombin has been shown to reduce fibrin clot formation. Additionally, the degradation of coagulation cascade components by secreted proteases or by acquired surface plasmin could also play a role in reducing clot formation, hence facilitating dissemination during infection. Interaction with host complement system regulators also plays a role in helping bacteria to evade the immune system, facilitating invasion. Interaction of Leptospira to cell receptors, such as cadherins, can contribute to investigate molecules that participate in virulence. To achieve a better understanding of the host-pathogen interaction, leptospiral mutagenesis tools have been developed and explored. This work presents several proteins that mediate binding to components of the ECM, plasma, components of the complement system and cells, to gather research achievements that can be helpful in better understanding the mechanisms of leptospiral-host interactions and discuss genetic manipulation for Leptospira spp. aimed at protein function validation.

Circulating Foamy Macrophages in the Golden Syrian Hamster (Mesocricetus auratus) Model of Leptospirosis.

Leptospirosis is a world-wide zoonotic disease caused by pathogenic Leptospira and can be asymptomatic or can cause clinical signs ranging from influenza-like to multi-organ failure and death in severe cases. While species and strain specificity can play a major role in disease presentation, the hamster is susceptible to most leptospiral infections and is the model of choice for vaccine efficacy testing. During evaluation of blood smears from hamsters challenged with different species and strains of Leptospira, a circulating population of large, mononuclear, lipid-filled cells, most similar to foamy macrophages (FMs), was detected. Circulating FMs were identified by Giemsa staining and verified by scanning and transmission electron microscopy. FMs were found in the circulating blood of all Leptospira-challenged hamsters, indicating that the finding was not species or strain specific, although higher numbers of FMs tended to correlate with severity of disease. The unique finding of circulating FMs in the hamster model of leptospirosis can yield additional insights into the pathogenesis of leptospirosis and other diseases that induce circulating FMs.

Distinct transcriptional profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures.

BACKGROUND: Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). METHODOLOGY/PRINCIPAL FINDINGS: L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. CONCLUSIONS/SIGNIFICANCE: Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets.

Evaluation of LipL32 and LigA/LigB Knockdown Mutants in Leptospira interrogans Serovar Copenhageni: Impacts to Proteome and Virulence.

Leptospirosis is a worldwide zoonosis caused by pathogenic species of the genus Leptospira. The recent application of CRISPR interference (CRISPRi) to Leptospira facilitates targeted gene silencing and provides a new tool to investigate pathogenic mechanisms of leptospirosis. CRISPRi relies on the expression of a catalytically "dead" Cas9 (dCas9) and a single-guide RNA (sgRNA). Previously, our group generated a LipL32 and a double LigA/LigB (LigAB) mutant, which, in the current study, are characterized by whole-cell proteomics in comparison with control leptospires harboring plasmid expressing dCas9 alone. Comparison of control and LigAB mutant leptospires identified 46 significantly differentially expressed (DE) proteins, including 27 proteins that were less abundant and 19 proteins that were more abundant in the LigAB mutant compared with the control. Comparison of the control and LipL32 mutant leptospires identified 243 DE proteins, of which 84 proteins were more abundant and 159 were less abundant in the LipL32 mutant strain. Significantly increased amounts of known virulence impactors and surface membrane receptors, including LipL45, LipL31, LigB, and LipL41, were identified. The virulence of LipL32 and LigAB mutants were evaluated in the hamster model of leptospirosis; the LigAB mutant was unable to cause acute disease although mutant leptospires could still be recovered from target organs, albeit at a significantly lower bacterial burden (<850 and <16-fold in liver and kidney, respectively, in comparison with control), indicating attenuation of virulence and a shift to chronic bacterial persistence. Notably, the LipL32 mutant displayed augmented virulence as evidenced by early onset of clinical symptoms and increased numbers of circulating foamy macrophages. Validation of LipL32 and LigAB mutants recovered from liver and kidney in the presence or absence of antibiotic selection revealed high plasmid stability and, by extension, gene silencing in vivo. Collectively, this work emphasizes the advantages and feasibility of using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host-pathogen interactions in animal models of leptospirosis. Importantly, it also provides insight into the requirements of LigA and LigB for acute disease and explores the impact of silencing expression of lipL32, which resulted in substantial changes in amounts of outer membrane proteins.

A Modified ELISA Method to Evaluate the Interaction of Schistosoma mansoni Proteins with Plasminogen.

An important aspect of host-pathogen interactions is the interference of secreted proteins with the fibrinolytic system. Herein, we describe a modified ELISA method used to evaluate the interaction of a recombinant Schistosoma mansoni protein with plasminogen (PLG). Using this protocol, we demonstrated that a secreted protein, recombinant venom allergen-like protein 18 (rSmVAL18) acts as a plasminogen receptor increasing its activation into plasmin in the presence of the urokinase-type plasminogen activator (uPA). PLG binding was determined by immobilizing human PLG in the plate and incubating with the recombinant protein; competitive binding with a lysine analog demonstrated the interaction of the protein lysine residues with PLG Kringle domains. To assess the activation of S. mansoni recombinant protein-bound PLG, the amidolytic activity of generated plasmin was measured using the D-Val-Leu-Lys 4-nitroanilide dihydrochloride substrate.

The interaction of two novel putative proteins of Leptospira interrogans with E-cadherin, plasminogen and complement components with potential role in bacterial infection.

Leptospirosis is a worldwide zoonosis caused by pathogenic species of Leptospira. Leptospires are able to adhere to exposed extracellular matrix in injured tissues and, once in the bloodstream, can survive the attack of the immune system and spread to colonize target organs. In this work, we report that two novel putative proteins, coded by the genes LIC11711 and LIC12587 of L. interrogans serovar Copenhageni are conserved among pathogenic strains, and probably exposed in the bacterial surface. Soluble recombinant proteins were expressed in Escherichia coli, purified and characterized. Both recombinant proteins bound to laminin and E-cadherin, suggesting an initial adhesion function in host epithelial cells. The recombinant protein LIC11711 (rLIC11711) was able to capture plasminogen (PLG) from normal human serum and convert to enzymatically active plasmin (PLA), in the presence of PLG activator. rLIC12587 (recombinant protein LIC12587) displayed a dose dependent and saturable interaction with components C7, C8, and C9 of the complement system, reducing the bactericidal effect of the complement. Binding to C9 may have consequences such as C9 polymerization inhibition, interfering with the membrane attack complex formation. Blocking LIC11711 and LIC12587 on bacterial cells by the respective antiserum reduced leptospiral cell viability when exposed to normal human serum (NHS). Both recombinant proteins could be recognized by serum samples of confirmed leptospirosis, but not of unrelated diseases, suggesting that the native proteins are immunogenic and expressed during leptospirosis. Taken together, our data suggest that these proteins may have a role in leptospiral pathogenesis, participating in immune evasion strategies.

Adjuvanted leptospiral vaccines: Challenges and future development of new leptospirosis vaccines.

Leptospirosis is a neglected infectious disease of global importance. Vaccination is the most viable strategy for the control of leptospirosis, but in spite of efforts for the development of an effective vaccine against the disease, few advances have been made, and to date, bacterin is the only option for prevention of leptospirosis. Bacterins are formulations based on inactivated leptospires that present a series of drawbacks, such as serovar-dependence and short-term immunity. Therefore, bacterins are not widely used in humans, and only Cuba, France and China have these vaccines licensed for at-risk populations. The development of recombinant DNA technology emerges as an alternative to solve the problem. Recombinant protein-based vaccines or DNA vaccines seem to be an attractive strategy, but the use of adjuvants is critical for achievement of a protective immune response. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells. In the last years, several components have been tested as adjuvants, such as aluminum salts, oil based-emulsion adjuvants, bacteria-derived components and liposomes. This review highlights the use of adjuvants in the multiple vaccine approaches that have been used for leptospirosis and their most important immunological aspects. Immune response data generated by these strategies can contribute to the understanding of the immune mechanisms involved in protection against leptospirosis, and consequently, the development of effective vaccines against this disease. This is the first review on leptospiral vaccines focusing on adjuvant aspects.

Evaluation of Lsa46 and Lsa77 Leptospiral Proteins for Their Immunoprotective Activities in Hamster Model of Leptospirosis.

Leptospirosis is a neglected tropical disease caused by pathogenic Leptospira spp. The lack of an effective vaccine favors the increase of the disease. Currently, surface-exposed proteins are the main targets for the search of vaccine candidates. In this study, we examined whether the surface Lsa46 and Lsa77 proteins, previously identified as laminin and plasminogen binding proteins, have the capacity of inducing protection and sterilizing immunity against challenge with virulent Leptospira in hamster model. Animals were subcutaneously immunized with Lsa46, Lsa77, or a combination of both in Alum adjuvant and challenged intraperitoneally with L. interrogans serovar Kennewicki strain Pomona Fromm. Hamster immunization with Lsa46 or Lsa77 or both promoted a strong IgG response. Th2- and Th1-biased immune responses were observed when Lsa46 and Lsa77 were individually administered, respectively, as detected by the IgG1/IgG2/3 ratio. Immunized hamsters with the combined proteins induced a Th1-biased immune response. Although the immunization with Lsa46 and Lsa77 stimulated protective immunity with reduction of bacterial burden, when compared to animals individually immunized with the proteins, the data was not statistically significant. Thus, although promising, more studies are needed before the role of these proteins in stimulating sterilizing immunity in mammals is conclusively determined.