Nucleotide-induced ClpC oligomerization and its non-preferential association with ClpP isoforms of pathogenic Leptospira.

Bacterial caseinolytic protease-chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class-I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide-induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non-preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade ß-casein or FITC-casein in an energy-independent manner. Adding either ATP or ATPγS further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of hetero-tetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

Proteomic profile of naturally released extracellular vesicles secreted from Leptospira interrogans serovar Pomona in response to temperature and osmotic stresses.

Bacterial extracellular vesicles (EVs) are generally formed by pinching off outer membrane leaflets while simultaneously releasing multiple active molecules into the external environment. In this study, we aimed to identify the protein cargo of leptospiral EVs released from intact leptospires grown under three different conditions: EMJH medium at 30 °C, temperature shifted to 37 °C, and physiologic osmolarity (EMJH medium with 120 mM NaCl). The naturally released EVs observed under transmission electron microscopy were spherical in shape with an approximate diameter of 80-100 nm. Quantitative proteomics and bioinformatic analysis indicated that the EVs were formed primarily from the outer membrane and the cytoplasm. The main functional COG categories of proteins carried in leptospiral EVs might be involved in cell growth, survival and adaptation, and pathogenicity. Relative to their abundance in EVs grown in EMJH medium at 30 °C, 39 and 69 proteins exhibited significant changes in response to the temperature shift and the osmotic change, respectively. During exposure to both stresses, Leptospira secreted several multifunctional proteins via EVs, while preserving certain virulence proteins within whole cells. Therefore, leptospiral EVs may serve as a decoy structure for host responses, whereas some virulence factors necessary for direct interaction with the host environment are reserved in leptospiral cells. This knowledge will be useful for understanding the pathogenesis of leptospirosis and developing as one of vaccine platforms against leptospirosis in the future.

Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans.

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently.

Leptospiral cell wall hydrolase (LIC_10271) binding peptidoglycan, lipopolysaccharide, and laminin and the protein show LysM and M23 domains are co-existing in pathogenic species.

Leptospirosis, a global reemerging zoonosis caused by the spirochete Leptospira, has severe human and veterinary implications. Cell wall hydrolase (LIC_10271) with LytM (peptidase M23) and LysM domains are found to be associated with various pathogenic bacteria. These domains regulate effects on extracellular matrix and biofilm components, which promote cell wall remodeling and pathogen dissemination in the host. In this study, we present the cloning, expression, purification, and characterization of LIC_10271. To determine the localization of LIC_10271 within the inner membrane of Leptospira, Triton X-114 subcellular fractionation and immunoblot studies were performed. Furthermore, r-LIC_10271 binds with peptidoglycan, lipopolysaccharide, and laminin in a dose-dependent manner. Analysis of the signal peptide, M23, and LysM domains revealed conservation primarily within the P1 group of Leptospira, which encompasses the most pathogenic species. Moreover, the presence of native-LIC_10271 in the inner membrane and the distribution of M23 and LysM domains across pathogenic strains indicates their potential involvement in the interaction between the host and Leptospira.

Crystal structure of a variable region segment of Leptospira host-interacting outer surface protein, LigA, reveals the orientation of Ig-like domains.

Leptospiral immunoglobulin-like (Lig) protein family is a surface-exposed protein from the pathogenic Leptospira. The Lig protein family has been identified as an essential virulence factor of L. interrogan. One of the family members, LigA, contains 13 homologous tandem repeats of bacterial Ig-like (Big) domains in its extracellular portion. It is crucial in binding with the host's Extracellular matrices (ECM) and complement factors. However, its vital role in the invasion and evasion of pathogenic Leptospira, structural details, and domain organization of the extracellular portion of this protein are not explored thoroughly. Here, we described the first high-resolution crystal structure of a variable region segment (LigA8-9) of LigA at 1.87 Å resolution. The structure showed some remarkably distinctive aspects compared with other closely related Immunoglobulin domains. The structure illustrated the relative orientation of two domains and highlighted the role of the linker region in the domain orientation. We also observed an apparent electron density of Ca2+ ions coordinated with a proper interacting geometry within the protein. Molecular dynamic simulations demonstrated the involvement of a linker salt bridge in providing rigidity between the two domains. Our study proposes an overall arrangement of Ig-like domains in the LigA protein. The structural understanding of the extracellular portion of LigA and its interaction with the ECM provides insight into developing new therapeutics directed toward leptospirosis.

Leptospiral imelysin (LIC_10713) is secretory, immunogenic and binds to laminin, fibronectin, and collagen IV.

Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira. Early and accurate diagnosis is the prime step in managing the disease. Secretory proteins of Leptospira remain distinguished for diagnosis due to their availability as soluble proteins in the serum and their interaction with the host immune response due to their extracellular presence. This study presents the cloning, expression, purification, and characterization of imelysin or LruB (LIC_10713), a putative leptospiral protein. We report that the localization of imelysin showed its presence in the inner membrane and in the culture supernatant. The imelysin was upregulated under in vitro physiological conditions of infection. The LIC_10713 interacted significantly with laminin, fibronectin, collagen type I, and collagen type IV in a dose-dependent manner. Phylogenetic analysis showed that LIC_10713 is predominately found in the pathogenic species of Leptospira, and the GxHxxE motif of imelysin-like proteins is represented as the amino acid sequence GWHAIE. Also, immunoglobulins in leptospirosis-infected patients recognize recombinant-LIC_10713 with 100% specificity and 90.9% sensitivity. The secretion nature, abundance, upregulation, binding to ECM components, and immunogenicity determine LIC_10713 as an important molecule that can be used as an anti-leptospirosis measure. KEY POINTS: • The imelysin-like protein (LIC_10713) of Leptospira is a secretory protein • The protein LIC_10713 can bind ECM molecules • The LIC_10713 is mainly found in pathogenic leptospires • The anti-LIC_10713 antibody from human serum can detect the r-LIC_10713.

Overcoming problems to produce the recombinant protein LipL21 of Leptospira interrogans.

The production of leptospiral recombinant proteins in the soluble form and in high yield from Escherichia coli is still a challenge. This work presents the cloning, expression and purification of the outer membrane protein of Leptospira interrogans, LipL21, which is considered an interesting target for vaccine and diagnostics development. The expression profile and yield of LipL21 was compared after cloning in the vectors pAE, pET28a and pET-SUMO, and it was observed that LipL21 was expressed in a low amount with pAE vector. By using the pET-28a vector, protein expression was increased, but the majority of the product was obtained as inclusion bodies. As a highlight, using a pET-SUMO vector was shown to overcome the problems of low expression and solubility of the lipoprotein LipL21.

Leptospira interrogans Prevents Macrophage Cell Death and Pyroptotic IL-1ß Release through Its Atypical Lipopolysaccharide.

Leptospira interrogans are bacteria that can infect all vertebrates and are responsible for leptospirosis, a neglected zoonosis. Some hosts, such as humans, are susceptible to the disease, whereas mice are resistant and get chronically colonized. Although leptospires escape recognition by some immune receptors, they activate the NOD-like receptor pyrin 3-inflammasome and trigger IL-1ß secretion. Classically, IL-1ß secretion is associated with lytic inflammatory cell death called pyroptosis, resulting from cytosolic LPS binding to inflammatory caspases, such as caspase 11. Interestingly, we showed that L. interrogans and Leptospira biflexa do not trigger cell death in either murine, human, hamster, or bovine macrophages, escaping both pyroptosis and apoptosis. We showed, in murine cells, that the mild IL-1ß secretion induced by leptospires occurred through nonlytic caspase 8-dependent gasdermin D pore formation and not through activation of caspase 11/noncanonical inflammasome. Strikingly, we demonstrated a potent antagonistic effect of pathogenic L. interrogans and their atypical LPS on spontaneous and Escherichia coli LPS-induced cell death. Indeed, LPS of L. interrogans efficiently prevents caspase 11 dimerization and subsequent massive gasdermin D cleavage. Finally, we showed that pyroptosis escape by leptospires prevents massive IL-1ß release, and we consistently found no major role of IL-1R in controlling experimental leptospirosis in vivo. Overall, to our knowledge, our findings described a novel mechanism by which leptospires dampen inflammation, thus potentially contributing to their stealthiness.

In silico identification and characterization of potential druggable targets among hypothetical proteins of Leptospira interrogans serovar Copenhageni: a comprehensive bioinformatics approach.

Leptospirosis is one of the neglected zoonosis, affecting human and animal populations worldwide. Reliable effective therapeutics and concerns to look for more research into the molecular analysis of its genome is therefore needed. In the genomic pool of the Leptospira interrogans many hypothetical proteins are still uncharacterized. In the current research, we performed extensive in silico analysis to prioritize the potential hypothetical proteins of L. interrogans serovar Copenhageni via stepwise reducing the available hypothetical proteins (Total 3606) of the assembly to only 15, based on non-homologous to homosapien, essential, functional, virulent, cellular localization. Out of them, only two proteins WP_000898918.1 (Hypothetical Protein 1) & WP_001014594.1 (Hypothetical Protein 2) were found druggable and involved in protein-protein interaction network. The 3 D structures of these two target proteins were predicted via ab initio homology modeling followed by structures refinement and validation, as no structures were available till date. The analysis also revealed that the functional domains, families and protein-protein interacting partners identified in both proteins are crucial for the survival of the bacteria. The binding cavities were predicted for both the proteins through blind and specific protein-ligand docking with their respective ligands and inhibitors and were found to be in accordance with the druggable sites predicted by DoGSiteScorer. The docking interactions were found within the active functional domains for both the proteins while for Hypothetical Protein 2, the same residues were involved in interactions with Cytidine-5'-triphosphate in blind and specific docking. Furthermore, the simulations of molecular dynamics and free binding energy revealed the stable substrate binding and efficient binding energies, and were in accordance to our docking results. The work predicted two unique hypothetical proteins of L. interrogans as a potential druggable targets for designing of inhibitors for them.Communicated by Ramaswamy H. Sarma.

Toll-like receptor 2-mediated induction of human beta-defensin 2 expression by Leptospira interrogans in human kidney cells.

BACKGROUND: Leptospirosis is a zoonotic disease caused by Leptospira interrogans. Severe leptospirosis is often accompanied by kidney dysfunction caused by chronic infection. The kidney pathology involves bacterial invasion and inflammation caused by pro-inflammatory cytokines. Human beta defensins (hBDs) are antimicrobial peptides induced by microbial infection and/or pro-inflammatory cytokines. One function of hBDs is the recruitment of immune cells that leads to inflammation. However, the expression of hBDs by kidney epithelium in response to pathogenic Leptospira has never been investigated. OBJECTIVE: To determine the expression of hBDs in human kidney epithelium responses to Leptospira. METHODS: Human kidney cells were infected with Leptospira interrogans serovar Autumnalis in the presence or absence of anti-TLR2 neutralizing antibody (Ab) for 6 hours. TLR2, hBDs and pro-inflammatory cytokines mRNA expressions were analyzed by quantitative polymerase chain reaction (qPCR). RESULTS: Pathogenic Leptospira upregulated the expressions of pro-inflammatory cytokines and hBD2, but not TLR2, hBD1 and hBD3 in kidney cells. The expressions of hBD2 and pro-inflammatory cytokines were inhibited in the presence of anti-hTLR2 neutralizing Ab. CONCLUSIONS: Our results provide the first evidence that pathogenic Leptospira induces hBD2 expression in kidney cells. The expressions of pro-inflammatory cytokines and hBD2 in the cells in response to pathogenic Leptospira are regulated by TLR2. Pro-inflammatory cytokines and hBD2 might be play role in recruitment of immune cells to the kidney and contribute to the development of inflammation-mediated tissue damage in the kidney. However, further study is needed to improve the understanding of the role of these molecules in immune response activation.

Host Cell Binding Mediated by Leptospira interrogans Adhesins.

Leptospirosis is a neglected infectious disease with global impact on both humans and animals. The increase in urban development without sanitation planning is one of the main reasons for the disease spreading. The symptoms are similar to those of flu-like diseases, such as dengue, yellow fever, and malaria, which can result in a misleading clinical diagnosis. The characterization of host-pathogen interactions is important in the development of new vaccines, treatments, and diagnostics. However, the pathogenesis of leptospirosis is not well understood, and many gaps remain to be addressed. Here, we aimed to determine if Leptospira strains, virulent, culture-attenuated, and saprophytic, and the major outer membrane proteins OmpL37, OmpL1, LipL21, LipL41, and LipL46 are able to adhere to different endothelial, epithelial and fibroblast cell lines in vitro. We showed that virulent leptospires robustly bind to all cells compared to the culture-attenuated and saprophytic lines. The recombinant proteins exhibited certain adhesion, but only OmpL1 and LipL41 were able to bind to several cell lines, either in monolayer or in cell suspension. Blocking OmpL1 with polyclonal antibodies caused a decrease in bacterial binding to cells, contrasting with an increase observed when anti-LipL41 antibodies were used. The adhesion of OmpL1 to HMEC-1 and EA.hy926 was inhibited when cells were pre-incubated with collagen IV, suggesting that both compete for the same cell receptor. We present here for the first time the interaction of five leptospiral outer membrane proteins with several cell lines, and we conclude that LipL41 and OmpL1 may have an impact on leptospiral adhesion to mammalian cells and may mediate the colonization process in leptospiral pathogenesis.

Identifying Leptospira interrogans putative virulence factors with a yeast protein expression screen.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: • High-throughput cloning and expression of leptospiral proteins in yeast. • Heterologous expression of nine leptospiral proteins inhibited yeast growth. • An uncharacterized protein LIC10280 maybe a putative VF for further validation.

Heterologous production of the adhesin LIC13411 from pathogenic Leptospira facilitates binding of non-pathogenic Leptospira in vitro and in vivo.

Leptospirosis is an important cause of morbidity and mortality worldwide. Disease severity ranges from asymptomatic colonization to widespread hemorrhage and multiorgan dysfunction. The causative agents, Leptospira spp., are zoonotic Gram-negative spirochetes. One important step in pathogenesis is binding of bacterial adhesins to host components. Previously our laboratory identified two L. interrogans candidate adhesins, LIC11574 and LIC13411, that bind to VE-cadherin in vitro. In the current study, we demonstrate the ability of two strains of pathogenic L. interrogans to disrupt the localization of VE-cadherin, a protein important to maintaining inter-endothelial junctions. Purified MBP-LIC11574 and MBP-LIC13411 bind human dermal microvascular endothelial cells in a pattern reminiscent of VE-cadherin, but do not disrupt VE-cadherin localization. Genes encoding the candidate adhesins from pathogenic Leptospira were cloned in an overexpression vector and introduced into non-pathogenic L. biflexa, creating gain-of-function strains producing LIC11574 or LIC13411. Protein production and localization to the outer membrane were confirmed by Triton X-114 fractionation. Although these strains do not disrupt VE-cadherin localization, production of LIC13411 increases binding of non-pathogenic Leptospira to human endothelial cells and specifically to VE-cadherin. In a short-term murine model of infection, LIC13411 production led to increased burdens of the non-pathogen in the lung, liver, kidney, and bladder. These data confirm the role of LIC13411 as an adhesin in Leptospira spp. and implicate it in dissemination to multiple organs. Importantly, anti-adhesin therapy has been shown to have many benefits over classical antibiotics. Taken together, this work provides novel insight into the pathogenesis of Leptospira spp. and identifies LIC13411 as a potential prophylactic and therapeutic target.

ErpY-like Lipoprotein of Leptospira Outsmarts Host Complement Regulation by Acquiring Complement Regulators, Activating Alternative Pathways, and Intervening in the Membrane Attack Complex.

The survival of pathogenic Leptospira in the host depends on its proficiency to circumvent the immune response. These pathogens evade the complement system in serum by enticing and amassing the serum complement regulators onto their surface. ErpY-like lipoprotein, a surface-exposed protein of Leptospira spp., is conserved in the pathogenic Leptospira serovars. The recombinant form of this protein interacts with multiple extracellular matrix (ECM) components and serum proteins such as soluble complement regulators factor H (FH) and factor I (FI). Here, we document that the supplementation of rErpY-like protein (10 µg/mL) in human serum inhibits complement-mediated bacterial cell lysis and augments the viability of Escherichia coli and saprophytic Leptospira biflexa by more than two-fold. Complement regulators FH and FI, when bound to rErpY-like protein, preserve their respective cofactor and protease activity and cleave the complement component C3b. The supplementation of rErpY-like protein (40 µg/mL) in serum ensued in an ∼90% reduction of membrane attack complex (C5b-9/MAC) deposition through the alternative pathway (AP) of complement activation. However, rErpY-like protein could moderately reduce (∼16%) MAC deposition in serum through the classical pathway (CP). In addition, the rErpY-like protein solely initiated the AP, suggesting its role in the rapid consumption and depletion of the complement components. Blocking the pathogenic Leptospira interrogans surface with anti-rErpY-like antibodies resulted in an increase in MAC formation on the bacterial surface, indicating a specific role of the ErpY-like lipoprotein in complement-mediated immune evasion. This study underscores the role of the ErpY-like lipoprotein of Leptospira in complement evasion.

Functional characterization of monothiol and dithiol glutaredoxins from Leptospira interrogans.

Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans.

Caracterização funcional de proteínas hipotéticas de Leptospira interrogans como adesinas e potenciais antígenos vacinais e para diagnóstico / Functional characterization of hypothetical proteins in Leptospira interrogans as adhesins and potential vaccine and diagnostic antigens

Leptospira spp. constitui um grupo de bactérias espiroquetas gram-negativas englobando espécies saprofíticas, intermediárias e patogênicas, sendo as últimas agentes causadores da leptospirose, doença zoonótica de alcance mundial e endêmica em regiões tropicais em desenvolvimento. O crescente número de espécies identificadas de leptospiras destaca ainda mais sua diversidade genética e mecanismos de virulência únicos, muitos deles com função ainda desconhecida. Esforços para o desenvolvimento de novas vacinas com proteção cruzada e efeito duradouro revelaram possíveis candidatos vacinais que necessitam ser adequadamente validados, sendo assim, há ainda uma urgente necessidade de uma vacina universal contra a leptospirose capaz de controlar e reduzir os surtos cada vez mais frequentes da doença. Adesinas são importantes fatores de virulência em diversos patógenos, constituindo antígenos promissores para o desenvolvimento de vacinas contra a leptospirose, assim como para o desenvolvimento de métodos diagnósticos mais rápidos e precisos. Previamente, foram identificadas três proteínas hipotéticas conservadas em L. interrogans pela técnica de phage display, denominadas arbitrariamente como LepA069, LepA962 e LepA388. A expressão do gene codificador da proteína LepA069 apresentou aumento de aproximadamente 70 % em animais infectados por leptospiras virulentas, representando a primeira evidência funcional desta proteína ainda desconhecida. Porções recombinantes da lipoproteína hipotética LepA962 (LepA962_Nt e LepA962_Phg) foram obtidos, sendo demonstrada a forte interação da proteína LepA962_Phg, contendo a sequência identificada por phage display, com laminina, fibronectina plasmática, colágeno I e fibrinogênio de maneira dose-dependente. Adicionalmente, LepA962_Phg apresentou ligação às células VERO e à sua matriz extracelular secretada, e o soro obtido a partir desta proteína recombinante foi capaz de se ligar à superfície de leptospiras virulentas, indicando que LepA962_Phg pode representar um importante domínio de interação entre as leptospiras e seu hospedeiro. Finalmente, a proteína LepA388 pertencente a uma extensa família de proteínas modificadoras de virulência com função desconhecida (DUF_61), presente apenas nas leptospiras patogênicas mais virulentas, apresentou aumento na expressão de seu gene codificador em animais infectados por leptospiras virulentas de acordo com dados na literatura. Além disso, porções recombinantes da região Nterminal desta proteína apresentaram ligação a laminina, colágenos I e IV, vitronectina e fibronectinas plasmática e celular, principalmente considerando a sequência identificada por phage display. Estes dados reforçam as predições de modelos tridimensionais da proteína LepA388 e de outros membros da família DUF_61, as quais identificam domínios semelhantes a toxinas (como abrina e CARDS) responsáveis pela ligação e internalização celulares nos hospedeiros. Dados recentes sugerem um possível papel citotóxico desempenhado pelas proteínas desta família em leptospiras, as quais podem também ser consideradas potenciais candidatas vacinais e para diagnóstico da leptospirose, devido à sua distribuição restrita em espécies e cepas patogênicas de importância para saúde humana.

Leptospira spp. constitutes a group of gram-negative spirochete bacteria comprising saprophytic, intermediate and pathogenic species, the last being causative agents of leptospirosis, a zoonotic disease of worldwide extent and endemic in developing tropical regions. The growing number of identified leptospiral species further highlights their genetic diversity and unique virulence mechanisms, many of them with unknown function. Efforts to develop new vaccines with cross-protection and long-lasting effect have revealed possible vaccine candidates that need to be properly validated. Therefore, there is still an urgent need for a universal vaccine against leptospirosis capable of controlling and reducing the increasing outbreaks of the disease. Adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines against leptospirosis, as well as for the development of faster and more accurate diagnostic methods. Previously, three conserved hypothetical proteins in L. interrogans were identified by phage display technique, arbitrarily named as LepA069, LepA962 and LepA388. Expression of the LepA069 encoding gene showed an increase of approximately 70 % in animals infected by virulent leptospires, representing the first functional evidence of this still unknown protein. Recombinant portions of the hypothetical lipoprotein LepA962 (LepA962_Nt and LepA962_Phg) were obtained, demonstrating the strong interaction of the LepA962_Phg protein, containing the sequence identified by phage display, with laminin, plasma fibronectin, collagen I and fibrinogen in a dose-dependent manner. Furthermore, LepA962_Phg showed binding to VERO cells and its secreted extracellular matrix, and the serum obtained from this recombinant protein was able to bind to the surface of virulent leptospires, indicating that LepA962_Phg may represent an important domain of interaction between leptospires and its host. Finally, LepA388 protein belonging to an extensive family of virulence modifying proteins with unknown function (DUF_61), present only in the most virulent pathogenic leptospires, showed an increase in the expression of its encoding gene in animals infected by virulent leptospires according to data in literature. Moreover, recombinant portions of the N-terminal region of this protein showed binding to laminin, collagens I and IV, vitronectin and plasma and cell fibronectins, especially considering the sequence identified by phage display. These data support the predictions of three-dimensional models of the LepA388 protein and other members of the DUF_61 family, which identify toxin-like domains (such as abrin and CARDS) responsible for cellular binding and internalization in hosts. Recent data suggest a possible cytotoxic role played by proteins of this family in leptospires, which can also be considered potential vaccine candidates and antigens for diagnosis, due to their restricted distribution in pathogenic species and strains of importance to human health

A Novel Leptospira interrogans Protein LIC13086 Inhibits Fibrin Clot Formation and Interacts With Host Components.

Leptospirosis is a neglected zoonosis, caused by pathogenic spirochetes bacteria of the genus Leptospira. The molecular mechanisms of leptospirosis infection are complex, and it is becoming clear that leptospires express several functionally redundant proteins to invade, disseminate, and escape the host's immune response. Here, we describe a novel leptospiral protein encoded by the gene LIC13086 as an outer membrane protein. The recombinant protein LIC13086 can interact with the extracellular matrix component laminin and bind plasminogen, thus possibly participating during the adhesion process and dissemination. Also, by interacting with fibrinogen and plasma fibronectin, the protein LIC13086 probably has an inhibitory effect in the fibrin clot formation during the infection process. The newly characterized protein can also bind molecules of the complement system and the regulator C4BP and, thus, might have a role in the evasion mechanism of Leptospira. Taken together, our results suggest that the protein LIC13086 may have a multifunctional role in leptospiral pathogenesis, participating in host invasion, dissemination, and immune evasion processes.

Characterizing the transcripts of Leptospira CRISPR I-B array and its processing with endoribonuclease LinCas6.

In Leptospira interrogans serovar Copenhageni, the CRISPR-Cas I-B locus possesses a CRISPR array between the two independent cas-operons. Using the reverse transcription-PCR and the in vitro endoribonuclease assay with Cas6 of Leptospira (LinCas6), we account that the CRISPR is transcriptionally active and is conventionally processed. The LinCas6 specifically excises at one site within the synthetic cognate repeat RNA or the repeats of precursor-CRISPR RNA (pre-crRNA) in the sense direction. In contrast, the antisense repeat RNA is cleaved at multiple sites. LinCas6 functions as a single turnover endoribonuclease on its repeat RNA substrate, where substitution of one of predicted active site residues (His38) resulted in reduced activity. This study highlights the comprehensive understanding of the Leptospira CRISPR array transcription and its processing by LinCas6 that is central to RNA-mediated CRISPR-Cas I-B adaptive immunity.

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans.

In bacteria, DNA methylation can be facilitated by 'orphan' DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.

AAA+ Molecular Chaperone ClpB in Leptospira interrogans: Its Role and Significance in Leptospiral Virulence and Pathogenesis of Leptospirosis.

Bacterial ClpB is an ATP-dependent disaggregase that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and cooperates with the DnaK chaperone system in the reactivation of aggregated proteins, as well as promotes bacterial survival under adverse environmental conditions, including thermal and oxidative stresses. In addition, extensive evidence indicates that ClpB supports the virulence of numerous bacteria, including pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in animals and humans. However, the specific function of ClpB in leptospiral virulence still remains to be fully elucidated. Interestingly, ClpB was predicted as one of the L. interrogans hub proteins interacting with human proteins, and pathogen-host protein interactions are fundamental for successful invasion of the host immune system by bacteria. The aim of this review is to discuss the most important aspects of ClpB's function in L. interrogans, including contribution of ClpB to leptospiral virulence and pathogenesis of leptospirosis, a zoonotic disease with a significant impact on public health worldwide.