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.

Leptospiral LipL45 lipoprotein undergoes processing and shares structural similarities with bacterial sigma regulators.

Leptospirosis is a widespread zoonotic infectious disease of human and veterinary concern caused by pathogenic spirochetes of the genus Leptospira. To date, little progress towards understanding leptospiral pathogenesis and identification of virulence factors has been made, which is the main bottleneck for developing effective measures against the disease. Some leptospiral proteins, including LipL32, Lig proteins, LipL45, and LipL21, are being considered as potential virulence factors or vaccine candidates. However, their function remains to be established. LipL45 is the most expressed membrane lipoprotein in leptospires, upregulated when the bacteria are transferred to temperatures resembling the host, expressed during infection, suppressed after culture attenuation, and known to suffer processing in vivo and in vitro, generating fragments. Based on body of evidence, we hypothesized that the LipL45 processing might occur by an auto-cleavage event, deriving two fragments. The results presented here, based on bioinformatics, structure modeling analysis, and experimental data, corroborate that LipL45 processing probably includes a self-catalyzed non-proteolytic event and suggest the participation of LipL45 in cell-surface signaling pathways, as the protein shares structural similarities with bacterial sigma regulators. Our data indicate that LipL45 might play an important role in response to environmental conditions, with possible function in the adaptation to the host.

Generation of an RNA aptamer against LipL32 of Leptospira isolated by Tripartite-hybrid SELEX coupled with in-house Python-aided unbiased data sorting.

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira. The major hurdle of the diagnosis of Leptospirosis lies in the issues associated with current methods of detection, which are time-consuming, tedious and the need for sophisticated, special equipments. Restrategizing the diagnostics of Leptospirosis may involve considerations of the direct detection of the outer membrane protein, which can be faster, cost-saving and require fewer equipments. One such promising marker is LipL32, which is an antigen with high amino acid sequence conservation among all the pathogenic strains. In this study, we endeavored to isolate an aptamer against LipL32 protein via a modified SELEX strategy known as tripartite-hybrid SELEX, based on 3 different partitioning strategies. In this study, we also demonstrated the deconvolution of the candidate aptamers by using in-house Python-aided unbiased data sorting in examining multiple parameters to isolate potent aptamers. We have successfully generated an RNA aptamer against LipL32 of Leptospira, LepRapt-11, which is applicable in a simple direct ELASA for the detection of LipL32. LepRapt-11 can be a promising molecular recognition element for the diagnosis of leptospirosis by targeting LipL32.

Leptospiral LPS escapes mouse TLR4 internalization and TRIF­associated antimicrobial responses through O antigen and associated lipoproteins.

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.

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.

Transcriptomic signatures of exacerbated progression in leptospirosis subclinical chronic kidney disease with secondary nephrotoxic injury.

High-incidence regions of leptospirosis caused by Leptospira spp. coincide with chronic kidney disease. This study investigated whether asymptomatic leptospirosis is an emerging culprit that predisposes to progressive chronic kidney disease when superimposed on secondary nephrotoxic injury. Kidney histology/function and whole transcriptomic profiles were evaluated for Leptospira-infected C57/BL6 mice with adenine-induced kidney injury. The extent of tubulointerstitial kidney lesions and expression of inflammation/fibrosis genes in infected mice with low-dose (0.1%) adenine, particularly in high-dose (0.2%) adenine-fed superimposed on Leptospira-infected mice, were significantly increased compared with mice following infection or adenine diet alone, and the findings are consistent with renal transcriptome analysis. Pathway enrichment findings showed that integrin-ß- and fibronectin-encoding genes had distinct expression within the integrin-linked kinase-signaling pathway, which were upregulated in 0.2% adenine-fed Leptospira-infected mice but not in 0.2% adenine-fed mice, indicating that background subclinical Leptospiral infection indeed enhanced subsequent secondary nephrotoxic kidney injury and potential pathogenic molecules associated with secondary nephrotoxic leptospirosis. Comparative analysis of gene expression patterns with unilateral ureteric obstruction-induced mouse renal fibrosis and patients with chronic kidney disease showed that differentially expressed orthologous genes such as hemoglobin-α2, PDZ-binding kinase, and DNA topoisomerase II-α were identified in infected mice fed with low-dose and high-dose adenine, respectively, revealing differentially expressed signatures identical to those found in the datasets and may serve as markers of aggravated kidney progression. This study indicates that background subclinical leptospirosis, when subjected to various degrees of subsequent secondary nephrotoxic injury, may predispose to exacerbated fibrosis, mimicking the pathophysiological process of progressive chronic kidney disease.NEW & NOTEWORTHYLeptospira-infected mice followed by secondary nephrotoxic injury exacerbated immune/inflammatory responses and renal fibrosis. Comparison with the murine model revealed candidates involved in the progression of renal fibrosis in chronic kidney disease (CKD). Comparative transcriptome study suggests that secondary nephrotoxic injury in Leptospira-infected mice recapitulates the gene expression signatures found in CKD patients. This study indicates that secondary nephrotoxic injury may exacerbate CKD in chronic Leptospira infection implicating in the progression of CKD of unknown etiology.

Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection.

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with Lactobacillus plantarum confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train ex vivo human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis.

Crystal structure of Leptospira leucine-rich repeat 20 reveals a novel E-cadherin binding protein to induce NGAL expression in HK2 cells.

Leptospirosis is the most common zoonotic disease caused by pathogenic Leptospira, which is classified into three groups according to virulence. Its pathogenic and intermediate species contain leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic strains. In this study, we presented the crystal structure of LSS_11580 (rLRR20) from pathogenic L. santarosai serovar Shermani. X-ray diffraction at a resolution of 1.99 Šrevealed a horseshoe-shaped structure containing seven α-helices and five ß-sheets. Affinity assays indicated that rLRR20 interacts with E-cadherin on the cell surface. Interestingly, its binds to the extracellular (EC) 1 domain in human epithelial (E)-cadherin, which is responsible for binding to another E-cadherin molecule in neighboring cells. Several charged residues on the concave face of LRR20 were predicted to interact with EC1 domain. In the affinity assays, these charged residues were replaced by alanine, and their affinities to E-cadherin were measured. Three vital residues and mutation variants of LRR20, namely D56A, E59A, and E123A, demonstrated significantly reduced affinity to E-cadherin compared with the control. Besides, we also demonstrated that rLRR20 induced the expression of neutrophil gelatinase-associated lipocalin (NGAL) in HK2 cells. The low ability of the three mutation variants to induce NGAL expression further demonstrates this induction. The present findings indicate that LRR20 from pathogenic Leptospira binds to E-cadherin and interacts with its EC1 domain. In addition, its induction of NGAL expression in HK2 cells is associated with acute kidney injury in human.

Crosstalk between E-Cadherin/ß-Catenin and NF-κB Signaling Pathways: The Regulation of Host-Pathogen Interaction during Leptospirosis.

Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor ß-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/ß-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.

Chaperonomics in leptospirosis.

INTRODUCTION: Knowledge of the function of molecular chaperones is required for a better understanding of cellular proteostasis. Nevertheless, such information is currently dispersed as most of previous studies investigated chaperones on a single-angle basis. Recently, a new subdiscipline of chaperonology, namely 'chaperonomics' (defined as 'systematic analysis of chaperone genes, transcripts, proteins, or their interaction networks using omics technologies'), has been emerging to better understand biological, physiological, and pathological roles of chaperones. Areas covered: This review provides broad overviews of bacterial chaperones, heat shock proteins (HSPs), and leptospirosis, and then focuses on recent progress of chaperonomics applied to define roles of HSPs in various pathogenic and saprophytic leptospiral species and serovars. Expert commentary: Comprehensive analysis of leptospiral chaperones/HSPs using a chaperonomics approach holds great promise for better understanding of functional roles of chaperones/HSPs in bacterial survival and disease pathogenesis. Moreover, this new approach may also lead to further development of chaperones/HSPs-based diagnostics and/or vaccine discovery for leptospirosis.

Characterizing interactions of Leptospira interrogans with proximal renal tubule epithelial cells.

BACKGROUND: Leptospira interrogans is a pathogenic, spirochetal bacterium that is responsible for leptospirosis, an emerging worldwide zoonosis. Leptospires colonize the renal proximal tubules and chronically infect the kidney. Live bacteria are excreted into urine, contaminating the environment. While it is well known that leptospires can persist in the kidneys without signs of disease for several months, the interactions of leptospires with the proximal renal epithelial tubule cells that allow the chronic renal colonization have not been elucidated yet. In the present study, we compared the interactions between a virulent, low passage (LP) strain and a cultured-attenuated, high passage (HP) strain with renal proximal tubule epithelial cells (RPTECs) to elucidate the strategies used by Leptospira to colonize the kidney. RESULTS: Kinetics analysis of kidney colonization in a mouse model of chronic infection performed by quantitative real-time PCR and immunofluorescence, showed that the LP strain reached the kidney by 3 days post infection (pi) and attached to the basal membrane side of the renal epithelial cells. At 10 days pi, some leptospires were attached to the luminal side of the tubular epithelia and the number of colonizing leptospires gradually increased. On the other hand, the HP strain was cleared during hematogenous dissemination and did not colonize the kidney. Transmission electron microscopy analysis of LP-infected kidneys at 25 days pi showed aggregated leptospires and membrane vesicles attached to the epithelial brush border. Leptospiral kidney colonization altered the organization of the RPTEC brush border. An in vitro model of infection using TCMK-1 cells, showed that leptospiral infection induced a host stress response, which is delayed in LP-infected cells. CONCLUSIONS: After hematogenous dissemination, leptospires create protective and replicative niches in the base membrane and luminal sides of the RPTECs. During the long-term colonization, leptospires attached to the RPTEC brush borders and membrane vesicles might be involved in the formation of a biofilm-like structure in vivo. Our results also suggested that the virulent strain is able to manipulate host cell stress responses to promote renal colonization.

Mammalian cell entry (Mce) protein of Leptospira interrogans binds extracellular matrix components, plasminogen and ß2 integrin.

A severe re-emergingzoonosis, leptospirosis, is caused by pathogenic spirochetes of the genus Leptospira. Several studies have identified leptospiral surface proteins with the ability to bind ECM and plasma components, which could mediate adhesion and invasion through the hosts. It has been shown that Mce of pathogenic Leptospira spp. is an RGD (Arg-Gly-Asp)-motif-dependent virulence factor, responsible for infection of cells and animals. In the present article, we decided to further study the repertoire of the Mce activities in leptospiral biological properties. We report that the recombinant Mce is a broad-spectrum ECM-binding protein, capable of interacting with laminin, cellular and plasma fibronectin and collagen IV. Dose--r-esponse interaction was observed for all the components, fulfilling ligand--receptor requirements. Mce is a PLG binding protein capable to recruit this component from NHS, generating PLA in the presence of PLG activator. Binding of Mce was also observed with the leukocyte cell receptors αLß2 [(CD11a/CD18)-LFA-1] and αMß2 [(CD11b/CD18)-Mac-1], suggesting the involvement of this protein in the host immune response. Indeed, virulent Leptospira L1-130 was capable of binding both integrins, whereas culture-attenuated M-20 strain only bind to αMß2 [(CD11b/CD18)-Mac-1]. To the best of our knowledge, this is the first work to describe that Mce surface protein could mediate the attachment of Leptospira interrogans to human cell receptors αLß2(CD11a/CD18) and αMß2(CD11b/CD18).

Outbreak of leptospirosis during a scout camp in the Luxembourg Belgian province, Belgium, summer 2012.

An outbreak of leptospirosis occurred in the South of Belgium, during August 2012, in teenagers who participated in two consecutive adventure scout camps near the Semois river. Among the symptomatic patient population (ten scouts), clinical manifestations included headache (70%), myalgia (50%), fever (50%), bilateral conjunctival injection (50%), general malaise (30%), vomiting (20%), anorexia (20%) and cough (20%). Some of the cases presented elevated blood creatinine (40%), or proteinuria (30%). Three patients were confirmed by serology and one by polymerase chain reaction. Potential risk factors included direct contact with a muskrat and indirect contact with potentially contaminated environments including the river water. Prospective environmental investigation carried out near the river banks 2 weeks after the outbreak identified Ondatra zibethicus (muskrat) as one Leptospira sp. reservoir.

Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway.

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease.

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection.

Downregulation of the Na/K-ATPase pump by leptospiral glycolipoprotein activates the NLRP3 inflammasome.

Leptospira interrogans is responsible for a zoonotic disease known to induce severe kidney dysfunction and inflammation. In this work, we demonstrate that L. interrogans induces NLRP3 inflammasome-dependent secretion of IL-1ß through the alteration of potassium transport in bone marrow-derived macrophages. Lysosome destabilization also contributed to the IL-1ß production upon stimulation with live, but not dead, bacteria. Using bone marrow-derived macrophages from various TLRs and nucleotide-binding oligomerization domain-deficient mice, we further determined that IL-1ß production was dependent on TLR2 and TLR4, suggesting a participation of the leptospiral LPS to this process. Hypokaliemia in leptospirosis has been linked to the presence of glycolipoprotein, a cell wall component of L. interrogans that is known to inhibit the expression and functions of the Na/K-ATPase pump. We show in this study that glycolipoprotein activates the inflammasome and synergizes with leptospiral LPS to produce IL-1ß, mimicking the effect of whole bacteria. These results were confirmed in vivo, as wild-type mice expressed more IL-1ß in the kidney than TLR2/4-deficient mice 3 d postinfection with L. interrogans. Collectively, these findings provide the first characterization, to our knowledge, of bacteria-induced activation of the NLRP3 inflammasome through the downregulation of a specific host potassium transporter.

Interaction of Leptospira interrogans with human proteolytic systems enhances dissemination through endothelial cells and protease levels.

We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date.

Impaired functions of human monocyte-derived dendritic cells and induction of regulatory T cells by pathogenic Leptospira.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. The disease outcome is influenced by the interplay between innate and adaptive immune responses. Dendritic cells (DCs) play a crucial role in shaping the adaptive immune response. A recent study revealed that pathogenic Leptospira limited the activation of human monocyte-derived dendritic cells (MoDCs) compared to non-pathogenic Leptospira, but their impact on T-cell responses has not been investigated. Our study is the first to explore how viable pathogenic and non-pathogenic Leptospira affect the interaction between human MoDCs and T cells. We found that MoDCs infected with pathogenic leptospires (L. interrogans serovar Pomona and a clinical isolate, MoDCs-P) exhibited lower levels of CD80 and CD83 expression, suggesting partially impaired MoDC maturation, induced regulatory T cells (Tregs) while failing to induce CD4+ T cell proliferation, compared to MoDCs infected with non-pathogenic leptospires (L. biflexa serovar Patoc and L. meyeri serovar Ranarum, MoDCs-NP). In contrast, non-pathogenic leptospires enhanced MoDC maturation and induced higher T cell proliferation including IFN-γ-producing CD4+ T cells, indicative of a Th1-type response. Furthermore, pathogenic leptospires induced higher MoDC apoptosis through a cysteine aspartic acid-specific protease-3 (caspase-3)-dependent pathway and upregulated expression of the prostaglandin-endoperoxide synthase 2 (PTGS2) gene. Notably, prostaglandin E2 (PGE2), a product of the PTGS2 pathway, was found at higher levels in the sera of patients with acute leptospirosis and in the supernatant of MoDCs-P, possibly contributing to Treg induction, compared to those of healthy donors and MoDCs-NP, respectively. In conclusion, this study reveals a novel immunosuppressive strategy employed by pathogenic Leptospira to evade host immunity by partially impairing MoDC maturation and inducing Tregs. These findings deepen our understanding of leptospirosis pathogenesis in humans and may provide a novel strategy to modulate DCs for the prevention and treatment of the disease.

Crystal structure of Leptospira LSS_01692 reveals a dimeric structure and induces inflammatory responses through Toll-like receptor 2-dependent NF-κB and MAPK signal transduction pathways.

Leptospirosis is a commonly overlooked zoonotic disease that occurs in tropical and subtropical regions. Recent studies have divided the Leptospira spp. into three groups based on virulence, including pathogenic, intermediate, and saprophytic species. Pathogenic species express a protein family with leucine-rich repeat (LRR) domains, which are less expressed or absent in nonpathogenic species, highlighting the importance of this protein family in leptospirosis. However, the role of LRR domain proteins in the pathogenesis of leptospirosis is still unknown and requires further investigation. In this study, the 3D structure of LSS_01692 (rLRR38) was obtained using X-ray crystallography at a resolution of 3.2 Å. The results showed that rLRR38 forms a typical horseshoe structure with 11 α-helices and 11 ß-sheets and an antiparallel dimeric structure. The interactions of rLRR38 with extracellular matrix and cell surface receptors were evaluated using ELISA and single-molecule atomic force microscopy. The results showed that rLRR38 interacted with fibronectin, collagen IV, and Toll-like receptor 2 (TLR2). Incubating HK2 cells with rLRR38 induced two downstream inflammation responses (IL-6 and MCP-1) in the TLR2 signal transduction pathway. The TLR2-TLR1 complex showed the most significant upregulation effects under rLRR38 treatment. Inhibitors also significantly inhibited nuclear factor κB and mitogen-activated protein kinases signals transduction under rLRR38 stimulation. In conclusion, rLRR38 was determined to be a novel LRR domain protein in 3D structure and demonstrated as a TLR2-binding protein that induces inflammatory responses. These structural and functional studies provide a deeper understanding of the pathogenesis of leptospirosis.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system.