Search

Background The life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are currently circulating worldwide due to the fastidious nature of the bacteria and the difficulty to isolate cultures. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Our understanding of the genetic diversity of strains during human infection is therefore limited. Methods Here, we carried out hybridization capture followed by Illumina sequencing of the core genome directly from 20 clinical samples that were PCR positive for pathogenic Leptospira to elucidate the genetic diversity of currently circulating Leptospira strains in mainland France. Results Capture with RNA probes covering the L. interrogans core genome resulted in a 72 to 13,000-fold increase in pathogen reads relative to standard sequencing without capture. Variant analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genomes was then carried out to determine the genotype of the infecting strain. For samples with sufficient coverage (19/20 samples with coverage > 8×), we could unambiguously identify L. interrogans serovars Icterohaemorrhagiae and Copenhageni (14 samples), L. kirschneri serovar Grippotyphosa (4 samples), and L. interrogans serovar Pyrogenes (1 sample) as the infecting strains. Conclusions We obtained high-quality genomic data with suitable coverage for confident core genome genotyping of the agent of leptospirosis for most of our clinical samples. The recovery of the genome of the serovars Icterohaemorrhagiae and Copenhageni directly from multiple clinical samples revealed low adaptive diversification of the core genes during human infection. The ability to generate culture-free genomic data opens new opportunities for better understanding of the epidemiology of this fastidious pathogen and pathogenesis of this neglected disease.

International audience; Reactive oxygen species (ROS) are byproducts of oxygen metabolism produced by virtually all organisms living in an oxic environment. ROS are also produced by phagocytic cells in response to microorganism invasion. These highly reactive molecules can damage cellular constituents (proteins, DNA, and lipids) and exhibit antimicrobial activities when present in sufficient amount. Consequently, microorganisms have evolved defense mechanisms to counteract ROS-induced oxidative damage. Leptospira are diderm bacteria form the Spirochaetes phylum. This genus is diverse, encompassing both free-living non-pathogenic bacteria as well as pathogenic species responsible for leptospirosis, a widespread zoonotic disease. All leptospires are exposed to ROS in the environment, but only pathogenic species are well-equipped to sustain the oxidative stress encountered inside their hosts during infection. Importantly, this ability plays a pivotal role in Leptospira virulence. In this review, we describe the ROS encountered by Leptospira in their different ecological niches and outline the repertoire of defense mechanisms identified so far in these bacteria to scavenge deadly ROS. We also review the mechanisms controlling the expression of these antioxidants systems and recent advances in understanding the contribution of Peroxide Stress Regulators in Leptospira adaptation to oxidative stress.

International audience; Spirochaetes, a phylum that includes medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, are in many ways highly unique bacteria. Their cell morphology, subcellular organization, and metabolism reveal atypical features. Spirochetal motility is also singular, dependent on the presence of periplasmic flagella or endoflagella, inserted subterminally at cell poles and not penetrating the outer membrane and elongating outside the cell as in enterobacteria. In this review we present a comprehensive comparative genomics analysis of endoflagellar systems in spirochetes, highlighting recent findings on the flagellar basal body and filament. Continued progress in understanding the function and architecture of spirochetal flagella is uncovering paradigm-shifting mechanisms of bacterial motility.

International audience; Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG- mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG- mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG- and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG- mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.

Leptospira interrogansare pathogenic bacteria responsible for leptospirosis, a zoonosis impacting 1 million peopleperyear worldwide. Leptospires can infect all vertebrates, but not all hosts develop similar symptoms. Human and cattle may suffer from mild to acute illnesses and are therefore considered as sensitive to leptospirosis. In contrast, mice and rats remain asymptomatic upon infection, although they get chronically colonized in their kidneys. Upon infection, leptospires are stealth pathogens that partially escape the recognition by the host innate immune system. Although leptospires are mainly extracellular bacteria, it was suggested that they could also replicate within macrophages. However, contradictory data in the current literature led us to reevaluate these findings. Using a gentamicin–protection assay coupled to high-content (HC) microscopy, we observed that leptospires were internalizedin vivoupon peritoneal infection of C57BL/6J mice. Additionally, three different serotypes of pathogenicL. interrogansand the saprophyticL. biflexaactively infected both human (PMA differentiated) THP1 and mouse RAW264.7 macrophage cell lines. Next, we assessed the intracellular fate of leptospires using bioluminescent strains, and we observed a drastic reduction in the leptospiral intracellular load between 3 h and 6 h post-infection, suggesting that leptospires do not replicate within these cells. Surprisingly, the classical macrophage microbicidal mechanisms (phagocytosis, autophagy, TLR–mediated ROS, and RNS production) were not responsible for the observed decrease. Finally, we demonstrated that the reduction in the intracellular load was associated with an increase of the bacteria in the supernatant, suggesting that leptospires exit both human and murine macrophages. Overall, our study reevaluated the intracellular fate of leptospires and favors an active entrance followed by a rapid exit, suggesting that leptospires do not have an intracellular lifestyle in macrophages.

Leptospira strains were isolated from freshwater sampled at four sites in Algeria and characterized by whole-genome sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The cells were spiral-shaped and motile. Phylogenetic and MALDI-TOF MS analyses showed that the strains can be clearly distinguished from the other described species in the genus Leptospir a, therefore representing two novel species of the pathogen subclade P1 and two novel species of the saprophyte subclade S1. The names Leptospira ainlahdjerensis sp. nov. (type strain 201903070T=KIT0297T=CIP111912T), Leptospira ainazelensis sp. nov. (201903071T=KIT0298T=CIP111913T), Leptospira abararensis sp. nov. (201903074T=KIT0299T=CIP111914T) and Leptospira chreensis (201903075T=KIT0300T=CIP111915T) are proposed.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.Author summaryLeptospirosis is a widespread infectious disease responsible for over one million of severe cases and 60 000 fatalities annually worldwide. This neglected and emerging disease has a worldwide distribution, but it mostly affects populations from developing countries in sub-tropical areas. The causative agents of leptospirosis are pathogenic bacterial Leptospira spp. There is a considerable deficit in our knowledge of these atypical bacteria, including their virulence mechanisms. In addition to the Leptospira PerRA regulator that represses defenses against peroxide, we have identified and characterized a second PerR regulator in pathogenic Leptospira species (PerRB) that participates in Leptospira tolerance to superoxide. Phenotypic and transcriptomic analyses of single PerRA and PerRB mutants suggest that the two PerRs fulfill distinct functions in the adaptation to oxidative stress. Concomitant inactivation of PerRA and PerRB resulted in a higher tolerance to both peroxide and superoxide. Moreover, the perRAperRB mutant lost its virulence. Major changes in gene expression, including a decreased expression of several virulence factors, were observed in the double perRAperRB mutant. Our study suggests that PerRA and PerRB cooperate to orchestrate a complex regulatory network involved in Leptospira virulence.

Background Although Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease. Methodology/Principal findings We used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017. Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains. Conclusions/Significance The present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data., Author summary Pathogenic Leptospira are the causative agents for leptospirosis, a neglected and emerging zoonosis occurring worldwide. In this study, we investigated the genetic diversity of Leptospira strains isolated from patients over a 12-year period in Lao PDR. Our genome analysis revealed a high diversity of Leptospira genotypes in this endemic country for leptospirosis, including a group of clonal strains which was responsible for a large outbreak in neighboring Thailand. Further progress in our understanding of the epidemiology of Leptospira circulating genotypes should contribute to the implementation of prevention and intervention measures to reduce the risk of leptospirosis transmission.

Leptospirabacteria comprise numerous species, several of which cause serious disease to a broad range of hosts including humans. These spirochetes exhibit large intraspecific variation, resulting in complex tabulations of serogroups/serovars that crisscross the species classification. Serovar identity, linked to biological/clinical phenotypes, depends on the structure of surface-exposed LPS. Many LPS biosynthesis–encoding genes reside within the chromosomicrfbgene cluster. However, the genetic basis of intraspecies variability is not fully understood, constraining diagnostics/typing methods to cumbersome serologic procedures. We now show that the gene content of therfbcluster strongly correlates withLeptospiraserovar designation. Whole-genome sequencing of pathogenicL. noguchii, including strains of different serogroups, reveals that therfbcluster undergoes extensive horizontal gene transfer. Therfbclusters from severalLeptospiraspecies disclose a univocal correspondence between gene composition and serovar identity. This work paves the way to genetic typing ofLeptospiraserovars, and to pinpointing specific genes within the distinctrfbclusters, encoding host-specific virulence traits. Further research shall unveil the molecular mechanism ofrfbtransfer amongLeptospirastrains and species.

International audience; Bacterial zoonotic diseases such as leptospirosis, Q fever, melioidosis, spotted fever group rickettsioses, and brucellosis are increasingly recognized causes of non-malaria acute fevers. However, though readily treatable with antibiotics, these diseases are commonly misdiagnosed resulting in poor outcomes in patients. There is a considerable deficit in the understanding of basic aspects of the epidemiology of these neglected diseases and diagnostic tests for these zoonotic bacterial pathogens are not always available in resource-poor settings. Raising awareness about these emerging bacterial zoonoses is directly beneficial to the patients by allowing a test-and-treat approach and is essential to control these life-threatening diseases.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverish populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans . Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans , the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, to the loss of Leptospira virulence. Interestingly, this correlated with major changes in gene and non-coding RNA expression, only observed in the double perRAperRB mutant. Notably, several virulence-associated genes ( clpB , ligA/B , and lvrAB ) were repressed. By obtaining the first double mutant in a pathogenic Leptospira strain, our study has uncovered for the first time the interplay of two PerRs, not only in the adaptation of Leptospira to oxidative stress, but also in their virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.

Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira. Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira, allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes.

Leptospira dzianensis and Leptospira putramalaysiae were recently described as novel species and published almost concurrently with Leptospira yasudae and Leptospira stimsonii . Genome comparisons based on average nucleotide identity of the type strain genomes indicate that L. dzianensis and L. putramalaysiae are conspecific with L. yasudae and L. stimsonii , respectively. Based on the rules of priority, L. dzianensis should be reclassified as a later heterotypic synonym of L. yasudae , and L. putramalaysiae should be reclassified as a later heterotypic synonym of L. stimsonii .

International audience; Leptospirosis is a waterborne zoonosis (60,000 infections and 1 million deaths annually). Knowledge about the disease in the urban context is surprisingly rare, especially in Africa. Here, we provide the first study of leptospires in waters within an African city. A simple centrifugation-based method was developed to screen waterborne leptospires from remote or poorly areas. Major ions, trace elements, stable isotopes and pathogenic Leptospira were then seasonally investigated in 193 water samples from three neighborhoods of Cotonou (Benin) with different socio-environmental and hydrographic characteristics. Firstly, no leptospire was detected in tap waters. Secondly, although surface contamination cannot be excluded, one groundwater well was found leptospire positive. Thirdly, pathogenic Leptospira mainly contaminated surface waters of temporary and permanent ponds (9.5% and 27.3% of total prevalence, respectively). Isotopic signatures suggest that leptospires occurred in pond waters formed at the beginning of the rainy season following low to moderate rainfall events. Nevertheless, Leptospira-containing waters possess physico-chemical characteristics that are similar to the spectrum of waters sampled throughout the three sites, thus suggesting that Cotonou waters are widely compatible with Leptospira survival. The frequent contact with water exposes Cotonou inhabitants to the risk of leptospirosis which deserves more attention from public health authorities.

Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains.

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.

Background Leptospirosis is a widespread zoonosis with global impact, particularly among vulnerable populations in resource-poor settings in tropical countries. Rodents have been considered to be the main reservoir of the disease; however, a wide variety of mammals can act as hosts as well. Here we examine the genetic diversity of Leptospira strains from biological samples of patients and animals in French Polynesia (FP) from 2011 to 2019. Methodology/Principal findings From 2011 to 2019, we have collected 444 blood samples from patients diagnosed as having leptospirosis. The limited volume of clinical material and low amount of leptospiral DNA in blood samples led us to develop a nested PCR targeting the secY locus that enabled us to amplify and sequence 244 samples (55%). In addition, 20 Leptospira strains recovered from the blood of patients from 2002 to 2011 were sequenced and fully characterized at the serogroup level and used as reference strains for the association of different phylogenetic branches with respective serogroups. The secY sequences were compared with publicly available sequences from patients and animal reservoirs in FP (n = 79). We identified rats as the main source of infection for L. borgpetersenii serogroup Ballum and L. interrogans serogroup Icterohaemorrhagiae, dogs as the main source of infection for L. interrogans serogroup Australis, and farm pigs as the main source of infection for L. interrogans serogroups Pomona or Canicola. L. interrogans was associated with the most severe infections with 10 and 5 fatal cases due to serogroups Icterohaemorrhagiae and Australis, respectively. Mortality was significantly associated with older age (p-value < 0.001). Conclusions/Significance We described the population dynamics of leptospires circulating among patients in FP, including two patients who were reinfected with unrelated Leptospira genotypes, and clarified the local role of the animal reservoirs in the transmission route of leptospirosis to humans. Routine Leptospira genotyping directly on biological samples should allow the epidemiological follow-up of circulating strains and assess the impact of control interventions on disease transmission., Author summary Pathogenic Leptospira are the causative agents for leptospirosis, a neglected zoonosis occurring worldwide with the highest incidence of the disease found in the Pacific region, including French Polynesia (FP). In this study, a nested PCR of blood specimens allowed a high isolation frequency of Leptospira DNA among patients with febrile illness in FP. Infecting Leptospira isolates were characterized by sequencing of the secY gene from 244 clinical samples from 2011 to 2019. The genotypes have been assigned to Leptospira species and serogroups by comparing the secY sequences with the genomes of strains isolated prior to 2011 in FP. This study therefore enables to follow the distribution of genotypes/serogroups over a 18-year time period. Leptospiral sequences from patients were also compared with secY sequences from different animal reservoirs from a previous study in FP showing that not only rats but also dogs and pigs might play an important role in the circulation of pathogenic Leptospira strains in FP. Further progress in our understanding of the epidemiology of Leptospira circulating genotypes should contribute to the implementation of prevention and intervention measures to reduce the risk of leptospirosis transmission in FP.

Leptospira (L.) interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagellae that allow their motility. L. interrogans are stealth pathogens that escape the innate immune recognition of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, which senses peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by qPCR. We did not identify any protective or inflammatory role of murine TLR5 for controlling pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5 signaling. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, but did not, or barely induced stimulation via murine TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, after using antimicrobial peptides to destabilize live leptospires, we detected TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in the flagellin proteins, we further showed that neither FlaA nor Fcp participated in the recognition by TLR5, suggesting a role for the FlaB. FlaB have structural homology to Salmonella FliC, and possess conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaB, but not the FlaA subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagellae, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape the TLR5 recognition and the induced immune response.

Leptospirosis is a ubiquitous zoonotic disease and a major clinical challenge owing to the multitude of clinical presentations and manifestations that are possibly attributable to the diversity of Leptospira, the understanding of which is key to study the epidemiology of this emerging global disease threat. Sri Lanka is a hotspot for leptospirosis with high levels of endemicity as well as annual epidemics. We carried out a prospective study of Leptospira diversity in Sri Lanka, covering the full range of climatic zones, geography, and clinical severity. Samples were collected for leptospiral culture from 1,192 patients from 15 of 25 districts in Sri Lanka over two and half years. Twenty-five isolates belonging to four pathogenic Leptospira species were identified: L. interrogans, L. borgpetersenii, L. weilii, and L. kirschneri. At least six serogroups were identified among the isolates: Autumnalis (6), Pyrogenes (4), Icterohaemorrhagiae (2), Celledoni (1), Grippotyphosa (2) and Bataviae (1). Seven isolates did not agglutinate using available antisera panels, suggesting new serogroups. Isolates were sequenced using an Illumina platform. These data add 25 new core genome sequence types and were clustered in 15 clonal groups, including 12 new clonal groups. L. borgpetersenii was found only in the dry zone and L. weilii only in the wet zone. Acute kidney injury and cardiovascular involvement were seen only with L. interrogans infections. Thrombocytopenia and liver impairment were seen in both L. interrogans and L. borgpetersenii infections. The inadequate sensitivity of culture isolation to identify infecting Leptospira species underscores the need for culture-independent typing methods for Leptospira., Author summary There is a huge diversity in pathogenic Leptospira species worldwide, and our knowledge of the currently circulating species is deficient owing to limited isolation and identification of Leptospira species from endemic countries. This prospective study reveals the wide pathogen diversity that causes human leptospirosis in Sri Lanka, representing four species, more than six serogroups, and fifteen clonal groups. Further, the different geographic and climatic zone distributions and clinical manifestations observed underscores the need for prospective studies to expand the molecular epidemiological approaches to combat leptospirosis.

International audience; Leptospira spp. are morphologically and phylogenetically unique Bacteria that are ubiquitous in the environment. Pathogenic species are the agents of leptospirosis, an emerging zoonotic disease transmitted to both human and animals from the environment contaminated with the urine of reservoir animals. The taxonomy of Leptospira has recently undergone extensive revisions with the use of whole-genome sequences, thus expanding considerably the number of species.

Cite this protocol as:Grillová L., Picardeau M. (2020) Core Genome Multi-locus Sequence Typing Analyses of Leptospira spp. Using the Bacterial Isolate Genome Sequence Database. In: Koizumi N., Picardeau M. (eds) Leptospira spp.. Methods in Molecular Biology, vol 2134. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0459-5_2; International audience; With the advent of whole-genome sequencing (WGS), comparative analysis has led to the use of core genome MLST (cgMLST) schemes for the high-resolution reproducible typing of bacterial isolates. In cgMLST, hundreds of loci are used for gene-by-gene comparisons of assembled genomes for studying the genetic diversity of clinically important pathogens. Combination of the cgMLST data and metadata of the isolates is useful for epidemiological investigations.Here we present a cgMLST scheme for the high-resolution typing of isolates from the whole Leptospira genus, enabling identification at the level of species, clades, clonal groups, and sequence types. We show several examples how the cgMLST Leptospira database, which is a publicly available web-based database, can be used for the analyses of WGS data of Leptospira isolates. This effort was undertaken in order to facilitate international collaborations and support the global surveillance of leptospirosis.

Cite this protocol as:Schiettekatte O., Bourhy P. (2020) Isolation, Purification, and Characterization of Leptophages. In: Koizumi N., Picardeau M. (eds) Leptospira spp.. Methods in Molecular Biology, vol 2134. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0459-5_7; International audience; To date, only three bacteriophages of leptospires-leptophages-are known. Nonetheless, numerous prophages have been found in the genus, especially in the genomes of pathogenic species. Thus, some laboratories attempt to isolate leptophage particles from environmental samples or following mitomycin C induction of bacterial cultures. Here, we propose multiple procedures to isolate, purify, and characterize bacteriophages, based on protocols used for LE3 and LE4 characterization.

Cite this protocol as:Zavala-Alvarado C., Benaroudj N. (2020) The Single-Step Method of RNA Purification Applied to Leptospira. In: Koizumi N., Picardeau M. (eds) Leptospira spp.. Methods in Molecular Biology, vol 2134. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0459-5_5; International audience; Establishing a rapid method to obtain pure and intact RNA molecules has revolutionized the field of RNA biology, enabling laboratories to routinely perform RNA analysis such as Northern blot, reverse transcriptase quantitative PCR and RNA sequencing. Here, we describe an application of the effective single-step method of RNA extraction (or guanidinium thiocyanate-phenol-chloroform extraction) applied to Leptospira species. This method is based on the powerful ability of guanidinium thiocyanate to inactivate RNases and on the different solubility of RNA and DNA in acidic phenol. This method allows one to reproducibly obtain total RNAs with high yield and integrity, as determined by capillary electrophoresis, suitable for the RNA sequencing technology.

International audience; Measuring viability is an important and necessary assessment in studying microorganisms. Several methods can be applied to Leptospira spp, each with advantages and inconveniencies. Here, we describe the traditional colony-forming unit method, together with two other methods based respectively on the reducing capacity of live cells (Alamar Blue Assay) and differential staining of live and dead cells (LIVE/DEAD BacLight). The Alamar Blue Assay uses the blue reagent resazurin, which can be reduced into the pink reagent resorufin by live cell oxidoreductases. Production of resorufin can be quantified by absorbance or fluorescence reading. The LIVE/DEAD BacLight assay uses a mixture of two nucleic acid dyes (Syto9 and propidium iodide) that differentially penetrate and stain nucleic acid of cells with decreased membrane integrity. The colony-forming unit method is labor-intensive but the most sensitive and linear method. The two other methods are not laborious and well-adapted to high throughput studies but the range of detection and linearity is limited.

Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled ‘endoflagella’ that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirocheteLeptospiraby integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of the spirochete flagellum.One Sentence summaryThe corkscrew-like motility of Spirochete bacteria is enabled by a unique, asymmetrically constructed flagellum that wraps around the cell body within the periplasm.

International audience; Leptospirosis is a neglected disease causing severe infections in humans and animals. Due in part to misdiagnosis, this infectious disease results in nearly 60,000 deaths per year around the globe. This study represents the first effort to describe the diversity of pathogenic Leptospira in Cuba based on whole-genome sequencing. We have collected nineteen whole-blood samples from patients that were diagnosed as having leptospirosis between 2008 and 2012 in Cuba. In addition, we have enhanced our sample set by three historical strains that were used for the development of a human vaccine in 1990s. The Leptospira strains were grown and serotyped by the microscopic agglutination test, and the draft genomes were generated by NGS (Illumina). Subsequently, the core genomes were analyzed and compared to the genetic data available from other Caribbean islands and countries in Central America. Core genome Multi-locus Sequence Typing (cgMLST) revealed four different core genome clonal groups (cgCGs), with the highest number of samples belonging to L. interrogans, followed by L. borgpetersenii and L. kirschneri. All cgCGs that were found in Cuba have been also identified from multiple origins across the globe, except in neighbor countries and Central America. Serotyping divided the samples into the serogroups Canicola, Ballum and Pomona. The most frequent cgCGs, cgCG28, associated with serogroup Canicola, and cgCG15, associated with serogroup Ballum, have also been identified from samples isolated from dogs, rodents, and pigs; suggesting that these hosts represent the major source of human infection in Cuba. The vaccine strains did not significantly differ from the recent patient isolates. However, the increasing prevalence of samples belonging to the serogroup Ballum combined with the fact that the available vaccine in Cuba represents inactivated Leptospira belonging to serogroups other than Ballum, should be a valuable information for the National and Regional Leptospirosis Control Programs.

International audience; Leptospirosis is a zoonotic disease with worldwide distribution. In French Guiana, a French overseas department of South America, this bacterial infection is endemic with the increase of human cases since the last 5 years. Nevertheless, the epidemiological data on the circulating infecting strain remains scarce due to the lack of specific symptoms and the used diagnostic approaches. We report a severe case of leptospirosis in a 52-years-old male, working as a street cleaner, hospitalized at the Intensive Care Unit of city capital hospital of French Guiana because of hemodynamic, neurological, renal, and respiratory failure. At ICU admission, the patient was comatose, his temperature was 37.3°C, heart rate 104 beats per minute, blood pressure 84/45mmHg, and oxygen saturation 95% while under mechanical ventilation. Retrospective exploration using molecular and serological approaches from the samples allowed reporting an infection by Leptospira santarosai serogroup Sejroe serovar Hardjo. To our knowledge, this is the first human case infected with this species in French Guiana. Through these analyses, this report provides new epidemiological information about the Leptospira strains circulating in French Guiana. In particular, this emphasizes the importance of accurate diagnosis to characterize with more precision the circulating Leptospira strains in this department.

The zoonotic bacterium Leptospira interrogans is the aetiological agent of leptospirosis, a re-emerging infectious disease that is a growing public health concern. Most human cases of leptospirosis result from environmental infection. Biofilm formation and its contribution to the persistence of virulent leptospires in the environment or in the host have scarcely been addressed. Here, we examined spatial and time-domain changes in biofilm production by L. interrogans. Our observations showed that biofilm formation in L. interrogans is a highly dynamic process and leads to a polarized architecture. We notably found that the biofilm matrix is composed of extracellular DNA, which enhances the biofilm’s cohesiveness. By studying L. interrogans mutants with defective diguanylate cyclase and phosphodiesterase genes, we show that biofilm production is regulated by intracellular levels of bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and underpins the bacterium’s ability to withstand a wide variety of simulated environmental stresses. Our present results show how the c-di-GMP pathway regulates biofilm formation by L. interrogans, provide insights into the environmental persistence of L. interrogans and, more generally, highlight leptospirosis as an environment-borne threat to human health.

Leptospirosis is a zoonotic bacterial disease with a worldwide importance, mostly frequent in tropical and subtropical countries. In Côte d'Ivoire, little is known about leptospirosis and human data are sparse. This disease is usually misdiagnosed with other febrile illnesses, and determining high-risk areas could allow better management of this disease, leading to policies. This study aims to map leptospirosis exposure areas by determining geographic distribution of anti-Leptospira antibodies in humans in Côte d'Ivoire. A total of 384 serum samples were randomly selected in the national surveillance system for communicable diseases in 2014. All the 82 health districts were include in the study. Serums were screened by ELISA at Institut Pasteur de Côte d'Ivoire and confirmed by MAT in the National Reference Centre for leptospirosis in Institut Pasteur in Paris. In these samples, ELISA screened 90 specimens showing anti-Leptospira antibodies and 36 specimens were confirmed by MAT (9.4%). Observed cases were mostly located in health districts of the western and the southern parts of the country. People with anti-Leptospira antibodies had a mean age of 34.5 years old and a sex ratio of 2. This pattern corresponds to active low-income farmers working into agricultural fields. This study reveals circulation of leptospirosis in human population in Côte d'Ivoire. The disease seems to be more frequent in the western and the southern parts of the country. Active low-income farmers working into agricultural fields without personal protective gear could be one of the most at-risk populations.

International audience; Leptospirosis is a neglected zoonotic bacterial disease caused by pathogenic Leptospira spp. Only limited studies have been conducted on the presence of Leptospira spp. in rats in Antananarivo, the capital city of Madagascar. We assessed Leptospira prevalence in small mammals in urban areas of Antananarivo where sanitation is inadequate and there is risk of flooding during the rainy season. We captured rodents and shrews at two sites and examined kidney samples from 114 animals using culture and a real-time polymerase chain reaction (PCR) assay specific to pathogenic Leptospira spp. We identified 23 positive samples containing Leptospira interrogans and Leptospira borgpetersenii, with a high prevalence in Rattus norvegicus (44.9%). Our results indicate that small mammals, in particular R. norvegicus, present a major public health risk for acquiring leptospirosis in Antananarivo.

We would like to thank Prof. Nicholas Robert Thomson (Wellcome Sanger Institute) for his valuable insights during the preparation of the manuscript. We also thank Robert Anthony Gaultney for his assistance with the English revision of the manuscript.; International audience; Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), remains an important public health problem with an increasing worldwide prevalence. Despite recent advances in in vitro cultivation, genetic variability of this pathogen during infection is poorly understood. Here, we present contemporary and geographically diverse complete treponemal genome sequences isolated directly from patients using a methyl-directed enrichment prior to sequencing. This approach reveals that approximately 50% of the genetic diversity found in TPA is driven by inter- and/or intra-strain recombination events, particularly in strains belonging to one of the defined genetic groups of syphilis treponemes: Nichols-like strains. Recombinant loci were found to encode putative outer-membrane proteins and the recombination variability was almost exclusively found in regions predicted to be at the host-pathogen interface. Genetic recombination has been considered to be a rare event in treponemes, yet our study unexpectedly showed that it occurs at a significant level and may have important impacts in the biology of this pathogen, especially as these events occur primarily in the outer membrane proteins. This study reveals the existence of strains with different repertoires of surface-exposed antigens circulating in the current human population, which should be taken into account during syphilis vaccine development.

International audience; Purpose: To report the incidence, risk factors, clinical presentation, and outcome predictors of severe leptospirosis requiring intensive care unit (ICU) admission in a temperate zone.Methods: LEPTOREA was a retrospective multicentre study conducted in 79 ICUs in metropolitan France. Consecutive adults admitted to the ICU for proven severe leptospirosis from January 2012 to September 2016 were included. Multiple correspondence analysis (MCA) and hierarchical classification on principal components (HCPC) were performed to distinguish different clinical phenotypes.Results: The 160 included patients (0.04% of all ICU admissions) had median values of 54 years [38-65] for age, 40 [28-58] for the SAPSII, and 11 [8-14] for the SOFA score. Hospital mortality was 9% and was associated with older age; worse SOFA score and early need for endotracheal ventilation and/or renal replacement therapy; chronic alcohol abuse and worse hepatic dysfunction; confusion; and higher leucocyte count. Four phenotypes were identified: moderately severe leptospirosis (n = 34, 21%) with less organ failure and better outcomes; hepato-renal leptospirosis (n = 101, 63%) with prominent liver and kidney dysfunction; neurological leptospirosis (n = 8, 5%) with the most severe organ failures and highest mortality; and respiratory leptospirosis (n = 17, 11%) with pulmonary haemorrhage. The main risk factors for leptospirosis contamination were contact with animals, contact with river or lake water, and specific occupations.Conclusions: Severe leptospirosis was an uncommon reason for ICU admission in metropolitan France and carried a lower mortality rate than expected based on the high severity and organ-failure scores. The identification in our population of several clinical presentations may help clinicians establish an appropriate index of suspicion for severe leptospirosis.

The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species., Author summary Leptospirosis which is an emerging zoonotic disease worldwide, is transmitted to humans through contact with soils or surface waters contaminated with the urine of reservoir animals. Species of Leptospira, which include infectious and non-infectious strains, are ubiquitous in the environment. In this study we have sequenced the genomes of strains of Leptospira isolated from several environmental sources worldwide. Comparison of these genomes with other members of the Leptospira genus revealed the existence of 30 novel Leptospira species. A comparative genomic analysis of species of the genus allowed us to identify genes or genome features that are specific of infectious species, providing insights into virulence evolution in these atypical bacteria but also allow refinement of the Leptospira classification.

Leptospira is a highly heterogeneous bacterial genus that can be divided into three evolutionary lineages and >300 serovars. The causative agents of leptospirosis are responsible of an emerging zoonotic disease worldwide. To advance our understanding of the biodiversity of Leptospira strains at the global level, we evaluated the performance of whole-genome sequencing (WGS) as a genus-wide strain classification and genotyping tool. Herein we propose a set of 545 highly conserved loci as a core genome MLST (cgMLST) genotyping scheme applicable to the entire Leptospira genus, including non-pathogenic species. Evaluation of cgMLST genotyping was undertaken with 509 genomes, including 327 newly sequenced genomes, from diverse species, sources and geographical locations. Phylogenetic analysis showed that cgMLST defines species, clades, subclades, clonal groups and cgMLST sequence types (cgST), with high precision and robustness to missing data. Novel Leptospira species, including a novel subclade named S2 (saprophytes 2), were identified. We defined clonal groups (CG) optimally using a single-linkage clustering threshold of 40 allelic mismatches. While some CGs such as L. interrogans CG6 (serogroup Icterohaemorrhagiae) are globally distributed, others are geographically restricted. cgMLST was congruent with classical MLST schemes, but had greatly improved resolution and broader applicability. Single nucleotide polymorphisms within single cgST groups was limited to, Author summary Leptospirosis, caused by pathogenic Leptospira strains, is an emerging bacterial zoonotic disease mostly affecting humans in tropical countries. Despite its public health importance, little is known about the strains that are circulating worldwide due to the lack of a universal common language on strain types. In this work we describe a new strain genotyping and classification system that is highly standardized, thus facilitating global collaboration, and that can discriminate all members of the Leptospira genus at high resolution. We then examine the genetic diversity of Leptospira strains from different origins. This study provides a framework for optimizing diagnostic methods and epidemiological surveillance of leptospirosis.

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the β-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with worldwide prevalence. Several different molecular typing schemes are currently available for this pathogen. To enable population biology studies of the syphilis agent and for epidemiological surveillance at the global scale, a harmonized typing tool needs to be introduced. Recently, we published a new multi-locus sequence typing (MLST) with the potential to significantly enhance the epidemiological data in several aspects (e.g., distinguishing genetically different clades of syphilis, subtyping inside these clades, and finally, distinguishing different subspecies of non-cultivable pathogenic treponemes). In this short report, we introduce the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. Moreover, we have summarized epidemiological data of all treponemal strains (n = 358) with available DNA sequences in typing loci and found several association between genetic groups and characteristics of patients. This study proposes the establishment of a single MLST of T. p. pallidum and encourages researchers and public health communities to use this PubMLST database as a universal tool for molecular typing studies of the syphilis pathogen.

International audience

Correction de l'article pasteur-02547654: https://hal-pasteur.archives-ouvertes.fr/pasteur-02547654; International audience

International audience; Leptospirosis is a neglected zoonosis with worldwide distribution. The causative agents are spirochete bacteria of the Leptospira genus, displaying huge diversity of serovars, the identity of which is critical for effective diagnosis and vaccination purposes. Among many other mammalian species, Leptospira infects cattle, eliciting acute signs in calves, and chronic disease in adult animals often leading to abortions. In South America, and including in Uruguay, beef and dairy export are leading sources of national income. Despite the importance of bovine health, food safety, and bovine-related dissemination of leptospirosis to humans, extremely limited information is available as to the identity of Leptospira species and serovars infecting cattle in Uruguay and the South American subcontinent. Here we report a multicentric 3-year study resulting in the isolation and detailed characterization of 40 strains of Leptospira spp. obtained from infected cattle. Combined serologic and molecular typing identified these isolates as L. interrogans serogroup Pomona serovar Kennewicki (20 strains), L. interrogans serogroup Canicola serovar Canicola (1 strain), L. borgpetersenii serogroup Sejroe serovar Hardjo (10 strains) and L. noguchii (9 strains). The latter showed remarkable phenotypic and genetic variability, belonging to 6 distinct serogroups, including 3 that did not react with a large panel of reference serogrouping antisera. Approximately 20% of cattle sampled in the field were found to be shedding pathogenic Leptospira in their urine, uncovering a threat for public health that is being largely neglected. The two L. interrogans serovars that we isolated from cattle displayed identical genetic signatures to those of human isolates that had previously been obtained from leptospirosis patients. This report of local Leptospira strains shall improve diagnostic tools and the understanding of leptospirosis epidemiology in South America. These strains could also be used as new components within bacterin vaccines to protect against the pathogenic Leptospira strains that are actually circulating, a direct measure to reduce the risk of human leptospirosis.

International audience

International audience; Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira. Recently, there has been a renewed interest in bacteriophages for their potential use as alternatives to conventional antibiotics 1 , and also in understanding their contribution in evolution of bacteria 2,3. In addition, phages could be used to develop new genetic tools such as replicative vectors from different compatibility groups and phage-delivery systems. Unfortunately, little is known about the diversity of phages among the genus Leptospira. Leptospira are ubiquitous organisms that are found as free-living saprophytes in environmental water and soil, or as pathogens that can cause acute or chronic infections in animals. A third group that is composed of intermediate species (in regards to their pathogenesis) of Leptospira, is phylogenetically closely related to the pathogenic species and can cause mild infections 4. Leptospirosis is an emerging waterborne zoonosis which results in more than one million human cases a year with a fatality rate frequently exceeding 10% 5. To the best of our knowledge, the only phages that have been isolated, purified, and phenotypically characterized in the genus Leptospira are: vB_LbiM_LE1 (renamed 6 , and abbreviated LE1), vB_LbiM_LE3 (LE3), and vB_LbiM_LE4 (LE4) 7. These tailed phages have been isolated from urban sewage and infect the saprophyte L. biflexa. The LE1 temperate phage genome was previously sequenced 8 and an Escherichia coli-L. biflexa shuttle vector was generated by cloning the replication origin of LE1 9. Until now, the virulent phages LE3 and LE4 have not been further characterized at the genomic and proteomic levels. In addition to these three phages, phage-like particles were also observed following mitomycin C induction of a pathogenic strain carrying a circular plasmid with phage-related genes, but these phage-like particles were not purified 10. Recently, comparative analyses of genome sequences have suggested the existence of prophages and genomic islands within the genus Leptospira 11,12. Putative prophages are found in infectious Leptospira species, including pathogenic and intermediate species, and absent in saprophytic Leptospira species, suggesting that phages may have had a major role in the emergence of the pathogens and/or in the acquisition of virulence factors. Analysis of the complete genomes of 20 Leptospira species led to the description of several predicted prophage regions 11 , including LE1-like and Mu-like prophages. In addition, the existence of two other groups of prophages have been proposed: a 22-kb region which was initially described in L. interrogans serovar Lai but that is present in most

International audience; Leptospirosis is a worldwide zoonosis, responsible for more than 1 million cases and 60,000 deaths every year. Among the 13 pathogenic species of the genus Leptospira, serovars belonging to L. interrogans serogroup Icterohaemorrhagiae are considered to be the most virulent strains, and responsible for majority of the reported severe cases. Serovars Copenhageni and Icterohaemorrhagiae are major representatives of this serogroup and despite their public health relevance, little is known regarding the genetic differences between these two serovars. In this study, we analyzed the genome sequences of 67 isolates belonging to L. interrogans serovars Copenhageni and Icterohaemorrhagiae to investigate the influence of spatial and temporal variations on DNA sequence diversity. Out of the 1072 SNPs identified, 276 were in non-coding regions and 796 in coding regions. Indel analyses identified 258 indels, out of which 191 were found in coding regions and 67 in non-coding regions. Our phylogenetic analyses based on SNP dataset revealed that both serovars are closely related but showed distinct spatial clustering. However, likelihood ratio test of the indel data statistically confirmed the presence of a frameshift mutation within a homopolymeric tract of lic12008 gene (related to LPS biosynthesis) in all the L. interrogans serovar Icterohaemorrhagiae strains but not in the Copenhageni strains. Therefore, this internal indel identified can genetically distinguish L. interrogans serovar Copenhageni from serovar Icterohaemorrhagiae with high discriminatory power. To our knowledge, this is the first study to identify global sequence variations (SNPs and Indels) in L. interrogans serovars Copenhageni and Icterohaemorrhagiae.

International audience; Pathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine-rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X-ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E- and VE-cadherins. These results provide biochemical and cellular evidences of LRR protein-mediated host-pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species.

International audience; Leptospirosis is an important environmental disease and a major threat to human health causing at least 1 million clinical infections annually. There has recently been a growing interest in understanding the environmental lifestyle of Leptospira. However, Leptospira isolation from complex environmental samples is difficult and time-consuming and few tools are available to identify Leptospira isolates at the species level. Here, we propose a polyphasic isolation and identification scheme, which might prove useful to recover and identify environmental isolates and select those to be submitted to whole-genome sequencing. Using this approach, we recently described 12 novel Leptospira species for which we propose names. We also show that MALDI-ToF MS allows rapid and reliable identification and provide an extensive database of Leptospira MALDI-ToF mass spectra, which will be valuable to researchers in the leptospirosis community for species identification. Lastly, we also re-evaluate some of the current techniques for the molecular diagnosis of leptospirosis taking into account the extensive and recently revealed biodiversity of Leptospira in the environment. In conclusion, we describe our method for isolating Leptospira from the environment, confirm the usefulness of mass spectrometry for species identification and propose names for 12 novel species. This also offers the opportunity to refine current molecular diagnostic tools.

International audience; Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira. In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one (lvrB) or both genes (lvrA/B) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium.

International audience; Leptospirosis affects 1 million and kills 60,000 people annually, but it remains poorly documented in Africa. We aim to describe the large West African Conurbation Corridor where the omnipresence of slums, water and close animal/human interactions may result in high leptospiral risk. Though scarce, data from this region point towards the wide circulation of pathogenic leptospires in the urban environment as well as in humans. However, because of the absence of reliable surveillance systems together with lack of awareness, the absence of reference laboratory and/or a high number of infected people showing only mild manifestations, it is likely that the burden is much higher. We believe raising awareness of leptospirosis may have a positive impact on many vulnerable African city dwellers, as the disease is a preventable and treatable.

Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral ‘pathogens’ cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.

International audience; Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress.

Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses., Author summary Leptospirosis is a widespread zoonosis caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). L. interrogans are primarily extracellular pathogens although some reports suggest they may replicate within macrophages. In humans, leptospirosis can cause mild or severe disease, potentially leading to death, although rats or mice, which constitute the reservoir, are asymptomatic carriers. Host defense mechanisms involved in leptospirosis remain poorly understood. Toll-Like Receptor (TLR)2 and TLR4 are crucial for the clearance of L. interrogans, but the role of the cytosolic NOD receptors in leptospirosis is unknown. Here, we report that pathogenic leptospires escape the sensing of bacterial peptidoglycan through the NOD response. We found that an outer membrane lipoprotein of L. interrogans binds to and protects the peptidoglycan from degradation into muropeptides, thereby blocking signaling through NOD proteins. Moreover, in absence of this lipoprotein, the peptidoglycan of L. interrogans is properly sensed by human NOD1 but not by murine NOD1. This is due to the near absence of muramyl tetrapeptide, the murine NOD1 agonist, in the peptidoglycan of pathogenic leptospires. These novel mechanisms of NOD avoidance may facilitate the escape of leptospires from the innate immune system of their hosts.

International audience; A strain of Leptospira kirschneri (serogroup Grippotyphosa) was cultured from urine of a mare post-abortion in Brazil and characterized by serogrouping, multiple-locus variable-number tandem repeat analysis, PGFE, and sequencing of genes rrs and secY. Strains of L. kirschneri have apparently never been recovered from horses in tropical area, only in Europe and USA. Knowledge of local epidemiology is important to interpret genetic profiles of leptospires circulating in an area.