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Precise and timely diagnosis is essential to prevent severe outcomes of leptospirosis in humans and animals. Existing diagnostic methods face challenges and limitations, underscoring the need for novel, field-applicable screening, and diagnostic tests/assays. This study evaluates the diagnostic utility of a recombinant ErpY-like lipoprotein (rErpY-LIC11966) in a latex agglutination test (LAT) for diagnosis of animal leptospirosis. The ErpY gene sequence from Leptospira interrogans serovar Pomona, excluding the signal peptide, was amplified, cloned into the pETite vector, and expressed in Escherichia coli. The expressed rErpY (∼16 kDa) was characterized by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western blot using Leptospira-specific standard sera. To assess the diagnostic potential of rErpY, Ni-NTA affinity-purified protein was used to sensitize latex-coated beads (0.8 µm colour beads), which were then employed in the LAT for standardization and optimization with standard positive and negative sera. For evaluation, the rErpY-LAT was tested on serum samples from 177 suspected animal cases and compared to the microscopic agglutination test. It showed a relative diagnostic sensitivity of 90.6%, a specificity of 89.1%, and an overall accuracy of 90%. This study proposes rErpY-LAT as a field testing/screening diagnostic tool for preliminary serodiagnosis of leptospirosis, highlighting the potential of recombinant protein-based assays to address current diagnostic challenges., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Leptospirosis is a widespread zoonosis caused by bacteria of the genus Leptospira. Although crucial to mitigate the disease risk, basic epidemiological information is lacking, such as the identities of Leptospira maintenance hosts. The raccoon ( Procyon lotor ), an alien invasive species in France, could pose a public health risk if it carries pathogenic Leptospira. We investigated the rate and type (selective vs. unselective) of Leptospira carriage in the two main raccoon populations in France. Out of the 141 raccoons collected, seven (5%) tested quantitative PCR positive, targeting lfb1 gene, based on kidney, lung, and urine samples. Phylogenetic analysis revealed the presence of three different L. interrogans clusters. The results suggest that raccoons were more likely accidental hosts and made only a limited contribution to Leptospira maintenance.

Bovine leptospirosis is a worldwide disease that causes reproductive issues, including early embryonic death, stillbirths and infertility, which result in significant economic losses. Although bovine leptospirosis is well-documented in cows, the role of bulls in harbouring and potentially transmitting genital leptospirosis has been largely neglected. We examined genital and blood samples from 16 slaughtered bulls using microscopic agglutination testing (MAT), polymerase chain reaction (PCR) and sequencing of amplicons. Our results showed that 81.2% of bulls were seroreactive, and 75% were genitally infected. The amplicons displayed an identity greater than 99% with Leptospira interrogans strains from the Sejroe serogroup, specifically serovar Hardjoprajitno. This study demonstrates that bulls can harbour in their genital tract the same leptospires associated with reproductive syndromes in cows from the same geographic region, highlighting the importance of bulls in maintaining and transmitting Sejroe serogroup strains associated with reproductive disease., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Previous in vitro works focusing on virulence determinants of the spirochete Leptospira implicated metalloproteinases as putative contributing factors to the pathogenicity of these bacteria. Those proteins have the capacity to degrade extracellular matrix components (ECM) and proteins of host's innate immunity, notably effectors of the complement system. In this study, we gained further knowledge on the role of leptolysin, one of the leptospiral-secreted metalloproteinases, previously described as having a broad substrate specificity. We demonstrated that a proportion of human patients with mild leptospirosis evaluated in the current study produced antibodies that recognize leptolysin, thus indicating that the protease is expressed during host infection. Using recombinant protein and a knockout mutant strain, Manilae leptolysin - , we determined that leptolysin contributes to Leptospira interrogans serum resistance in vitro, likely by proteolysis of complement molecules of the alternative, the classical, the lectin, and the terminal pathways. Furthermore, in a hamster model of infection, the mutant strain retained virulence; however, infected animals had lower bacterial loads in their kidneys. Further studies are necessary to better understand the role and potential redundancy of metalloproteinases on the pathogenicity of this important neglected disease., (© 2024 John Wiley & Sons Ltd.)

Pathogenic bacteria Leptospira spp. are commonly associated with bovine leptospirosis, characterized chiefly by chronic and subclinical reproductive disorders. Strains from the Sejroe serogroup play a significant role in these chronic genital infections known as Bovine Genital Leptospirosis (BGL), which notably impact cattle health. This study aims to deepen our understanding of BGL by investigating the genetic diversity, geographical distribution, and specific anatomical sites of infection of the causative agents. Initially, uterine fragments and cervicovaginal mucus were collected from 47 cows and subjected to PCR targeting the lipL32 gene. Positive samples in lipL32-PCR (9 samples) underwent genotyping based on the secY gene. Subsequently, sequences were aligned with GenBank entries (108 sequences) and analyzed in silico. All nine sequences from this study were identified as L. interrogans with an identity >99 % to serogroup Sejroe reference strains (Norma and L53). In the broader analysis, the most prevalent species observed was L. borgpetersenii, followed by L. interrogans and L. santarosai. The haplotype network of L. interrogans revealed that haplogroups B and C exclusively included L. interrogans strains of genital origin, while haplogroup A encompassed strains from renal sources as well. These findings underscore the significance of the L. borgpetersenii genotype Hardjobovis and L. interrogans genotype Hardjoprajitno as the predominant circulating strains and highlight the existence of distinct haplogroups of pathogenic leptospires originating from genital sources. We advocate for the use of secY as an effective genetic marker for Leptospira spp. and stress the necessity for additional research prioritizing the genital tract. The outcomes of this study contribute to the development of improved control measures for chronic cattle diseases and provide valuable guidance for future investigations., Competing Interests: Declaration of Competing Interest None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leptospirosis is a bacterial disease of worldwide distribution with relevant implications for animal and human health. Different large wild carnivore species can act as reservoirs of this zoonotic pathogen. This study aimed to evaluate the circulation of Leptospira spp. in free-ranging wolves (Canis lupus) from southern Europe. A total of 281 kidney samples of wolves from Spain and Italy were collected between 2017 and 2023. The presence of Leptospira DNA was analysed by real-time PCR and phylogenetic analyses were carried out using a Bayesian approach. The overall prevalence was 3.2 % (9/281; 95 %CI: 1.1-5.3). Leptospira DNA was detected in nine of the 180 wolves from Spain (5.0 %; 95 %CI: 1.8-8.2), but not in the Italian wolf population (0 %; 0/101). Molecular analyses revealed high homology between the sequences obtained in the present study and isolates of Leptospira interrogans and Leptospira borgpetersenii from different rodent and domestic ungulate species. Our results provide evidence of a low and spatially heterogeneous circulation of this pathogen in wolf populations of southern Europe. The detection of zoonotic Leptospira species in this survey supports the need to consider wolf populations in monitoring programs for leptospirosis with a One Health approach., Competing Interests: Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Terrestrial and aquatic environments contaminated with animal urine may contribute to the transmission of Leptospira, a causative agent of leptospirosis in humans and wild/domesticated animals. Although enormous amounts of work have been done decoding the ecophysiology, the factors governing the cell growth and virulence in Leptospires derived from environmental samples still remain elusive. Here, we show oxidation of a wide array of organic acids including acetoacetate by a new strain of Leptospira interrogans designated as KeTo, isolated from a sewage sample originating from a wildlife enclosure located at Mangalore, India. We further demonstrate the susceptibility of KeTo to ethyl ester of acetoacetate (ethyl acetoacetate, EA). A 4.7 Mbp genome of KeTo shared the highest relatedness to pathogenic L. interrogans RGA T (99.3%), followed by L. kirschneri 3522C T (91.3%) and other related species of Leptospira (80.8‒74.3%), and harbored genes encoding acetoacetyl-CoA synthetase and acetoacetate decarboxylase respectively involved in the acetoacetate utilization and acetone formation. In line with this, strain KeTo oxidized acetoacetate when supplied as a sole carbon. Aqueous EA suppressed biofilm formation (p < 0.0001) of KeTo in basal Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Similarly, significant inhibition in the growth/biofilm of Leptospira was recorded in semisolid EMJH with/without blood supplementation when exposed to volatile EA. The extent of ketone body oxidation and susceptibility to EA was found to vary with strain as evident through the analysis of L. interrogans serogroup Australis sv. Australis strain Ballico and L. interrogans serogroup Icterohaemorrhagiae sv. Lai Like strain AF61. In conclusion, our study demonstrated the ketone body metabolic ability and susceptibility to an esterified derivative of a major ketone body in the tested strains of L. interrogans. Molecular aspects governing EA-driven growth inhibition warrant further investigations to develop optimal therapeutics for leptospirosis., (© 2024. The Author(s).)

In an integrated swine farm with 135 sows in Nagasaki Prefecture, premature births and stillbirths were observed in six sows in June and July 2023, and their etiology was investigated. Leptospiral flaB gene was detected in the kidney of one stillborn fetus and the placenta of its sow by nested PCR. Multilocus sequence typing revealed that the infecting strain was Leptospira interrogans ST118. In addition, leptospiral antigens were detected in lesions of the above tissues by immunohistochemical staining. The examined sow and several other sows in the farm had antibodies against serogroup Hebdomadis. These results suggested that L. interrogans serogroup Hebdomadis ST118 was the causative agent of premature births and stillbirths that occurred in this farm.

Life-threatening Leptospira interrogans navigate a dual existence: surviving in the environment and infecting mammalian hosts. Biofilm formation is presumably an important survival strategy to achieve this process. Understanding the relation between biofilm and virulence might improve our comprehension of leptospirosis epidemiology. Our study focused on elucidating Leptospira's adaptations and regulations involved in such complex microenvironments. To determine the transcriptional profile of Leptospira in biofilm, we compared the transcriptomes in late biofilms and in exponential planktonic cultures. While genes for motility, energy production, and metabolism were downregulated, those governing general stress response, defense against metal stress, and redox homeostasis showed a significant upsurge, hinting at a tailored defensive strategy against stress. Further, despite a reduced metabolic state, biofilm disruption swiftly restored metabolic activity. Crucially, bacteria in late biofilms or resulting from biofilm disruption retained virulence in an animal model. In summary, our study highlights Leptospira's adaptive equilibrium in biofilms: minimizing energy expenditure, potentially aiding in withstanding stresses while maintaining pathogenicity. These insights are important for explaining the survival strategies of Leptospira, revealing that a biofilm lifestyle may confer an advantage in maintaining virulence, an understanding essential for managing leptospirosis across both environmental and mammalian reservoirs., (© 2024. The Author(s).)

Pathogenic spirochetes of the genus Leptospira are the causative agent of leptospirosis, a widely disseminated zoonosis that affects humans and animals. The ability of leptospires to quickly cross host barriers causing infection is not yet fully understood. Thus, understanding the mechanisms of pathogenicity is important to combat leptospiral infection. Outer membrane proteins are interesting targets to study as they are able to interact with host molecules. Proteins containing leucine-rich repeat (LRR) domains are characterized by the presence of multiple regions containing leucine residues and they have putative functions related to host-pathogen interactions. Hence, the present study aimed to clone and express the recombinant protein encoded by the LIC11098 gene, an LRR protein of L. interrogans serovar Copenhageni. In silico analyses predicted that the target protein is conserved among pathogenic strains of Leptospira, having a signal peptide and multiple LRR domains. The DNA sequence encoding the LRR protein was cloned in frame into the pAE vector, expressed without mutations in Escherichia coli and purified by His-tag chromatography. Circular dichroism (CD) spectrum showed that the recombinant protein was predominantly composed of β-sheets. A dose-dependent interaction was observed with cellular and plasma fibronectins, laminin and the complement system component C9, suggesting a possible role of the protein encoded by LIC11098 gene at the initial stages of infection., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Leptospirosis, a neglected zoonotic disease, adversely affects animal, human health, and socioeconomic conditions, particularly in developing countries like Nigeria. This study aimed to determine the occurrence and molecular identification of pathogenic Leptospira spp. among abattoir workers, cattle, and rats in Jos North, Plateau State, Nigeria. Using a cross-sectional study design, a total of 394 samples were collected, including 149 urine samples from abattoir workers, 125 urine samples from cattle bladders, and 120 bladders from trapped rats. Samples were processed and cultured in Ellinghausen McCullough Johnson Harrison (EMJH) medium and examined under a darkfield microscope. Positive cultures were confirmed using the Microscopic Agglutination Test (MAT) and nested Polymerase Chain Reaction (N-PCR) targeted the 16 S rDNA gene. Results revealed a prevalence of 33.76 % for Leptospira spp. across all samples, with the highest occurrence in abattoir workers (13.96 %), followed by rats (13.45 %), and cattle (6.35 %). The MAT showed L. interrogans serovar Hardjo str. Hardjoprajitno as the most prevalent serotype (41.61 %), followed by L. interrogans serovar Icterohaemorrhagiae str. RGA (34.31 %). N-PCR confirmed the presence of pathogenic Leptospira spp., showing bands of 1200 bp. Phylogenetic analysis of the 16 S rDNA gene sequences revealed close similarities to known pathogenic Leptospira strains from Brazil and the USA. The study underscores the significant public health risk posed by leptospirosis in Jos North and highlights the need for improved diagnostic capabilities, increased awareness, and effective control measures to mitigate the disease burden. Enhanced surveillance and preventive strategies are crucial to protect both animal and human health in the region., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three 'noNterm' Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions., (© 2024. The Author(s).)

Leptospirosis is an infectious disease with a worldwide distribution, which represents a major challenge in animal production across developing countries, mainly in tropical areas. Horses are particularly susceptible to the disease, presenting manifestations ranging from subclinical to the development of uveitis that compromises the visual health of the animals. In recent years, serological studies have been carried out in equid populations from America, demonstrating high exposure. For this reason, the aim of this study was to demonstrate microbiologically and molecularly the presence of the members of the genus Leptospira in urine samples from equids in an endemic state of leptospirosis in Mexico, and to detect the serological presence of anti-Leptospira antibodies in the sampled animals. For this reason, blood and urine samples were collected from 28 horses and one mule from three localities in the state of Veracruz, Mexico. Urine samples were inoculated in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and the recovered isolates were typed using a short Multi Locus Sequence Typing scheme. Amplifications of the expected size were subjected to sequencing, and the recovered sequences were compared with those of reference deposited in GenBank using the BLAST tool. To identify their phylogenetic position, we performed a phylogenetic reconstruction using the maximum likelihood method. Additionally, Microscopic Agglutination test was performed on the serum samples to identify anti-Leptospira antibodies. We recovered 16 urine isolates which tested positive for the presence of Leptospira DNA. The phylogenetic reconstruction and the MLST analysis confirmed the presence of several genotypes of Leptospira interrogans and Leptospira santarosai. An overall serological frequency of 97.1 % was detected. Our results represent the first record of the presence of Leptospira through bacteriological isolates in equids from Mexico., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Leptospirosis is a common but underdiagnosed zoonosis. We conducted a 1-year prospective study in La Guaira State, Venezuela, analyzing 71 hospitalized patients who had possible leptospirosis and sampling local rodents and dairy cows. Leptospira rrs gene PCR test results were positive in blood or urine samples from 37/71 patients. Leptospira spp. were isolated from cultured blood or urine samples of 36/71 patients; 29 had L. interrogans, 3 L. noguchii, and 4 L. venezuelensis. Conjunctival suffusion was the most distinguishing clinical sign, many patients had liver involvement, and 8/30 patients with L. interrogans infections died. The Leptospira spp. found in humans were also isolated from local rodents; L. interrogans and L. venezuelensis were isolated from cows on a nearby, rodent-infested farm. Phylogenetic clustering of L. venezuelensis isolates suggested a recently expanded outbreak strain spread by rodents. Increased awareness of leptospirosis prevalence and rapid diagnostic tests are needed to improve patient outcomes.

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals., (© 2024. The Author(s).)

Background: Rodents are recognized as major reservoirs of numerous zoonotic pathogens and are involved in the transmission and maintenance of infectious diseases. Furthermore, despite their importance, diseases transmitted by rodents have been neglected. To date, there have been limited epidemiological studies on rodents, and information regarding their involvement in infectious diseases in the Republic of Korea (ROK) is still scarce., Methodology/principal Findings: We investigated rodent-borne pathogens using nested PCR/RT-PCR from 156 rodents including 151 Apodemus agrarius and 5 Rattus norvegicus from 27 regions in eight provinces across the ROK between March 2019 and November 2020. Spleen, kidney, and blood samples were used to detect Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato group, Coxiella burnetii, Leptospira interrogans, and severe fever with thrombocytopenia syndrome virus (SFTSV). Of the 156 rodents, 73 (46.8%) were infected with Bartonella spp., 25 (16.0%) with C. burnetii, 24 (15.4%) with L. interrogans, 21 (13.5%) with A. phagocytophilum, 9 (5.8%) with SFTSV, and 5 (3.2%) with Borrelia afzelii. Co-infections with two and three pathogens were detected in 33 (21.1%) and 11 rodents (7.1%), respectively. A. phagocytophilum was detected in all regions, showing a widespread occurrence in the ROK. The infection rates of Bartonella spp. were 83.3% for B. grahamii and 16.7% for B. taylorii., Conclusions/significance: To the best of our knowledge, this is the first report of C. burnetii and SFTSV infections in rodents in the ROK. This study also provides the first description of various rodent-borne pathogens through an extensive epidemiological survey in the ROK. These results suggest that rodents harbor various pathogens that pose a potential threat to public health in the ROK. Our findings provide useful information on the occurrence and distribution of zoonotic pathogens disseminated among rodents and emphasize the urgent need for rapid diagnosis, prevention, and control strategies for these zoonotic diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Choi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Leptospirosis is a zoonotic disease caused by the pathogenic spirochaetes of the genus Leptospira. It is a public health concern in the Pacific Islands and is considered endemic in Palau. However, information on the genotypes and serotypes of causative Leptospira spp. in the country is limited. In this study, we isolated leptospires and detected antileptospiral antibodies in dogs and pigs. The isolates were characterized using a serological method and whole-genome sequencing. Leptospira interrogans was isolated from five of the 20 symptomatic dogs and one of the 58 healthy pigs. Their serogroups were identified as Icterohaemorrhagiae and Pyrogenes; however, the serogroup of one isolate could not be determined. Anti-Leptospira antibodies were detected in 14.4% (26/181) of the dogs and 20% (10/50) of the pigs. The reactive serogroups in dogs and pigs were almost identical, except for the Panama serogroup. Core genome multilocus sequence typing revealed that five of the six core genome sequence types (cgSTs) were newly identified in this study. The cgSTs from the serogroup Icterohaemorrhagiae isolates belonged to the same group as the Copenhageni and Icterohaemorrhagiae serovars isolated in other countries, whereas no similar cgSTs were identified in the Pyrogenes or unidentified serogroup strains. We demonstrated a high incidence of canine and porcine leptospirosis and identified new L. interrogans genotypes (cgSTs) circulating in Palau. Further investigations are needed to determine whether dogs and pigs serve as maintenance hosts for newly identified L. interrogans genotypes and whether they pose a risk of leptospirosis transmission to humans.

ECF (extracytoplasmic function) σ factors, members of the σ 70 -family, are the largest class of alternative σ factors which are stimulated in the presence of specific signals and direct RNA polymerase to transcribe a defined subset of genes. Thanks to them, bacterial pathogens can effectively reprogram their gene expression and, consequently, survive in the host and establish infection in a relatively short time. The number of ECF σ factors encoded within bacterial genomes is different depending on a given species and it reflects the likelihood that these bacteria will encounter harsh environmental conditions. The genome of L. interrogans, a zoonotic pathogen responsible for leptospirosis, is predicted to encode 11 ECF σ E -type factors, but none of them have been characterized biochemically to date and their functions are still unknown. Here, we focused on one of the leptospiral ECF σ factors, namely LIC&#95;12757, which was previously found to be up-regulated at elevated temperatures and may be related to the expression of clpB encoding an important L. interrogans virulence factor. We report cloning of the coding sequence of the LIC&#95;12757 gene, its expression with the pET system and biochemical characterization of LIC&#95;12757. By performing EMSA and in vitro transcription assays, we provide strong evidence that LIC&#95;12757 indeed functions as a transcriptional factor that enables RNA polymerase to bind to the specific σ E -type promoter and to initiate transcription. Interestingly, we demonstrate that LIC&#95;12757 is autoregulated at the transcriptional level. Our study is a first step towards determining key aspects of LIC&#95;12757 function in pathogenic Leptospira., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Leptospira spp. are bacteria responsible for leptospirosis, a zoonotic disease with considerable impacts on the economy, animal health, and public health. This disease has a global distribution and is particularly prevalent in Brazil. Both rural and urban environments are habitats for Leptospira spp., which are primarily transmitted through contact with the urine of infected animals. Consequently, domestic and wild species can harbor these prokaryotes and serve as infection sources for other hosts. In the context of wild animals, there is a dearth of molecular studies elucidating the roles of various animal and bacterial species in the epidemiology of leptospirosis. Therefore, this study aimed to evaluate the presence of Leptospira spp. DNA in different species of free-living and captive wild animals and to assess the phylogenetic relationships of the identified microorganisms in Rio Grande do Sul, Brazil. The samples were evaluated for the presence of the gene lipL32 by polymerase chain reaction (PCR) and sequencing of the amplified fragment after which phylogenetic analyzes were carried out. DNA from Leptospira spp. was extracted from kidney tissue from wild animals (Mammalia class). Pathogenic Leptospira spp. DNA was detected in 9.6% (11/114) of the samples, originating from nine species of wild animals, including the white-eared opossum (Didelphis albiventris), skunk (Conepatus chinga), geoffroy's cat (Leopardus geoffroyi), margay (Leopardus wiedii), pampas fox (Lycalopex gymnocercus), capybara (Hydrochoerus hydrochaeris), common marmoset (Callithrix jacchus), neotropical river otter (Lontra longicaudis), and european hare (Lepus europaeus). Phylogenetic analysis revealed the presence of Leptospira borgpetersenii and Leptospira interrogans in these animals. This research is the first study contributing to the epidemiology of leptospirosis by identifying L. borgpetersenii and L. interrogans in free-living and captive wild animals in Rio Grande do Sul, Brazil, potentially acting as bacterial reservoirs. Additionally, our findings can inform sanitary measures for controlling and preventing the disease, thereby safeguarding public health., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The World Health Organization classifies leptospirosis as a significant public health concern, predominantly affecting impoverished and unsanitary regions. By using the Pensacola Bay System as a case study, this study examines the underappreciated susceptibility of developed subtropical coastal ecosystems such as the Pensacola Bay System to neglected zoonotic pathogens such as Leptospira . We analyzed 132 water samples collected over 12 months from 44 distinct locations with high levels of Escherichia coli (>410 most probable number/100 mL). Fecal indicator bacteria (FIB) concentrations were assessed using IDEXX Colilert-18 and Enterolert-18, and an analysis of water physiochemical characteristics and rainfall intensity was conducted. The LipL32 gene was used as a quantitative polymerase chain reaction (qPCR) indicator to identify the distribution of Leptospira interrogans . The results revealed 12 instances of the presence of L. interrogans at sites with high FIB over various land cover and aquatic ecosystem types. Independent of specific rainfall events, a seasonal relationship between precipitation and elevated rates of fecal bacteria and leptospirosis was found. These findings highlight qPCR's utility in identifying pathogens in aquatic environments and the widespread conditions where it can be found in natural and developed areas., Competing Interests: The authors declare there is no conflict., (© 2024 The Authors This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).)

Antimicrobial resistance (AMR) is one of the global health challenges of the 21st century. Data regarding AMR mechanisms in Leptospira interrogans , the causative agents of leptospirosis, have been relatively limited. Therefore, our study aimed to identify resistance genes and explore potential resistance mechanisms specific to particular serovars. We conducted a comprehensive analysis of 98 Leptospira strains, representing 10 common serovars, using whole-genome sequencing (WGS) FASTA files. Employing the PATRIC tool from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), we scrutinized the genomes for AMR genes. Our investigation revealed 32 genes associated with AMR, with 20 key genes consistently prevalent across most strains. Notably, we identified unique efflux pump systems in serovar Pomona, indicating distinctive resistance mechanisms in this serovar. In summary, our findings shed light on the genetic landscape of AMR in Leptospira, uncovering both common and serovar-specific resistance elements. The presence of unique efflux pump systems in serovar Pomona introduces a novel dimension to our understanding of resistance mechanisms. These insights underscore the importance of tailored intervention strategies and collaborative efforts between human and veterinary healthcare professionals, as well as environmental scientists, to address the complex dynamics of leptospirosis and its implications for antibiotic resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Petakh and Kamyshnyi.)

Bacterial motility is often a crucial virulence factor for pathogenic species. A common approach to study bacterial motility is fluorescent labeling, which allows detection of individual bacterial cells in a population or in host tissues. However, the use of fluorescent labeling can be hampered by protein expression stability and/or interference with bacterial physiology. Here, we apply machine learning to microscopic image analysis for label-free motion tracking of the zoonotic bacterium Leptospira interrogans on cultured animal cells. We use various leptospiral strains isolated from a human patient or animals, as well as mutant strains. Strains associated with severe disease, and mutant strains lacking outer membrane proteins (OMPs), tend to display fast mobility and reduced adherence on cultured kidney cells. Our method does not require fluorescent labeling or genetic manipulation, and thus could be applied to study motility of many other bacterial species., (© 2023. The Author(s).)

Leptospirosis is one of the neglected zoonosis, affecting human and animal populations worldwide. Reliable effective therapeutics and concerns to look for more research into the molecular analysis of its genome is therefore needed. In the genomic pool of the Leptospira interrogans many hypothetical proteins are still uncharacterized. In the current research, we performed extensive in silico analysis to prioritize the potential hypothetical proteins of L. interrogans serovar Copenhageni via stepwise reducing the available hypothetical proteins (Total 3606) of the assembly to only 15, based on non-homologous to homosapien, essential, functional, virulent, cellular localization. Out of them, only two proteins WP&#95;000898918.1 (Hypothetical Protein 1) & WP&#95;001014594.1 (Hypothetical Protein 2) were found druggable and involved in protein-protein interaction network. The 3 D structures of these two target proteins were predicted via ab initio homology modeling followed by structures refinement and validation, as no structures were available till date. The analysis also revealed that the functional domains, families and protein-protein interacting partners identified in both proteins are crucial for the survival of the bacteria. The binding cavities were predicted for both the proteins through blind and specific protein-ligand docking with their respective ligands and inhibitors and were found to be in accordance with the druggable sites predicted by DoGSiteScorer. The docking interactions were found within the active functional domains for both the proteins while for Hypothetical Protein 2, the same residues were involved in interactions with Cytidine-5'-triphosphate in blind and specific docking. Furthermore, the simulations of molecular dynamics and free binding energy revealed the stable substrate binding and efficient binding energies, and were in accordance to our docking results. The work predicted two unique hypothetical proteins of L. interrogans as a potential druggable targets for designing of inhibitors for them.Communicated by Ramaswamy H. Sarma.

Leptospira interrogans serovar Copenhageni's genome harbors two CRISPR-Cas systems belonging to subtypes I-B and I-C. However, in L. interrogans, the subtype I-C locus lacks an array component essential for assembling an interference complex. Thus, the reason for sustaining the expense of a cluster of cas genes (I-C) is obscure. Type I-C (previously Dvulg) is the only CRISPR subtype that engages Cas5c, a Cas5 variant, to process precursor CRISPR-RNA (pre-crRNA). In this study, thus, the recombinant Cas5c (rLinCas5c) of L.interrogans and its mutant variants were cloned, expressed, and purified. The purified rLinCas5c is illustrated as metal-independent, sequence, and size-specific cleavage on repeat RNA and pre-crRNA of subtype I-B or orphan CRISPR array. However, the Cas6-bound mature crRNA of subtype I-B fends off from the rLinCas5c activity. In addition, rLinCas5c holds metal and size-dependent DNase activity. The bioinformatics analysis of LinCas5c inferred that it belongs to the subgroup Cas5c-B. Substitution of Phe141 with a more conserved His residue and deletion of unique (β1'-β2') insertions usher a gain of rLinCas5c activity over nucleic acid. Overall, our results uncover the functional diversity of Cas5c ribonucleases and infer an incognito auxiliary role in the absence of a cognate CRISPR array., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Manish Kumar reports financial support was provided by India Ministry of Science & Technology Department of Biotechnology., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Leptospirosis, a global reemerging zoonosis caused by the spirochete Leptospira, has severe human and veterinary implications. Cell wall hydrolase (LIC&#95;10271) with LytM (peptidase M23) and LysM domains are found to be associated with various pathogenic bacteria. These domains regulate effects on extracellular matrix and biofilm components, which promote cell wall remodeling and pathogen dissemination in the host. In this study, we present the cloning, expression, purification, and characterization of LIC&#95;10271. To determine the localization of LIC&#95;10271 within the inner membrane of Leptospira, Triton X-114 subcellular fractionation and immunoblot studies were performed. Furthermore, r-LIC&#95;10271 binds with peptidoglycan, lipopolysaccharide, and laminin in a dose-dependent manner. Analysis of the signal peptide, M23, and LysM domains revealed conservation primarily within the P1 group of Leptospira, which encompasses the most pathogenic species. Moreover, the presence of native-LIC&#95;10271 in the inner membrane and the distribution of M23 and LysM domains across pathogenic strains indicates their potential involvement in the interaction between the host and Leptospira., Competing Interests: Declaration of competing interest The authors declare no conflict of interest in any aspect., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Introduction: A high incidence of human leptospirosis is recorded on Mayotte, an oceanic island located in southwestern Indian Ocean, but the severity of the disease appears relatively mild in terms of mortality rate and admission to the intensive care unit. It has been proposed that mild leptospirosis may result from a limited virulence of some of the occurring Leptospira species to which the population is exposed., Methods: Clinical and biological data of patients admitted to the Centre Hospitalier de Mayotte were collected and the infecting Leptospira species were determined through molecular typing., Results: Leptospira interrogans was detected in the minority of admitted patients but most of these patients suffered from severe forms, with 50% admitted to intensive care unit and suffering from organ failures. Nineteen percent of patients infected with Leptospira borgpetersenii were admitted to the intensive care, with 13% displaying organ failures, and one patient died. Leptospira mayottensis was found in 28% of the patients and not a single severe case was observed., Discussion: The distribution of Leptospira species in patients was not different from that reported 10-15 years ago and bacterial genotypes were very closely related to those previously reported. These results highlight the importance of the diversity of pathogenic Leptospira circulating on Mayotte island and are in keeping with distinct outcome of the disease depending on the infecting Leptospira . Altogether, presented data support that the infecting Leptospira species is an important driver of disease severity in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Rajaonarivelo, Desmoulin, Maillard, Collet, Baudino, Jaffar-Bandjee, Blondé, Raffray and Tortosa.)

The aim of this review is to summarize the current knowledge on the role of σ factors in a highly invasive spirochaete Leptospira interrogans responsible for leptospirosis that affects many mammals, including humans. This disease has a significant impact on public health and the economy worldwide. In bacteria, σ factors are the key regulators of gene expression at the transcriptional level and therefore play an important role in bacterial adaptative response to different environmental stimuli. These factors form a holoenzyme with the RNA polymerase core enzyme and then direct it to specific promoters, which results in turning on selected genes. Most bacteria possess several different σ factors that enable them to maintain basal gene expression, as well as to regulate gene expression in response to specific environmental signals. Recent comparative genomics and in silico genome-wide analyses have revealed that the L. interrogans genome, consisting of two circular chromosomes, encodes a total of 14 σ factors. Among them, there is one putative housekeeping σ 70 -like factor, and three types of alternative σ factors, i.e., one σ 54 , one σ 28 and 11 putative ECF (extracytoplasmic function) σ E -type factors. Here, characteristics of these putative σ factors and their possible role in the L. interrogans gene regulation (especially in this pathogen's adaptive response to various environmental conditions, an important determinant of leptospiral virulence), are presented., (© 2023 The Author. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Leptospirosis, a major zoonotic disease caused by pathogenic Leptospira spp. is recognized globally as an emerging zoonotic disease. Whole-genome sequencing reveals hidden messages about Leptospira's pathogenesis. We used Single Molecule Real-Time (SMRT) sequencing to obtain complete genome sequences of twelve L. interrogans isolates from febrile patients from Sri Lanka for a comparative whole genome sequencing study. The sequence data generated 12 genomes with a coverage greater than X600 with sizes ranging from 4.62 Mb to 5.16 Mb, and a G + C content ranging from 35.00% to 35.42%. The total number of coding sequences predicted by the NCBI (National Center for Biotechnology Information) genome assembly platform ranged from 3845 to 4621 for the twelve strains. Leptospira serogroup with similar-sized LPS biosynthetic loci that belonged to the same clade had a close relationship in the phylogenetic analysis. Nonetheless, variations in the genes encoding sugar biosynthesis were found in the serovar determinant region (rfb locus). Type I and Type III CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems were found in all of the strains. Genome BLAST Distance Phylogeny of these sequences allowed for detailed genomic strain typing. These findings may help us better understand the pathogenesis, develop a tools for early diagnosis, comparative genomic analysis and evolution of Leptospira., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Published by Elsevier B.V.)

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch's molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen's ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin&#95;I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin&#95;I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates into LinCas11b. A mutant variant LinCascade -Cas11b that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade -Cas11b rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2023. The Author(s).)

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

Leptospirosis is an infectious disease caused by Leptospira spp. and affects animals and humans. Reports of leptospirosis in bats have increased and prompted epidemiological research in Brazil. This study aimed to perform a molecular and epidemiological investigation of pathogenic Leptospira spp. in bat kidneys. The total DNA was extracted from 102 kidney samples from chiropterous of different species and cities in Rio Grande do Sul State (RS), Brazil. The polymerase chain reaction was used to amplify a fragment corresponding to lipL32 gene, which is only present in pathogenic Leptospira spp. lipL32 gene was detected in 22.5% (23/102) of the bat kidney tissues. Phylogenetic analysis showed that L. interrogans is circulating in bats in RS. Most species of the bats collected were insectivores. Pathogenic Leptospira spp. detection in bats demonstrated that these animals participate in the infection chain of leptospirosis and, therefore, may play as reservoirs and disseminators of this microorganism. Thus, it is important to monitor infectious agents, especially with zoonotic potential in bats., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A 2-year-old male mongoose-scat-detection dog was diagnosed with leptospirosis by urine PCR. The patient developed acute renal failure, hepatic dysfunction, and disseminated intravascular coagulation. Treatment with antibiotics was administered, including ampicillin and doxycycline, and supportive care management was provided. Seroconversion against serogroup Hebdomadis was observed on day 8. The leptospiral gene flaB was detected only in urine collected on day 1, from which Leptospira interrogans ST329 was identified by multilocus sequence typing using seven housekeeping genes. L. interrogans serogroup Hebdomadis ST329 has been isolated from mongooses and humans in Okinawa, Japan. This patient received early treatment with antibiotics, which may have contributed to the early recovery of renal function and removal of L. interrogans from kidney tissue.

Leptospirosis in ruminants causes reproductive failures leading to important economic losses. This study assessed the occurrence and genetically identified Leptospira spp. in the follicular fluid (FF) of naturally infected live cows. A total of 251 asymptomatic cows from different commercial dairy herds were subjected to ovum-pick up technique for follicular fluid sampling. PCR was performed for Leptospira spp. detection and phylogenetic analysis was later implemented for sequencing. From 251 samples analyzed, 67 (26.7 %) were lipL32-PCR positive, confirming the presence of leptospiral DNA on FF. Furthermore, it was possible to amplify and sequence nine strains after secY nested-PCR. All of them were identified as L. interrogans, with 100 % of identity with strains belonging to Sejroe serogroup. Our findings reveal a high occurrence of infection of Leptospira in the ovarium of asymptomatic cows, highlighting the importance of considering the silent leptospirosis syndrome when screening animals for assisted reproductive biotechniques., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Leptospirosis is an important cause of morbidity and mortality worldwide. Disease severity ranges from asymptomatic colonization to widespread hemorrhage and multiorgan dysfunction. The causative agents, Leptospira spp., are zoonotic Gram-negative spirochetes. One important step in pathogenesis is binding of bacterial adhesins to host components. Previously our laboratory identified two L. interrogans candidate adhesins, LIC11574 and LIC13411, that bind to VE-cadherin in vitro . In the current study, we demonstrate the ability of two strains of pathogenic L. interrogans to disrupt the localization of VE-cadherin, a protein important to maintaining inter-endothelial junctions. Purified MBP-LIC11574 and MBP-LIC13411 bind human dermal microvascular endothelial cells in a pattern reminiscent of VE-cadherin, but do not disrupt VE-cadherin localization. Genes encoding the candidate adhesins from pathogenic Leptospira were cloned in an overexpression vector and introduced into non-pathogenic L. biflexa , creating gain-of-function strains producing LIC11574 or LIC13411. Protein production and localization to the outer membrane were confirmed by Triton X-114 fractionation. Although these strains do not disrupt VE-cadherin localization, production of LIC13411 increases binding of non-pathogenic Leptospira to human endothelial cells and specifically to VE-cadherin. In a short-term murine model of infection, LIC13411 production led to increased burdens of the non-pathogen in the lung, liver, kidney, and bladder. These data confirm the role of LIC13411 as an adhesin in Leptospira spp. and implicate it in dissemination to multiple organs. Importantly, anti-adhesin therapy has been shown to have many benefits over classical antibiotics. Taken together, this work provides novel insight into the pathogenesis of Leptospira spp. and identifies LIC13411 as a potential prophylactic and therapeutic target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Surdel, Hahn, Anderson and Coburn.)

Leptospira interrogans serogroup Icterohaemorrhagiae is the predominant pathogen causing leptospirosis in China and is still used as the vaccine strain for the current human inactivated vaccine. Unlike the clade ST17, which is distributed worldwide, ST1 is the most prevalent in serogroup Icterohaemorrhagiae in China. To further characterize leptospiral pathogens, isobaric tags for relative and absolute quantitation and parallel reaction monitoring were used to analyze differences at the proteomic level between serogroup Icterohaemorrhagiae vaccine strain 56001 (ST1) and circulating isolate 200502 (ST17) from different periods. Two hundred and eighty-one proteins were differentially expressed between the circulating isolate and vaccine strain, of which 166 were upregulated (> 1.2-fold change, P < 0.05) and 115 (< 0.8-fold change, P < 0.05) were downregulated. Function prediction revealed that nine upregulated proteins were outer membrane proteins, including several known immunogenic and/or virulence-related proteins, such as ompL1, LipL71, and LipL41. Furthermore, important expression differences in carbohydrate, amino acid, and energy metabolism and transport proteins were identified between both strains from different clusters, suggesting that these differences may reflect metabolic diversity and the potential of the pathogens to adapt to different environments. In summary, our findings provide insights into a better understanding of the component strains of the Chinese human leptospirosis vaccine at the proteomic level. Additionally, these data facilitate evaluating the mechanisms by which pathogenic Leptospira species adapt to the host environment., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Analysis of Leptospira dissemination and colonization of sex organs in rodents is of significant value as it queries the possibility of mammal-to-mammal venereal transmission. The aim of our study was to evaluate the presence and viability of Leptospira interrogans in testes of mice using models of infection that we previously developed. Using sublethal and lethal doses of bioluminescent strains of L. interrogans serovars Manilae and Copenhageni, we visualized the presence of leptospires in testes of C57BL/6 mice as early as 30 min and up to days 3-4 postinfection. This was confirmed by qPCR for the Copenhageni serovar after lethal infection of C3H/HeJ mice. In this model, no histopathological changes were noticed in testis. We further studied persistence of serovar Copenhageni in C3H/HeJ testes after lethal and sublethal infection, with different doses of leptospires. No viable leptospires were recovered from testes of lethally infected mice. However, we found live culturable Leptospira in testes of 19/19 (100%) sublethally infected mice at the acute phase but not at 15 days postinfection, which corresponds to the chronic phase of renal colonization. The data suggest that colonization of testes with live and potentially infectious leptospires is transient and limited to the spirochetemic phase of infection. Further studies are necessary to evaluate if presence of Leptospira in testes of mice leads to excretion in semen and to venereal transmission to female mice. IMPORTANCE Analysis of venereal transmission of Leptospira is important to determine if direct animal to animal transmission occurs, which could impact measures to prevent and treat leptospirosis. The goal of this study was to determine if live Leptospira colonize mouse testes. We found that colonization of mouse testes with live Leptospira was transient and limited to the acute spirochetemic phase of infection and that transient colonization of the testes was insufficient to cause histopathological changes.

Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

In this study, we provide the first characterization of a chemoreceptor from Leptospira interrogans, the cause of leptospirosis. This receptor is related to the Aer2 receptors that have been studied in other bacteria. In those organisms, Aer2 is a soluble receptor with one or two PAS-heme domains and signals in response to O 2 binding. In contrast, L. interrogans Aer2 ( Li Aer2) is an unusual membrane-bound Aer2 with a periplasmic domain and three cytoplasmic PAS-heme domains. Each of the three PAS domains bound b -type heme via conserved Eη-His residues. They also bound O 2 and CO with similar affinities to each other and other PAS-heme domains. However, all three PAS domains were uniquely hexacoordinate in the deoxy-heme state, whereas other Aer2-PAS domains are pentacoordinate. Similar to other Aer2 receptors, Li Aer2 could hijack the E. coli chemotaxis pathway but only when it was expressed with an E. coli high-abundance chemoreceptor. Unexpectedly, the response was inverted relative to classic Aer2 receptors. That is, Li Aer2 caused E. coli to tumble (it was signal-on) in the absence of O 2 and to stop tumbling in its presence. Thus, an endogenous ligand in the deoxy-heme state was correlated with signal-on Li Aer2, and its displacement for gas-binding turned signaling off. This response also occurred in a soluble version of Li Aer2 lacking the periplasmic domain, transmembrane (TM) region, and first two PAS domains, meaning that PAS3 alone was sufficient for O 2 -mediated control. Future studies are needed to understand the unique signaling mechanisms of this unusual Aer2 receptor. IMPORTANCE Leptospira interrogans, the cause of the zoonotic infection leptospirosis, is found in soil and water contaminated with animal urine. L. interrogans survives in complex environments with the aid of 12 chemoreceptors, none of which has been explicitly studied. In this study, we characterized the first L. interrogans chemoreceptor, Li Aer2, and reported its unique characteristics. Li Aer2 is membrane-bound, has three cytoplasmic PAS-heme domains that each bound hexacoordinate b -type heme and O 2 turned Li Aer2 signaling off. An endogenous ligand in the deoxy-heme state was correlated with signal-on Li Aer2 and its displacement for O 2 -binding turned signaling off. Our study corroborated previous findings that Aer2 receptors are O 2 sensors, but also demonstrated that they do not all function the same way.

Canine leptospirosis is a worldwide zoonosis, varying from asymptomatic and chronic infections to clinical acute disease. In many parts of the world Leptospira interrogans serogroup Icterohaemorrhagiae strains have great epidemiological importance, being the most prevalent on dogs. The present study aims to characterize and compare strains/sequences belonging to the serogroup Icterohaemorrhagiae recovered from clinically ill and asymptomatic dogs. Based on secY gene sequences of L. interrogans serovar Icterohaemorrhagiae, we have studied genetic diversity of strains obtained from 13 dogs, including dogs with clinical signs of acute leptospirosis, asymptomatic dogs and animals with chronic kidney disease, all of them from the same geographical area, the state of Rio de Janeiro, Brazil. No genetic variations on secY gene were observed between strains/groups. No significant associations were observed between clinical status and age, sex or vaccinal status. The same strain leads to different clinical outcomes on canine leptospirosis. The answer for this will rise from deep studies regarding whole genomic sequencing of the strains, as well as proteomics. Those studies may provide key information for understanding of the clinical manifestation of the disease., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In this study, we genotyped samples from environmental reservoirs (surface water and soil), colonized rat specimens, and cases of human severe leptospirosis from an endemic urban slum in Brazil, to determine the molecular epidemiology of pathogenic Leptospira and identify pathways of leptospirosis infection. We identified a well-established population of Leptospira interrogans serovar Copenhageni common to human leptospirosis cases, and animal and environmental reservoirs. This finding provides genetic evidence for a potential environmental spillover pathway for rat-borne leptospirosis through the environment in this urban community and highlights the importance of environmental and social interventions to reduce spillover infections., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. A universal vaccine against leptospirosis is likely to require highly conserved epitopes from pathogenic leptospires that are exposed on the bacterial surface and that generate a protective and sterilizing immune response. Our group recently identified several genes predicted to encode TonB-dependent receptors (TBDR) in Leptospira interrogans using a reverse vaccinology approach. Three leptospiral TBDRs were previously described and partially characterized as ferric-citrate, hemin, and cobalamin transporters. In the current study, we designed a fusion protein composed of predicted surface-exposed epitopes from three conserved leptospiral TBDRs. Based on their three-dimensional structural models and the prediction of immunogenic regions, nine putative surface-exposed fragments were selected to compose a recombinant chimeric protein. A Mycobacterium bovis BCG strain expressing this chimeric antigen encoded in the pUP500/P pAN mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with an endpoint dose of L. interrogans (p < 0.001). No animal survived in the negative control group. Immunization with our recombinant BCG elicited a humoral immune response against leptospiral TBDRs, as demonstrated by ELISA and immunoblot. No leptospiral DNA was detected by lipL32 qPCR in the kidneys of vaccinated hamsters. Similarly, no growth was observed in macerated kidney cultures from the same animals, suggesting the induction of a sterilizing immune response. Design of new vaccine antigens based on the structure of outer membrane proteins is a promising approach to overcome the impact of leptospirosis by vaccination. KEY POINTS: • Predicted surface-exposed epitopes were identified in three leptospiral TBDRs. • An M. bovis BCG strain expressing a chimeric protein (rTBDRchi) was constructed. • Hamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Leptospira interrogans, the causative agent of most cases of human leptospirosis, must respond to myriad environmental signals during its free-living and pathogenic lifestyles. Previously, we compared L. interrogans cultivated in vitro and in vivo using a dialysis membrane chamber (DMC) peritoneal implant model. From these studies emerged the importance of genes encoding the Peroxide responsive regulators PerRA and PerRB. First described in in Bacillus subtilis, PerRs are widespread in Gram-negative and -positive bacteria, where regulate the expression of gene products involved in detoxification of reactive oxygen species and virulence. Using perRA and perRB single and double mutants, we establish that L. interrogans requires at least one functional PerR for infectivity and renal colonization in a reservoir host. Our finding that the perRA/B double mutant survives at wild-type levels in DMCs is noteworthy as it demonstrates that the loss of virulence is not due to a metabolic lesion (i.e., metal starvation) but instead reflects dysregulation of virulence-related gene products. Comparative RNA-Seq analyses of perRA, perRB and perRA/B mutants cultivated within DMCs identified 106 genes that are dysregulated in the double mutant, including ligA, ligB and lvrA/B sensory histidine kinases. Decreased expression of LigA and LigB in the perRA/B mutant was not due to loss of LvrAB signaling. The majority of genes in the perRA and perRB single and double mutant DMC regulons were differentially expressed only in vivo, highlighting the importance of host signals for regulating gene expression in L. interrogans. Importantly, the PerRA, PerRB and PerRA/B DMC regulons each contain multiple genes related to environmental sensing and/or transcriptional regulation. Collectively, our data suggest that PerRA and PerRB are part of a complex regulatory network that promotes host adaptation by L. interrogans within mammals., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis vaccines that elicit broad protection against a range of pathogenic Leptospira spp. would overcome a major drawback of currently licensed bacterin vaccines. Live attenuated vaccine produced from a lipopolysaccharide (LPS) mutant strain of L. interrogans serovar Manilae M1352 (Live M1352) stimulated better protective efficacy than heat killed M1352 (HK M1352) against a heterologous challenge with L. interrogans serovar Pomona. To identify antigens of Live M1352 potentially responsible for cross protection, in vivo-induced antigen technology (IVIAT), a powerful tool to identify in vivo-induced (ivi) genes expressed during infection, was employed in this study. Pooled sera from hamsters immunized with Live M1352 were sequentially adsorbed with various preparations of in vitro grown M1352. The pre-adsorbed sera were used to screen a genomic expression library of M1352. Nineteen strongly reactive clones were selected for DNA sequencing. These ivi genes are conserved in most Leptospira strains. Four selected genes including LIMLP&#95;04965 (tolB), LIMLP&#95;01535, LIMLP&#95;06785 (fliI), and LIMLP&#95;14930 were confirmed for their upregulated expression in kidneys of infected hamsters by RT-qPCR, suggesting their role in leptospiral infection. These ivi proteins represent potential targets for vaccine candidates that warrant further investigation for their protective efficacy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rattus norvegicus (Norway rat) is the main reservoir host of pathogenic Leptospira, the causative agent of leptospirosis, in urban environments. Pathogenic Leptospira forms biofilms in the environment, possibly contributing for bacterial survival and maintenance. Nonetheless, biofilms have not yet been studied in natural animal reservoirs presenting leptospiral renal carriage. Here, we described biofilm formation by pathogenic Leptospira inside the renal tubules of R. norvegicus naturally infected and captured in an urban slum endemic for leptospirosis. From the 65 rats carrying Leptospira in their kidneys, 24 (37%) presented biofilms inside the renal tubules. The intensity of leptospiral colonization in the renal tubules (OR: 1.00; 95% CI 1.05-1.1) and the type of occlusion pattern of the colonized renal tubules (OR: 3.46; 95% CI 1.20-9.98) were independently associated with the presence of Leptospira biofilm. Our data showed that Leptospira interrogans produce biofilms during renal chronic colonization in rat reservoirs, suggesting a possible role for leptospiral biofilms in the pathogenesis of leptospirosis and bacterial carriage in host reservoirs., Competing Interests: The authors have declared that no competing interests exist.

Indian Ocean islands are endemic areas for human and animal leptospirosis. Maintenance host species for Leptospira spp. have still not been completely elucidated, and recently the role of cats (Felis catus) has been questioned. This cross-sectional study aims to determine whether cats are part of the maintenance community of different strains of Leptospira spp. in Reunion Island. The prevalence of Leptospira infection in an opportunistic sample of stray and domestic cats (n = 92) from Reunion Island has been studied using serological (microagglutination test) and molecular detection (polymerase chain reaction (PCR)). The results revealed a seroprevalence of 37.0% (34/92) (cut-off 1:40) without a significant difference in the living conditions of animals. The predominant serogroup was Icterohaemorrhagiae, but Ballum, Cynopteri and Australis were also detected. Using PCR, 28.6% (12/42) of stray cats were tested positive. Leptospiral DNA was detected in renal tissue, urine and blood of respectively 14.3% (6/42), 10.3% (4/39) and 11.9% (5/42) of stray cats, but 0% (0/3), 0% (0/50) and 0% (0/36) of domestic cats (P = non-applicable, P = 0,038, P = 0,058 respectively). Partial rrs gene (16S rRNA) sequencing identified Leptospira interrogans in all PCR-positive samples. Our study confirms that renal carriage and urinary shedding are possible, positioning cats, and especially stray cats as potential actors within the maintenance community of L. interrogans in Reunion Island.

Leptospirosis, a zoonotic disease with worldwide distribution, is caused by spirochetes of the genus Leptospira. In dogs, this disease is frequently misdiagnosed. Few studies have attempted to associate the detection of Leptospira spp. infection with clinicopathological and renal histopathological findings using a multidisciplinary approach. The present study isolated and characterized Leptospira spp. obtained from naturally infected dogs and described relevant clinical and histopathological findings. Blood and urine were collected from 57 dogs with clinical symptomatology suggestive of leptospirosis; 38 cases were confirmed by PCR in urine or by culture or microscopic agglutination testing (titers ≥800). A total of 12 strains of pathogenic Leptospira were isolated from the studied dogs (seven in blood, four in urine and one in both blood and urine samples). All isolates were characterized as Leptospira interrogans serogroup Icterohaemorrhagiae. Of the confirmed cases, almost one-third of the animals had been vaccinated. Our analysis of laboratory testing revealed that azotemia and proteinuria were statistically significant predictors of infection. The main histopathological findings seen in kidney tissues were necrosis, degeneration, tubular regeneration, mononuclear inflammatory infiltrate and congestion. A multidisciplinary approach involving clinicopathological and histopathological characterization of renal involvement can aid in the identification of acute leptospirosis infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In Leptospira interrogans serovar Copenhageni, the CRISPR-Cas I-B locus possesses a CRISPR array between the two independent cas-operons. Using the reverse transcription-PCR and the in vitro endoribonuclease assay with Cas6 of Leptospira (LinCas6), we account that the CRISPR is transcriptionally active and is conventionally processed. The LinCas6 specifically excises at one site within the synthetic cognate repeat RNA or the repeats of precursor-CRISPR RNA (pre-crRNA) in the sense direction. In contrast, the antisense repeat RNA is cleaved at multiple sites. LinCas6 functions as a single turnover endoribonuclease on its repeat RNA substrate, where substitution of one of predicted active site residues (His38) resulted in reduced activity. This study highlights the comprehensive understanding of the Leptospira CRISPR array transcription and its processing by LinCas6 that is central to RNA-mediated CRISPR-Cas I-B adaptive immunity., (Copyright © 2021 Elsevier B.V. All rights reserved.)