Ketone body oxidation and susceptibility to ethyl acetoacetate in a novel hemolytic multidrug-resistant strain Leptospira interrogans KeTo originated from sewage water.

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 RGAT (99.3%), followed by L. kirschneri 3522CT (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.

Leptospira interrogans biofilm transcriptome highlights adaption to starvation and general stress while maintaining virulence.

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.

In silico analysis and functional characterization of a leucine-rich repeat protein of Leptospira interrogans.

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 ß-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.

A probable cluster of premature birth and stillbirth caused by Leptospira interrogans serogroup Hebdomadis in an integrated swine farm in Nagasaki Prefecture, Japan.

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.

Leptospira interrogans encodes a canonical BamA and three novel noNterm Omp85 outer membrane protein paralogs.

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.

Pathogenic Leptospira interrogans in Jos North Abattoir, Nigeria: Occurrence, serology, and molecular characterization.

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.

Molecular surveillance of zoonotic pathogens from wild rodents in the Republic of Korea.

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.

Outbreak of Intermediate Species Leptospira venezuelensis Spread by Rodents to Cows and Humans in L. interrogans-Endemic Region, Venezuela.

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.

Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential.

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.

Spatiotemporal assessment of pathogenic Leptospira in subtropical coastal watersheds.

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.

Bacteriological and Serological Investigation of Leptospirosis in Dogs and Pigs in Palau.

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.

Isolation and molecular identification of Leptospira santarosai and Leptospira interrogans in equines from eastern Mexico.

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.

Leptospira borgptersenii and Leptospira interrogans identified in wild mammals in Rio Grande do Sul, Brazil.

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.

LIC_12757 from the pathogenic spirochaete Leptospira interrogans encodes an autoregulated ECF σE-type factor.

ECF (extracytoplasmic function) σ factors, members of the σ70-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 σE-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_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_12757 gene, its expression with the pET system and biochemical characterization of LIC_12757. By performing EMSA and in vitro transcription assays, we provide strong evidence that LIC_12757 indeed functions as a transcriptional factor that enables RNA polymerase to bind to the specific σE-type promoter and to initiate transcription. Interestingly, we demonstrate that LIC_12757 is autoregulated at the transcriptional level. Our study is a first step towards determining key aspects of LIC_12757 function in pathogenic Leptospira.

AMR mechanisms in L. interrogans serovars: a comprehensive study.

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.

Machine learning-based motion tracking reveals an inverse correlation between adhesivity and surface motility of the leptospirosis spirochete.

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.

Clinical manifestations of human leptospirosis: bacteria matter.

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.

Differential processing of CRISPR RNA by LinCas5c and LinCas6 of Leptospira.

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 (ß1'-ß2') 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.

Sigma factors of RNA polymerase in the pathogenic spirochaete Leptospira interrogans, the causative agent of leptospirosis.

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 σ70 -like factor, and three types of alternative σ factors, i.e., one σ54 , one σ28 and 11 putative ECF (extracytoplasmic function) σE -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.

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

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