Spirochetes Of Leptospira Research Articles

Leptospira Leptolysin Contributes to Serum Resistance but Is Not Essential for Acute Infection.

Previous invitro 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 invitro, 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.

Evolutionary insights into the emergence of virulent Leptospira spirochetes.

Pathogenic Leptospira are spirochete bacteria which cause leptospirosis, a re-emerging zoonotic disease of global importance. Here, we use a recently described lineage of environmental-adapted leptospires, which are evolutionarily the closest relatives of the highly virulent Leptospira species, to explore the key phenotypic traits and genetic determinants of Leptospira virulence. Through a comprehensive approach integrating phylogenomic comparisons with in vitro and in vivo phenotyping studies, we show that the evolution towards pathogenicity is associated with both a decrease of the ability to survive in the environment and the acquisition of strategies that enable successful host colonization. This includes the evasion of the mammalian complement system and the adaptations to avoid activation of the innate immune cells by the highly-virulent Leptospira species (also called P1+ species), unlike other species belonging to the phylogenetically related P1- and P2 groups, as well as saprophytes. Moreover, our analysis reveals specific genetic determinants that have undergone positive selection during the course of evolution in Leptospira, contributing directly to virulence and host adaptation as demonstrated by gain-of-function and knock-down studies. Taken together, our findings define a new vision on Leptospira pathogenicity, identifying virulence attributes associated with clinically relevant species, and provide insights into the evolution and emergence of these life-threatening pathogens.

Epidemiology, Clinical and Laboratory Findings of Pediatric Leptospirosis in Southeastern Turkey

Background: Leptospirosis is a significant zoonotic infection caused by Leptospira spirochetes, which are distributed globally. Infection typically occurs through exposure to water or soil contaminated by the urine of mammals, including dogs, cattle, pigs, or rodents. Objectives: The objective of this study was to elucidate the epidemiological, clinical, and laboratory characteristics of leptospirosis cases in Turkey. Methods: In a retrospective review, we analyzed the clinical and laboratory data of all individuals under 18 years diagnosed with leptospirosis at a secondary care center in Southeast Turkey from the beginning of 2020 to the end of 2022. Results: Over the two-year period, 36 confirmed cases of leptospirosis in individuals under 18 were identified. All patients had been engaged in activities at wells in cornfields, assisting their families with irrigation or related to well operations. Of these patients, 3 (8%) resided in urban areas, while 33 (92%) lived in rural settings. None required intensive care, and there were no fatalities reported. The most common symptoms at presentation were fatigue (83%), fever (75%), and nausea/vomiting (75%). Laboratory tests revealed that all patients had positive C-reactive protein (CRP) levels, with a significant portion (61%) showing highly positive results (CRP > 100). Conclusions: In conclusion, leptospirosis should be considered by clinicians when evaluating patients, especially those with risk factors like occupational exposure. Prompt testing for the disease is advisable under these circumstances.

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

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

Signal-sensing triggers the shutdown of HemKR, regulating heme and iron metabolism in the spirochete Leptospira biflexa.

Heme and iron metabolic pathways are highly intertwined, both compounds being essential for key biological processes, yet becoming toxic if overabundant. Their concentrations are exquisitely regulated, including via dedicated two-component systems (TCSs) that sense signals and regulate adaptive responses. HemKR is a TCS present in both saprophytic and pathogenic Leptospira species, involved in the control of heme metabolism. However, the molecular means by which HemKR is switched on/off in a signal-dependent way, are still unknown. Moreover, a comprehensive list of HemKR-regulated genes, potentially overlapped with iron-responsive targets, is also missing. Using the saprophytic species Leptospira biflexa as a model, we now show that 5-aminolevulinic acid (ALA) triggers the shutdown of the HemKR pathway in live cells, and does so by stimulating the phosphatase activity of HemK towards phosphorylated HemR. Phospho~HemR dephosphorylation leads to differential expression of multiple genes, including of heme metabolism and transport systems. Besides the heme-biosynthetic genes hemA and the catabolic hmuO, which we had previously reported as phospho~HemR targets, we now extend the regulon identifying additional genes. Finally, we discover that HemR inactivation brings about an iron-deficit tolerant phenotype, synergistically with iron-responsive signaling systems. Future studies with pathogenic Leptospira will be able to confirm whether such tolerance to iron deprivation is conserved among Leptospira spp., in which case HemKR could play a vital role during infection where available iron is scarce. In sum, HemKR responds to abundance of porphyrin metabolites by shutting down and controlling heme homeostasis, while also contributing to integrate the regulation of heme and iron metabolism in the L. biflexa spirochete model.

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.

A predictive risk map for human leptospirosis guiding further investigations in brown rats and surface water

ABSTRACT Leptospirosis is a zoonosis caused by the spirochete Leptospira spp. It is often not clear why certain areas appear to be hotspots for human leptospirosis. Therefore, a predictive risk map for the Netherlands was developed and assessed, based on a random forest model for human leptospirosis incidence levels with various environmental factors and rat density as variables. Next, it was tested whether misclassifications of the risk map could be explained by the prevalence of Leptospira spp. in brown rats. Three recreational areas were chosen, and rats (≥25/location) were tested for Leptospira spp. Concurrently, it was investigated whether Leptospira spp. prevalence in brown rats was associated with Leptospira DNA concentration in surface water, to explore the usability of this parameter in future studies. Approximately 1 L of surface water sample was collected from 10 sites and was tested for Leptospira spp. Although the model predicted the locations of patients relatively well, this study showed that the prevalence of Leptospira spp. infection in rats may be an explaining variable that could improve the predictive model performance. Surface water samples were all negative, even if they had been taken at sites with a high Leptospira spp. prevalence in rats.

The structure of Leptospira interrogans GAPDH sheds light into an immunoevasion factor that can target the anaphylatoxin C5a of innate immunity.

Leptospirosis is a neglected worldwide zoonosis involving farm animals and domestic pets caused by the Gram-negative spirochete Leptospira interrogans. This bacterium deploys a variety of immune evasive mechanisms, some of them targeted at the complement system of the host's innate immunity. In this work, we have solved the X-ray crystallographic structure of L. interrogans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to 2.37-Å resolution, a glycolytic enzyme that has been shown to exhibit moonlighting functions that potentiate infectivity and immune evasion in various pathogenic organisms. Besides, we have characterized the enzyme's kinetic parameters toward the cognate substrates and have proven that the two natural products anacardic acid and curcumin are able to inhibit L. interrogans GAPDH at micromolar concentration through a noncompetitive inhibition modality. Furthermore, we have established that L. interrogans GAPDH can interact with the anaphylatoxin C5a of human innate immunity in vitro using bio-layer interferometry and a short-range cross-linking reagent that tethers free thiol groups in protein complexes. To shed light into the interaction between L. interrogans GAPDH and C5a, we have also carried out cross-link guided protein-protein docking. These results suggest that L. interrogans could be placed in the growing list of bacterial pathogens that exploit glycolytic enzymes as extracellular immune evasive factors. Analysis of the docking results indicates a low affinity interaction that is consistent with previous evidence, including known binding modes of other α-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system.

Genomic insights into the c-di-GMP signaling and biofilm development in the saprophytic spirochete Leptospira biflexa.

C-di-GMP is a bacterial second messenger with central role in biofilm formation. Spirochete bacteria from Leptospira genus present a wide diversity, with species of medical importance and environmental species, named as saprophytic. Leptospira form biofilms in the rat's reservoir kidneys and in the environment. Here, we performed genomic analyses to identify enzymatic and effector c-di-GMP proteins in the saprophytic biofilm-forming species Leptospira biflexa serovar Patoc. We identified 40 proteins through local alignments. Amongst them, 16 proteins are potentially functional diguanylate cyclases, phosphodiesterases, or hybrid proteins. We also identified nine effectors, including PilZ proteins. Enrichment analyses suggested that c-di-GMP interacts with cAMP signaling system, CsrA system, and flagella assembly regulation during biofilm development of L. biflexa. Finally, we identified eight proteins in the pathogen Leptospira interrogans serovar Copenhageni that share high similarity with L. biflexa c-di-GMP-related proteins. This work revealed proteins related to c-di-GMP turnover and cellular response in Leptospira and their potential roles during biofilm development.

Analysis of Adhesion and Surface Motility of a Spirochete Bacterium.

Spirochetes are Gram-negative bacteria with helical or flat wave morphology and move using flagella residing beneath the outer membrane. Most commonly, flagellated bacteria swim in liquid. Meanwhile, some species of spirochete not only swim but keep moving after adhering to solid surfaces, and such amphibious motility is believed to be significant for pathogenicity. This chapter focuses on the zoonotic spirochete Leptospira and describes the method for measuring the spirochete adhesion and surface motility.

Seroprevalence and Molecular Characterization of Leptospira spp. in Rats Captured near Pig Farms in Colombia.

Gram-negative spirochete Leptospira spp. causes leptospirosis. Leptospirosis is still a neglected disease, even though it can cause potentially fatal infections in a variety of species including humans. The purpose of this study was to determine the seroprevalence of leptospirosis in pig farm captured rodents and characterize the isolated samples. Rats were captured, sampled, and euthanized in the vicinity of pig farms to obtain serum for microagglutination tests (MAT) and kidney tissues for PCR amplification of the 16S rRNA and LipL32 genes. A fraction of the 16S rRNA PCR product was sequenced and phylogenetically analyzed. The results showed a Leptospira seroprevalence of 13.8% (77/555) among the 555 captured rats. PCR positivity for Leptospira spp. reached 31.2% (156/500), and the positivity for pathogenic Leptospira spp. was 4% (22/500). Phylogenetic analysis matched eight samples with L. interrogans serovar icterohaemorrhagiae and two with L. interrogans serovar pyrogenes. Two sequences were located within the pathogenic Leptospira clade but did not match with any specific strain. The seroprevalence found in the rats around swine farms indicates a potential risk of transmission to the pigs. The identification of pathogenic Leptospira outlines the importance of more research as well as updating the current strategies for the diagnosis, control, and prevention of porcine leptospirosis in Colombia.

A case report on leptospirosis with pseudohepatorenal syndrome

The spirochete Leptospira interrogans causes leptospirosis, a zoonosis. There are numerous clinical symptoms of leptospirosis. Clinical manifestations might range from a subclinical infection with a mild fever to severe clinical symptoms with jaundice and renal failure. Here, a case of leptospirosis with acute renal failure, thrombocytopenia, and hyperbilirubinemia is presented. A 51-year-old man presented with fever, body aches, muscle aches, colored urine, and jaundice for 2 days. On serology report, L. interrogans AB IGM-CARD was weekly positive. In the Renal function test, urea and creatinine values were high as well as the liver function test was abnormal. The patient was given 1 g of ceftriaxone intravenously and 100 mg of doxycycline orally. Hepatorenal treatment was supportive and the outcome was positive. It may have been overlooked when the diagnosis of leptospirosis was confirmed.

Evaluation of the impact of chemical control on the ecology of Rattus norvegicus of an urban community in Salvador, Brazil.

The presence of synanthropic rodents, such as Rattus norvegicus, in urban environments generates high costs of prophylaxis and control, in large part due to the environmental transmission of the pathogenic spirochete Leptospira interrogans, which causes leptospirosis. In Salvador, Brazil, The Center for Control of Zoonosis (CCZ) is responsible for planning and implementing Rodent Control Programs (RCP) which are based on chemical rodenticide. However, these strategies have not been standardized for use in developing countries. This study aimed to identify the effect of a chemical control campaign on the demographic variables of urban R. norvegicus, analyzing relative abundance, sex structure, body mass, and age of the population, as well as the characterization of spatial distribution among households, rodent capture campaigns and interventions. This study was carried out during 2015 in three valleys of an urban poor community in Salvador. Individuals of R. norvegicus were systematically captured before (Pre-intervention) and three months (1st post-intervention) and six months (2nd post-intervention) after a chemical control intervention conducted by the CCZ in two valleys of the study area while the third valley was not included in the intervention campaign and was used as a non-intervention reference. We used analysis of variance to determine if intervention affected demographic variables and chi-square to compare proportions of infested households (Rodent infestation index-PII). During the chemical intervention, 939 households were visited. In the pre-intervention campaign, an effort of 310 trap nights resulted in 43 rodents captured, and in the 1st and 2nd, post-intervention campaigns resulted in 47 rodents captured over 312 trap nights and 36 rodents captured over 324 traps-nights, respectively. The rodent infestation index (PII) points did not show a reduction between the period before the intervention and the two periods after the chemical intervention (70%, 72%, and 65%, respectively). Regarding relative abundances, there was no difference between valleys and period before and two periods after chemical intervention (trap success valley 1: 0,18; 0,19; 0,18 / Valley 3 0,15; 0,17; 0,13/ P>0,05). Other demographic results showed that there was no difference in demographic characteristics of the rodent population before and after the intervention, as well as there being no influence of the application of rodenticide on the areas of concentration of capture of rodents between the campaigns. Our study indicates that the chemical control was not effective in controlling the population of R. norvegicus and provides evidence of the need for re-evaluation of rodent control practices in urban poor community settings.

Molecular Diagnostic Methods for the Detection of Leptospirosis

Leptospirosis is a widespread infectious disease caused by the spirochete Leptospira. The clinical features of leptospirosis are fever, headache, vomiting, jaundice, and the acute form of the disease is commonly called Weil’s disease. The microscopic agglutination test (MAT) is a gold standard method used to detect leptospirosis. However, it requires 14 days of time and skilled personnel to detect leptospirosis. Various molecular methods were developed for the rapid detection process, including polymerase chain reaction (PCR), multiplex PCR, nested PCR, real-time PCR, and Loop-mediated isothermal amplification (LAMP). Other immuno-based biosensor kits are readily available for the diagnosis of leptospirosis. Though these methods claim to be highly sensitive and specific, each method has its drawbacks. This review discusses the different molecular diagnostic techniques applied for the diagnosis of leptospirosis; elaborating on each method’s sensitivity, specificity, and detection time and the different samples of water, blood, and urine used.

Assembly of Cas7 subunits of Leptospira on the mature crRNA of CRISPR-Cas I-B is modulated by divalent ions.

The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni. The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg2+ ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg2+ ions. The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg2+ ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (Kd=26.81mM) between rLinCas7 and Mg2+ ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7's activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.

Motility of the Zoonotic Spirochete Leptospira: Insight into Association with Pathogenicity.

If a bacterium has motility, it will use the ability to survive and thrive. For many pathogenic species, their motilities are a crucial virulence factor. The form of motility varies among the species. Some use flagella for swimming in liquid, and others use the cell-surface machinery to move over solid surfaces. Spirochetes are distinguished from other bacterial species by their helical or flat wave morphology and periplasmic flagella (PFs). It is believed that the rotation of PFs beneath the outer membrane causes transformation or rolling of the cell body, propelling the spirochetes. Interestingly, some spirochetal species exhibit motility both in liquid and over surfaces, but it is not fully unveiled how the spirochete pathogenicity involves such amphibious motility. This review focuses on the causative agent of zoonosis leptospirosis and discusses the significance of their motility in liquid and on surfaces, called crawling, as a virulence factor.

Weil's Disease

Leptospirosis is a zoonotic disease that predominantly occurs in tropical and subtropical areas. It is caused by the spirochete Leptospira interrogans complex and may lead to syndromes that can vary from a subclinical infection or a mild febrile infection to a severe icterohemorrhagic condition. This is a case report on a 52-year-old farmer who had presented with upper gastrointestinal (GI) bleed. He was initially thought to be a case of complications of Chronic Liver Disease. However, he was later found to have icterus, renal failure, and hemorrhagic manifestations and his IgM ELISA for leptospira was positive. It was later confirmed by MAT. He was treated with antibacterials and supportive measures and his clinical and biochemical parameters improved. The occurrence of Weil's disease in the month of November and its occurrence in the state of Assam, India was unpredictable and hence posed as a diagnostic dilemma. It is a curable disease and hence needs timely intervention and treatment. This highlights the need for a better understanding of the natural course of this disease and improvement of public health measures.

Severe leptospirosis complicated with multiorgan dysfunction successfully managed with plasma exchange: a case report

BackgroundLeptospirosis is a common zoonotic infection caused by the spirochete Leptospira. The disease is more prevalent in the tropics, causing subclinical to severe illness leading to high morbidity and mortality.Case presentationA 77-year-old healthy Sri Lankan man presented to the Teaching Hospital Peradeniya with severe leptospirosis complicated with acute kidney injury, pulmonary hemorrhages, myocarditis, and severe thrombocytopenia. He was deteriorating despite treatment with intravenous antibiotics and methylprednisolone boluses. He made a dramatic improvement with two cycles of plasma exchange.ConclusionTherapeutic plasma exchange is a life-saving treatment modality in severe leptospirosis with multiorgan failure.

Role of the major determinant of polar flagellation FlhG in the endoflagella-containing spirochete Leptospira.

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