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Inactivation of clpB in the pathogen Leptospira interrogans reduces virulence and resistance to stress conditions.

ABSTRACT: Leptospira interrogans is the causative agent of leptospirosis, which is an emerging zoonotic disease. Resistance to stress conditions is largely uncharacterized for this bacterium. We therefore decided to analyze a clpB mutant that we obtained by random transposon mutagenesis. The mutant did not produce any of the two isoforms of ClpB. The clpB mutant exhibited growth defects at 30° and 37°C and in poor nutrient medium and showed increased susceptibility to oxidative stress, whereas the genetically complemented strain was restored in ClpB expression and in vitro wild-type growth. We also showed that the clpB mutant was attenuated in virulence in an animal model of acute leptospirosis. Our findings demonstrate that ClpB is involved in the general stress response. The chaperone is also necessary, either directly or indirectly, for the virulence of the pathogen L. interrogans.

SUBMITTER: Lourdault K

PROVIDER: S-EPMC3165490 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Inactivation of clpB in the pathogen Leptospira interrogans reduces virulence and resistance to stress conditions.

Lourdault Kristel K Cerqueira Gustavo M GM Wunder Elsio A EA Picardeau Mathieu M

Infection and immunity 20110705 9

Leptospira interrogans is the causative agent of leptospirosis, which is an emerging zoonotic disease. Resistance to stress conditions is largely uncharacterized for this bacterium. We therefore decided to analyze a clpB mutant that we obtained by random transposon mutagenesis. The mutant did not produce any of the two isoforms of ClpB. The clpB mutant exhibited growth defects at 30° and 37°C and in poor nutrient medium and showed increased susceptibility to oxidative stress, whereas the genetic ...[more]

PMID: 21730091

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A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

Project description:Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

| S-EPMC4019197 | biostudies-literature

Leptospira interrogans catalase is required for resistance to H2O2 and for virulence.

Project description:Pathogenic Leptospira spp. are likely to encounter higher concentrations of reactive oxygen species induced by the host innate immune response. In this study, we characterized Leptospira interrogans catalase (KatE), the only annotated catalase found within pathogenic Leptospira species, by assessing its role in resistance to H(2)O(2)-induced oxidative stress and during infection in hamsters. Pathogenic L. interrogans bacteria had a 50-fold-higher survival rate under H(2)O(2)-induced oxidative stress than did saprophytic L. biflexa bacteria, and this was predominantly catalase dependent. We also characterized KatE, the only annotated catalase found within pathogenic Leptospira species. Catalase assays performed with recombinant KatE confirmed specific catalase activity, while protein fractionation experiments localized KatE to the bacterial periplasmic space. The insertional inactivation of katE in pathogenic Leptospira bacteria drastically diminished leptospiral viability in the presence of extracellular H(2)O(2) and reduced virulence in an acute-infection model. Combined, these results suggest that L. interrogans KatE confers in vivo resistance to reactive oxygen species induced by the host innate immune response.

| S-EPMC3486042 | biostudies-literature

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Project description:Systems biology conceptualizes biological systems as dynamic networks of interacting elements, whereby functionally important properties are thought to emerge from the structure of such networks. Owing to the ubiquitous role of complexes of interacting proteins in biological systems, their subunit composition and temporal and spatial arrangement within the cell are of particular interest. 'Visual proteomics' attempts to localize individual macromolecular complexes inside of intact cells by template matching reference structures into cryo-electron tomograms. Here we combined quantitative mass spectrometry and cryo-electron tomography to detect, count and localize specific protein complexes in the cytoplasm of the human pathogen Leptospira interrogans. We describe a scoring function for visual proteomics and assess its performance and accuracy under realistic conditions. We discuss current and general limitations of the approach, as well as expected improvements in the future.

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A pathogen-specific 4mC methyltransferase is critical for epigenetic regulation and virulence of Leptospira interrogans

Project description:Transcriptome analysis of a mutant in an orphan methyltranferase revealed global transcriptional regulation of 100 differentially expressed genes.

2020-10-09 | GSE138917 | GEO

Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

Project description:Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.

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The zoonotic pathogen Leptospira interrogans mitigates environmental stress through cyclic-di-GMP-controlled biofilm production.

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

| S-EPMC7293261 | biostudies-literature

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans.

Project description:In bacteria, DNA methylation can be facilitated by 'orphan' DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.

| S-EPMC7708080 | biostudies-literature

Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai.

Project description:BackgroundPathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown.ResultsA suicide plasmid for the fliY gene of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (bla) was constructed, and the inactivation of fliY gene in a mutant (fliY-) was confirmed by PCR and Western Blot analysis. The inactivation resulted in the mRNA absence of fliP and fliQ genes which are located downstream of the fliY gene in the same operon. The mutant displayed visibly weakened rotative motion in liquid medium and its migration on semisolid medium was also markedly attenuated compared to the wild-type strain. Compared to the wild-type strain, the mutant showed much lower levels of adhesion to murine macrophages and apoptosis-inducing ability, and its lethality to guinea pigs was also significantly decreased.ConclusionInactivation of fliY, by the method used in this paper, clearly had polar effects on downstream genes. The phentotypes observed, including lower pathogenicity, could be a consequence of fliY inactivation, but also a consequence of the polar effects.

| S-EPMC3224694 | biostudies-literature

Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans.

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| S-EPMC2723184 | biostudies-literature

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence.

Project description:Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model.

| S-EPMC4598399 | biostudies-literature

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