Heme rescues a two-component system Leptospira biflexa mutant

Unité de Biologie des Spirochètes, Institut Pasteur, Paris, France.
BMC Microbiology (Impact Factor: 2.73). 02/2008; 8(1):25. DOI: 10.1186/1471-2180-8-25
Source: PubMed


Heme is typically a major iron source for bacteria, but little is known about how bacteria of the Leptospira genus, composed of both saprophytic and pathogenic species, access heme.
In this study, we analysed a two-component system of the saprophyte Leptospira biflexa. In vitro phosphorylation and site-directed mutagenesis assays showed that Hklep is a histidine kinase which, after autophosphorylation of a conserved histidine, transfers the phosphate to an essential aspartate of the response regulator Rrlep. Hklep/Rrlep two-component system mutants were generated in L. biflexa. The mutants could only grow in medium supplemented with hemin or delta-aminolevulinic acid (ALA). In the pathogen L. interrogans, the hklep and rrlep orthologous genes are located between hemE and hemL genes, which encode proteins involved in heme biosynthesis. The L. biflexa hklep mutant could be complemented with a replicative plasmid harbouring the L. interrogans orthologous gene, suggesting that these two-component systems are functionally similar. By real-time quantitative reverse transcription-PCR, we also observed that this two-component system might influence the expression of heme biosynthetic genes.
These findings demonstrate that the Hklep/Rrlep regulatory system is critical for the in vitro growth of L. biflexa, and suggest that this two-component system is involved in a complex mechanism that regulates the heme biosynthetic pathway.

Download full-text


Available from: Jean-Michel Betton, Oct 05, 2015
11 Reads
  • Source
    • "Different genes with the same predicted function, such as putative metallopeptidases (LIC11149 and LIC10271), sensor or receiver proteins of two-component response regulators (LIC20012, LIC11201, LIC12807, LIC12979 and LIC13289), and adenylate/guanylate cyclase (LIC10900 and LIC11095) were found to be regulated in opposite directions. LIC20012, an ortholog of hklep encoding a sensor kinase of the Hklep/Rrlep two-component system involved in heme biosynthesis in L. biflexa [54], was down-regulated. However, an ortholog of rrlep regulator (LIC20013) was not differentially expressed. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Leptospirosis is a zoonosis of worldwide distribution caused by infection with pathogenic serovars of Leptospira spp. The most common species, L. interrogans, can survive in the environment for lengthy periods of time in between infection of mammalian hosts. Transmission of pathogenic Leptospira to humans mostly occurs through abraded skin or mucosal surfaces after direct or indirect contact with infected animals or contaminated soil or water. The spirochete then spreads hematogenously, resulting in multi-organ failure and death in severe cases. Previous DNA microarray studies have identified differentially expressed genes required for adaptation to temperature and osmolarity conditions inside the host compared to those of the environment. In order to identify genes involved in survival in the early spirochetemic phase of infection, we performed a transcriptional analysis of L. interrogans serovar Copenhageni upon exposure to serum in comparison with EMJH medium. One hundred and sixty-eight genes were found to be differentially expressed, of which 55 were up-regulated and 113 were down-regulated. Genes of known or predicted function accounted for 54.5 and 45.1% of up- and down-regulated genes, respectively. Most of the differentially expressed genes were predicted to be involved in transcriptional regulation, translational process, two-component signal transduction systems, cell or membrane biogenesis, and metabolic pathways. Our study showed global transcriptional changes of pathogenic Leptospira upon exposure to serum, representing a specific host environmental cue present in the bloodstream. The presence of serum led to a distinct pattern of gene expression in comparison to those of previous single-stimulus microarray studies on the effect of temperature and osmolarity upshift. The results provide insights into the pathogenesis of leptospirosis during the early bacteremic phase of infection.
    BMC Microbiology 01/2010; 10(1):31. DOI:10.1186/1471-2180-10-31 · 2.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The transposon TnSC189 was used to construct a mutant in the putative heme oxygenase gene hemO (LB186) of Leptospira interrogans. Unlike its parent strain, the mutant grew poorly in medium in which hemoglobin was the sole iron source. The putative heme oxygenase was over expressed in a His-tagged form, purified and was demonstrated to degrade heme in vitro. Unexpectedly, it was also found that the L. interrogans growth rate was significantly increased when medium was supplemented with hemoglobin, but only if ferrous iron sources were absent. This result was mirrored in the expression of some iron-related genes and suggests the presence of regulatory mechanisms detecting Fe2+ and hemoglobin. This is the first demonstration of a functional heme oxygenase from a spirochete.
    Microbes and Infection 06/2008; 10(7):791-7. DOI:10.1016/j.micinf.2008.04.010 · 2.86 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spcr) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization.
    Infection and immunity 10/2008; 76(12):5826-33. DOI:10.1128/IAI.00989-08 · 3.73 Impact Factor
Show more