The recent availability of the complete genome sequences of Leptospira interrogans, the agent of leptospirosis, has allowed the identification of several putative virulence factors. However, to our knowledge, attempts to carry out gene transfer in pathogenic Leptospira spp. have failed so far. In this study, we show that the Himar1 mariner transposon permits random mutagenesis in the pathogen L. interrogans. We have identified genes that have been interrupted by Himar1 insertion in 35 L. interrogans mutants. This approach of transposon mutagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of Leptospira, which remain largely unknown.
The spirochetes of the Leptospira genus contain saprophytic and pathogenic members, the latter being responsible for leptospirosis. Despite the recent sequencing of the genome of the pathogen L. interrogans, the slow growth of these bacteria, their virulence in humans, and a lack of genetic tools make it difficult to work with these pathogens. In contrast, the development of numerous genetic tools for the saprophyte L. biflexa enables its use as a model bacterium. Leptospira spp. require iron for growth. In this work, we show that Leptospira spp. can acquire iron from different sources, including siderophores. A comparative genome analysis of iron uptake systems and their regulation in the saprophyte L. biflexa and the pathogen L. interrogans is presented in this study. Our data indicated that, for instance, L. biflexa and L. interrogans contain 8 and 12 genes, respectively, whose products share homology with proteins that have been shown to be TonB-dependent receptors. We show that some genes involved in iron uptake were differentially expressed in response to iron. In addition, we were able to disrupt several putative genes involved in iron acquisition systems or iron regulation in L. biflexa. Comparative genomics, in combination with gene inactivation, gives us significant functional information on iron homeostasis in Leptospira spp.
The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.
Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.
The genus Leptospira belongs to the order Spirochaetales and is composed of both saprophytic and pathogenic members, such as Leptospira biflexa and L. interrogans, respectively. A major factor contributing to our ignorance of spirochetal biology has been the lack of methods available for genetic analysis of these organisms. In recent years, an E. coli‐L. biflexa shuttle vector has been constructed and a system for targeted mutagenesis and random transposon mutagenesis of the saprophyte L. biflexa has been developed. These studies enable the use of L. biflexa as a model bacterium among spirochetes.
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