Random Insertional Mutagenesis of Leptospira interrogans, the Agent of Leptospirosis, Using a mariner Transposon

Laboratoire des Spirochètes, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France.
Journal of Bacteriology (Impact Factor: 2.81). 06/2005; 187(9):3255-8. DOI: 10.1128/JB.187.9.3255-3258.2005
Source: PubMed


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.

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    • "This work has significantly improved the feasibility of manipulating genes in pathogenic strains. For instance, we first developed systems for targeted mutagenesis and random transposon mutagenesis in the saprophyte L. biflexa and then applied these approaches in the pathogen L. interrogans [5-7]. However, the introduction of exogenous genetic information into pathogenic strains by electroporation [8] or conjugation [9] is still hindered by poor transformation efficiencies. "
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    ABSTRACT: In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.
    Full-text · Article · Jun 2011 · BMC Microbiology
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    • "To definitively determine the importance of any candidate virulence attribute, the genetic basis of Leptospira species virulence will need to be determined at the level of gene function and regulation. Recent developments in the genetic manipulation of Leptospira species (Bauby et al., 2003; Bourhy et al., 2005; Louvel and Picardeau, 2007; Croda et al., 2008; Ko et al., 2009; Murray et al., 2009; Aviat et al., 2010; Poggi et al., 2010) will facilitate identification of genes whose products are essential for, or contribute to, pathogenesis of leptospirosis. In a groundbreaking demonstration of the power of this approach, the gene encoding an OmpA-like outer membrane protein, loa22, was demonstrated to contribute to the ability of L. interrogans serovar Lai to cause disease in hamsters and guinea pigs (Ristow et al., 2007). "
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    ABSTRACT: Since the 1980s, the incidence of severe pulmonary hemorrhage caused by Leptospira spp. infection has increased. The mild, non-specific symptoms or the more classical form of severe disease with hepatorenal manifestations, Weil's syndrome, predominate world-wide. However, several regions of the world have seen increases in numbers of patients with pulmonary hemorrhage attributed to leptospirosis. The reasons behind the emergence of this syndrome, which carries a high mortality rate, are not known. Several avenues for future research may shed light on the mechanisms involved in development of pulmonary hemorrhage, and inform targeted therapeutics to improve outcomes. Possibilities to consider include: (1) emergence of new bacterial strains, (2) acquisition of virulence traits by strains in the endemic regions, (3) changes in underlying health of the affected human populations, and (4) increased recognition of the syndrome and better record keeping by the medical and veterinary communities. Determining the causes of emerging clinical manifestations presents challenges and opportunities for potentially life-saving research into the pathogenesis of a number of infectious diseases, including leptospirosis.
    Full-text · Article · Jan 2011 · Frontiers in Cellular and Infection Microbiology
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    • "Transposon mutagenesis was conducted in L. interrogans sv. Manilae strain L495 as described previously (Bourhy et al., 2005; Murray et al., 2008; 2009a). In mutant M895 the transposon had inserted into the LPS synthesis locus within a gene named Lman_1456, homologous to la1641 of L. interrogans sv. "
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    ABSTRACT: Leptospira interrogans is the causative agent of leptospirosis. Lipopolysaccharide (LPS) is the major outer membrane component of L. interrogans. It is the dominant antigen recognized during infection and the basis for serological classification. The structure of LPS and its role in pathogenesis are unknown. We describe two defined mutants of L. interrogans serovar Manilae with transposon insertions in the LPS locus. Mutant M895 was disrupted in gene la1641 encoding a protein with no known homologues. M1352 was disrupted in a gene unique to serovar Manilae also encoding a protein of unknown function. M895 produced truncated LPS while M1352 showed little or no change in LPS molecular mass. Both mutants showed altered agglutination titres against rabbit antiserum and against a panel of LPS-specific monoclonal antibodies. The mutants were severely attenuated in virulence via the intraperitoneal route of infection, and were cleared from the host animal by 3 days after infection. M895 was also highly attenuated via the mucosal infection route. Resistance to complement in human serum was unaltered for both mutants. While complementation of mutants was not possible, the attenuation of two independently derived LPS mutants demonstrates for the first time that LPS plays an essential role leptospiral virulence.
    Full-text · Article · Nov 2010 · Molecular Microbiology
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