Targeted mutagenesis in pathogenic Leptospira species: Disruption of the LigB gene does not affect virulence in animal models of leptospirosis

Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil.
Infection and immunity (Impact Factor: 4.16). 10/2008; 76(12):5826-33. DOI: 10.1128/IAI.00989-08
Source: PubMed

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 (Spc(r)) 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.

Download full-text


Available from: Julio Croda, Aug 24, 2015
  • Source
    • "In the Rodent Order, the situation of the different species is contrasting. While some of the species, especially mice (Mus musculus) and rats (Rattus sp.), are known to be reservoirs of pathogenic leptospires (Levett 2001), others, such as hamsters (Mesocricetus auratus) or guinea pigs (Cavia porcellus), are very susceptible hosts and can be used as a pathogenic model for severe leptospirosis in human (Nally et al. 2004; Croda et al. 2008). Considering the aquatic rodents, and especially the Coypu (Myocastor coypus), it is not known to which host category they belong. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Leptospirosis is caused by pathogenic species of the Leptospira genus. Animals can have two roles in the epidemiological cycle: they can be an accidental host and suffer of the disease or a reservoir host which does not express any clinical sign and shed bacteria in their urine. Some of the most known reservoirs for leptospirosis are certain rodent species, but the situation is less clear for aquatic rodents, especially for coypu (Myocastor coypus). It has been shown that this species can have kidney carriage for leptospirosis, but the relationship between carriage and individuals or population health has not been investigated yet. We trapped 133 coypus in two wetlands in the East of France during 3 years. For each animal, a complete necropsy, leptospirosis serology, and a specific real-time quantitative PCR (qPCR) for pathogenic leptospires were performed; in addition, for some animals, a specific kidney culture for leptospires and histology on kidney were performed. In spite of a high seroprevalence (respectively 76 % and 64 %) and of a significant prevalence of kidney carriage in both areas (respectively 12.1 % and 8.0 % of positive qPCR on kidney), the trapped animals seemed in good health, and the population did not seem to be affected by the circulation of the bacteria. These findings are concurring arguments to consider coypu as a real reservoir for leptospirosis.
    European Journal of Wildlife Research 02/2014; 60(1). DOI:10.1007/s10344-013-0758-z · 1.21 Impact Factor
  • Source
    • "Apart from Fn, LigBCen2 also binds to elastin, laminin, collagen and Fg (Lin and Chang, 2007; 2008; Lin et al., 2009b,c). A recent study indicates that knockout of the C-terminal of the LigB gene does not affect the virulence of this organism (Croda et al., 2008). Therefore, the role that LigB plays in the pathogenesis of leptospirosis is still unclear. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Leptospira immunoglobulin-like (Lig) proteins including LigA and LigB are adhesins that bind to fibronectin, collagen, laminin and elastin. In addition, Lig proteins are fibrinogen (Fg)-binding proteins, although the physiological role of the Lig-Fg interaction is unclear. In this study, a previously identified Fg-binding region, LigBCen2 (amino acids 1014-1165 of LigB), has been further localized to LigBCen2R, which consists of the partial 11th and entire 12th Ig-like domain (amino acids 1014-1119). LigBCen2R was found to bind to the C-terminal αC domain of Fg (FgαCC; amino acids 392-644 in Fg α chain; isothermal titration calorimetry, K(D) = 0.375 µM; fluorescence spectrometry, K(D) = 0.364 µM). The quenching and blue shift observed for the maximum wavelength intensities of the tryptophan fluorescence spectra for FgαCCY570W upon LigBCen2RW1073C binding suggested an RGD motif close to the sole tryptophan on FgαCCY570W was buried in LigBCen2R upon saturation with FgαCC. A conformational change in LigBCen2R when bound to the FgαCC RGD motif blocked further binding to integrin α(IIb) β3 on platelets, thus preventing their aggregation. LigBCen2R binding to FgαCC reduced clot formation but did not affect plasminogen and tissue-type plasminogen activator interactions with FgαCC. This study is the first to report that a spirochaetal protein binds to the C-terminal αC domain of Fg, which regulates thrombosis and fibrinolysis, and may help explain the pulmonary haemorrhage and thrombocytopenia seen in clinical cases of leptospirosis.
    Molecular Microbiology 02/2011; 79(4):1063-76. DOI:10.1111/j.1365-2958.2010.07510.x · 5.03 Impact Factor
  • Source
    • "The development of a transposon mutagenesis system has allowed production of defined mutants (Bourhy et al., 2005). Application of mutagenesis has shown that Loa22 and HemO are required for full virulence (Ristow et al., 2007; Murray et al., 2008; 2009b), and found that the outer membranes proteins LipL32 and LigB are not essential for Leptospira to cause either acute disease or renal colonization (Croda et al., 2008; Murray et al., 2009a,c). "
    [Show abstract] [Hide abstract]
    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.
    Molecular Microbiology 11/2010; 78(3):701-9. DOI:10.1111/j.1365-2958.2010.07360.x · 5.03 Impact Factor
Show more