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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.

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Available from: Julio Croda, Aug 24, 2015
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    • "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. "
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    • "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. "
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    • "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). "
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