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: 3.73). 10/2008; 76(12):5826-33. DOI: 10.1128/IAI.00989-08
Source: PubMed


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, Sep 30, 2015
10 Reads
  • 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 · 1.63 Impact Factor
  • Source
    • "However, some proteins that bear the hallmarks of virulence factors (such as abundance, absence in saprophytic Leptospira spp., cellular location, expression profiles, predicted function and in vitro characterized functions ) are not required for virulence (reviewed by Adler et al., 2011). These include the extracellular matrix binding proteins LipL32 and LigB (Croda et al., 2008; Murray et al., 2009c) and the abundant surface lipoprotein LipL41 (King et al., 2013). These findings highlight the existing lack of understanding of leptospiral virulence and demonstrate the importance of improving the molecular characterization of the more than 40 % of leptospiral proteins for which no function has been determined (Adler et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The molecular basis for leptospirosis remains poorly understood, with no efficient methods available for screening libraries of mutants for attenuation. We have analysed the attenuation of leptospiral transposon mutants in vivo using a high-throughput method by infecting animals with pooled sets of transposon mutants. A total of 95 mutants was analysed by this method in the hamster model of acute infection, and one mutant was identified as attenuated (M1233, lb058 mutant). All virulence factors identified in Leptospira to date have been characterised in the acute model of infection, neglecting the carrier host. To address this, a BALB/c mouse colonisation model was established. The lb058 mutant and two mutants defective in LPS synthesis were colonisation-deficient in the mouse model. By applying the high-throughput screening method a further five colonisation-deficient mutants were identified for the mouse model; these included two mutants in genes encoding proteins with a predicted role in iron uptake (LB191/HbpA and LB194). Two attenuated mutants had transposon insertions in either la0589 or la2786 (encoding proteins of unknown function). The final attenuated mutant had an unexpected deletion of five genes (la0969-la0975) at the point of transposon insertion. This is the first description of defined, colonisation-deficient mutants in a carrier host for Leptospira. These mutants were either not attenuated, or only weakly attenuated, in the hamster model of acute leptospirosis, thus illustrating different factors that may be required in the carrier and acute models of leptospiral infection. High-throughput screening can reduce the number of animals used in virulence studies and increase the capacity to screen mutants for attenuation, thereby enhancing the likelihood of detecting unique virulence factors. A comparison of virulence factors required in the carrier and acute models of infection will help to unravel colonisation and dissemination mechanisms of leptospirosis.
    Journal of Medical Microbiology 06/2013; 62(Pt_10). DOI:10.1099/jmm.0.058586-0 · 2.25 Impact Factor
  • Source
    • "(vol/vol) Tween 20 with 5% (wt/vol) nonfat dry milk. The blots were washed, incubated for 1 h at room temperature with a 1,000-fold dilution of mouse ascites containing MAb to the LigB identical repeat region (LigA/B) [6] and probed with goat anti-mouse conjugated to alkaline phosphatase (Sigma). Immunoblots were developed in a nitroblue tetrazolium--5-bromo-4-chloro-3-indolylphosphate (BCIP) solution (Bio-Rad). "
    [Show abstract] [Hide abstract]
    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.
    BMC Microbiology 06/2011; 11(1):129. DOI:10.1186/1471-2180-11-129 · 2.73 Impact Factor
Show more