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

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.

FIG. 1.

Disruption of ligB in L. interrogans strain Fiocruz L1-130. (A) Schematic representation of the genotype of the parental (Fiocruz wt) and ligB (Fiocruz KO2) mutant strains. The LigB protein has a tripartite structure which includes an N-terminal identical repeat region, a nonidentical repeat region, and the C-terminal region. The vertical bars indicate EcoRI restriction sites. The locations of primers used to check for allelic exchange, as well as the expected sizes of amplified products, are indicated. (B) Map of the pB2SK plasmid, in which the Spc^r cassette was inserted between EcoRI sites in the B2 region of ligB. (C) PCR amplification of chromosomal DNA from the L. interrogans wild-type strain (lane 1), L. biflexa strain Patoc1 (lane 2), the L. interrogans ligB KO2 mutant (lane 3), and the L. interrogans ligB KO2 mutant reisolated from hamsters after infection (lane 4) with primers B2EF and B2ER, as shown in panel A. (D) Western blot of LigA and LigB expression in the L. interrogans wild-type strain (lane 1), L. biflexa strain Patoc1 (lane 2), the L. interrogans ligB mutant (lane 3), and the L. interrogans ligB mutant which was reisolated from hamsters (lane 4). Blots were also probed with LipL41 antiserum as a reference. (E) Immunofluorescence assays were performed with L. interrogans wild-type (wt) and ligB mutant (KO2) strains. Strains were labeled with antibodies against LigANI (α-LigA0), LigBNI (α-LigB), and a control. Alexa- and fluorescein isothiocyanate-conjugated secondary antibodies were used to detect surface-bound antibodies to LigANI and LigBNI, respectively. A DAPI counterstain was used to document the presence of leptospires. The photomicrograph show the results of one of three representative experiments.

FIG. 2.

Pathology in hamsters infected with the ligB mutant. (A) Gross appearance of hamsters infected with the wild-type (wt) and mutant ligB mutant (KO2) strains and a representative uninfected control hamster. (B) Livers and kidneys from hamsters infected with the wild-type and ligB mutant strains of L. interrogans. Tissues were stained with hematoxylin and eosin (HE) (magnification, ×400), and the immunohistochemistry analysis was performed with antiserum specific for LipL32 (IHC) (magnification, ×1,000).

FIG. 3.

Adherence of the L. interrogans ligB mutant to MDCK epithelial cells. The adherence of the L. interrogans wild-type (A) and ligB mutant KO2 (B) strains to MDCK epithelial cells was examined. Anti-Salmonella OmpA MAb was used as a control (C). Representative micrographs obtained by fluorescence microscopy are shown. (D) Attachment ratios (means ± standard deviations) determined using 10 random fields. wt, wild type.