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Infection and Immunity, December 2008, p. 5826-5833, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00989-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Targeted Mutagenesis in Pathogenic Leptospira Species: Disruption of the LigB Gene Does Not Affect Virulence in Animal Models of Leptospirosis{triangledown}

Julio Croda,1,2 Claudio Pereira Figueira,1 Elsio A. Wunder Jr.,1 Cleiton S. Santos,1 Mitermayer G. Reis,1 Albert I. Ko,1,3* and Mathieu Picardeau4

Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil,1 Laboratory of Pathology of Transmissible Diseases, Department of Pathology, Medicine School, São Paulo University, São Paulo, Brazil,2 Division of International Medicine and Infectious Disease, Weill Medical College of Cornell University, New York, New York,3 Unité de Biologie des Spirochètes, Institut Pasteur, Paris, France4

Received 7 August 2008/ Returned for modification 27 August 2008/ Accepted 16 September 2008

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 (Spcr) 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.

* Corresponding author. Mailing address: Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz/MS, Rua Waldemar Falcão, 121, Salvador, Bahia 40296-710, Brazil. Phone: 55 71 3176 2302. Fax: 55 71 3176 2281. E-mail: aik2001{at}med.cornell.edu

{triangledown} Published ahead of print on 22 September 2008.

Editor: A. Camilli

Infection and Immunity, December 2008, p. 5826-5833, Vol. 76, No. 12
0019-9567/08/$08.00+0     doi:10.1128/IAI.00989-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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