Reintroduction of the PLB1 gene into Candida albicans restores virulence in vivo

Center for Medical Mycology, University Hospitals of Cleveland and Department of Dermatology, Case Western Reserve University, Cleveland, OH 44106-5028, USA 1 Department of Microbiology and Immunology, Medical College of Ohio, Toledo, OH 43614-5806, USA 2 Author for correspondence: Mahmoud A. Ghannoum. Tel: +1 216 844 8580. Fax: +1 216 844 1076. e-mail: mag3{at}po.cwru.edu Phospholipases have been proposed to contribute to the virulence of Candida albicans . Recently, a candidal strain deleted for PLB1 , the gene encoding the predominant phospholipase B (Plb1) secreted by C. albicans , was constructed and its virulence in an intravenous murine model of disseminated candidiasis was evaluated. In the present study, the PLB1 gene was reintroduced back into the plb1 null mutant to generate the revertant strain, which showed similar growth and morphology to its isogenic parent strain. Virulence of the revertant strain was found to be comparable to that of the parent strain in an intravenous murine model of disseminated candidiasis. To compare the abilities of the plb1 null mutant, the revertant and the isogenic parent strains to cross the gastrointestinal (GI) tract and cause systemic infection, an oral–intragastric infant mouse model of candidiasis was used. Histological examinations and analysis of c.f.u. of the pathogen in liver homogenates revealed that the parental and revertant strains were able to invade and traverse the GI mucosa to a significantly greater extent than the plb1 null mutant. Immunofluorescence and immunoelectron microscopic studies of infected host tissue using anti-Plb1 antibody showed that Plb1 is secreted during invasion of the gastric mucosa by the parental and revertant strains. In contrast, little or no labelling was observed in the null mutant strain. The results indicate that the Plb1 secreted by C. albicans enhances the ability of this organism to cross the GI tract and disseminate haematogenously. These studies provide unequivocal evidence supporting a role for Plb1 during the course of infection by C. albicans . Keywords: phospholipase B, virulence factor, candidal transmigration, in vivo localization of Plb1 Abbreviations: GI, gastrointestinal; PAS, periodic acid–Schiff reagent a These authors contributed equally to this work.