Molecular analysis of chitin synthase genes of Paracoccidioides brasiliensis
Two main characteristics make the fungal cell wall a good target for the development of antifungal antibiotics: its integrity is essential for the survival of the fungus, and it is mainly composed of polysaccharides which are not found in animal cells (Cabib et al., 1988). As chitin is the second major component in the cell wall of the pathogenic yeast form in P. brasiliensis and its content is almost three times higher than in the mycelial cell wall (Kanetsuna et al, 1969; San-Blas, 1985), its biosynthesis is an attractive target for the use of antifungal antibiotics against this medically important fungus in Central and South America. In the present work, a first step has been taken in trying to understand the biosynthesis of chitin in P. brasiliensis through the identification and cloning of fungal genes. Five different chitin synthase genes namely PbCHSl, PbCHS2, PbCHS3, PbCHS4 and PbCHS5, were identified during the course of the present work in the dimorphic fungal human pathogen P. brasiliensis, strain IVIC Pb73. The identification of these genes was accomplished through PCR amplification by using primers designed on regions of high homology amongst fungal chitin synthases (Bowen et al., 1992; Mellado et al., 1995). The complete nucleotide sequence was obtained for PbCHS2. Analysis of the expression of PbCHSl, PbCHSl, PbCHS4 and PbCHSS by northern hybridisation suggested that these genes may be regulated during dimorphism, and appear to be preferentially expressed in the mycelial form of the fungus. The use of RFLP analysis as a typing method for P. brasiliensis strains presented 100% typeability, 100% in vitro reproducibility and a discriminatory power between 0.972 and 1, depending on the restriction enzyme used. Computational analysis by using evolutionary distances of the RPLP patterns obtained for 10 P. brasiliensis isolates from different endemic areas suggested a relationship between genetic identity of the isolates and the endemic area from where they were isolated.