Isolation of the CHS4 gene of Paracoccidioides brasiliensis and its accommodation in a new class of chitin synthases.

The nucleotide sequence of a chitin synthase gene (PbrCHS4) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. A homology search with the deduced amino acid sequence of PbrChs4 (1744 aa) reveals the presence of two distinct domains, an N-terminal domain showing up to 30% homology to myosin motor-like domains and a C-terminal domain with up to 68% homology to chitin synthases, as has been reported for some class V chitin synthases. However, unlike class V chitin synthases with myosin motor-like domains, PbrChs4 does not present characteristic signatures of myosin motor-like domains. Also, although the Chs domain presents the closest homology to other fungal class V enzymes, it is low enough to consider PbrChs4 as belonging to a new class, which we propose as class VII.

Differential expression of chitin synthase genes during temperature-induced dimorphic transitions in Paracoccidioides brasiliensis.

Fragments of five genes encoding chitin synthase enzymes were identified in the dimorphic fungus Paracoccidioides brasiliensis by polymerase chain reaction (PCR) amplification of conserved CHS gene domains. These represent several classes of enzyme: PbrCHS1, class I; PbrCHS2, class II; PbrCHS3, class IV; and PbrCHS4 and PbrCHS5, class V. Expression of these genes during the temperature regulated dimorphic transition from yeast to mycelium and from mycelium to yeast was determined by Northern analysis. One gene (PbrCHS3) was not expressed at detectable levels. The others were regulated by morphology and/or by the growth phase of the organism. Despite the fact that yeast cells contain more chitin than hyphal cells, the levels of mRNA for PbrCHS1, PbrCHS2, PbrCHS4, and PbrCHS5 were higher in hyphal cells than in yeast cells. This supports observations in other fungi that transcript levels often do not correlate with chitin content and that post-transcriptional regulation of CHS gene expression is important for morphogenesis.

RFLP analysis reveals marked geographical isolation between strains of Paracoccidioides brasiliensis.

Restriction fragment length polymorphism (RFLP) was performed on 32 isolates of the pathogenic fungus Paracoccidioides brasiliensis from geographically separated regions of South America. The use of HinfI and HincII gave clear RFLP patterns, for which high discriminatory indices could be calculated. Computational analysis of the RFLP patterns for the 32 isolates suggested that at least five groups of strains existed, each of which was geographically distinct and corresponded closely with present country borders. These results underline the belief that P. brasiliensis infections are acquired from exogenous sources and that this fungus occupies specialist endemic niches within the natural environment.

Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis.

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus.