Molecular analysis of cell cycle genes in Cryptococcus neoformans

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Abstract

Cryptococcus neoformans is a pathogenic yeast belonging to Basidiomycetes, a taxonomic Class phylogenetically remote from the rest of the pathogenic yeasts (Ascomycetes). C. neoformans causes fatal meningoencephalitis primarily in patients with T- lymphocyte dysfunction and is the only species in the genus Cryptococcus that has optimum growth at temperatures higher than 30C. Although extensive studies on the molecular bases of cell cycle and morphogenesis have been carried out in ascomycetous yeasts, such studies have not been conducted in C. neoformans. This project was initiated to study the molecular genetics of cell cycle associated genes in C. neoformans. In 1999, we cloned the CCN1 gene from a serotype A strain of C. neoformans (B-4551) that exhibited altered cell morphology at 35 degree celsius and failed to multiply at 37 C. This strain caused chronic granulomatouse lesions in the nasal cavity of a cat without spreading to other sites due to its inability to grow at 37 C. This year we tested this strain (B-4551) for its ability to cause systemic infection in a murine model and characterized the CCN1 gene and the mutations that could be of responsible for the temperature sensitive phenotype. The strain was unable to cause systemic infection in mice. The CCN1 gene was found to encode a protein containing 16 tetratricopeptide repeats (TPR) of 34 amino acids each. It showed high sequence similarity with two known essential genes, CRN and CLF1, belonging to the TPR family. The CRN of Drosophila is involved in neurogenesis and the CLF1(S. cerevisiae homolog of CRN) is required for the assembly of spliceosomes. Comparisons between the genomic sequence of the CCN1 gene from both a wild type and strain B-4551 revealed the presence of five mutations, three consecutive nucleotides deletions and two missence mutations in the coding region. Site directed mutagenesis indicated that deletion of three consecutive nucleotides, corresponding to the highly conserved 8th amino acid of TPR7, was responsible for the temperature sensitivity of B-4551.The strain B-4551 complemented with wild type CCN1 restored its ability to grow at 37 C and caused fatal systemic infection in mice. These results indicated that CCN1 is essential for C. neoformans for growth at the body temperature and causes systemic infection in warm-blooded hosts.