Molecular analysis of CAP59 gene sequences from five serotypes of Cryptococcus neoformans

Abstract

The nucleotide sequences of CAP59 genes from five serotypes of Cryptococcus neoformans were analyzed for their phylogenetic relationships. Approximately 600-bp genomic DNA fragments of the CAP59 gene were amplified from each isolate by PCR and sequenced. The CAP59 nucleotide sequences of C. neoformans showed more than 90% similarity among the five serotypes. By phylogenetic analysis, their sequences were divided into three clusters: serotypes A and AD, serotypes B and C, and serotype D. In addition, the results of reduced amino acid sequences were similar to the nucleotide sequence data. These data revealed that serotype AD was genetically close to serotype A rather than serotype D, although it had been considered to be a mixed type of serotype A and D by serological analysis. Furthermore, the nucleotide sequences of the serotype B and C isolates of C. neoformans were very similar to each other. These results indicated that serotype B and C isolates belonging to C. neoformans var. gattii were genetically homogeneous and closely related. The molecular analysis of the CAP59 gene will provide useful information for the differentiation of serotypes of C. neoformans and for an understanding of their phylogenetic relationships.

FIG. 1

The CAP59 gene has five exons. The longest exon, a 597-bp fragment containing about 30% of the coding sequence of the gene, was amplified.

FIG. 2

Alignment of CAP59 sequences of C. neoformans. The sequences of five serotypes of the yeast were aligned by using the Clustal W computer programs. The asterisks indicate conserved serotype D sequence (DDBJ-EMBL-GenBank accession no. L26508).

FIG. 3

Phylogenetic tree showing relationships of CAP59 gene fragments from five serotypes of C. neoformans based on DNA sequences. The DNA sequences were compared by Clustal W multiple sequence alignment programs, and a phylogenetic tree was constructed by TREEVIEW. Bootstrap analysis was performed on 1,000 random samples. The numbers at the branches were determined by bootstrap analysis, indicating the times in 1,000 repeat samples in a monophylogenic grouping.

FIG. 4

A phylogenetic tree showing relationships of CAP59 gene fragments from five serotypes of C. neoformans based on reduced amino acid sequences. The reduced amino acid sequences were compared by Clustal W multiple sequence alignment programs, and a phylogenetic tree was constructed by TREEVIEW. Bootstrap analysis was performed on 1,000 random samples. The numbers at the branches were determined by bootstrap analysis, indicating the times in 1,000 repeat samples in a monophylogenic grouping.