Genetic relationship between Cryptococcus neoformans var. neoformans strains of serotypes A and D

Abstract

Cryptococcus neoformans serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference C. neoformans strains belonging to serotypes A and D by analyzing their URA5 sequences and restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently of the basis of the nucleotide sequences of their URA5 genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 +/- 1.7 and 7.2 +/- 3.4, respectively (P < 0.0001), and between serotypes A and D of 41.9 +/- 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of C. neoformans var. neoformans and suggest that these serotypes could probably be considered different varieties of C. neoformans.

FIG. 1

CNRE-1 RFLP patterns obtained from reference strains of C. neoformans of serotypes A and D. The serotype of each strain is indicated in parentheses. The numbers on the left are in kilobases.

FIG. 2

Dendrogram generated from the Dice coefficients computed from the CNRE-1 patterns for reference strains of C. neoformans and recent isolates from Brazil (C and E isolates) and New York City (J isolates). The serotypes and binding patterns obtained by IF with MAb 13F1 are presented on the right.

FIG. 3

Partial nucleotide sequence of the URA5 genes from reference strains of C. neoformans serotypes A and D. Serotypes are indicated in parentheses. A hyphen indicates that the base is identical to that in strain 3501 (14). A space implies that the base was not present in the allele. Lowercase letters indicate synonymous substitutions. The complete sequences are available in GenBank under the accession numbers listed in Table 1.

FIG. 4

Relationship of reference strains of C. neoformans serotypes A and D and recent isolates from Brazil and New York City obtained from phylogenetic analysis of URA5 sequence data. The tree was obtained by use of DNAPENNY and CONSENSE of the PHYLIP program. The numbers at each branch point were generated by use of SEQBOOT and indicate the percentage of bootstrap replications. C. neoformans var. gattii was designated the outgroup. The serotypes and binding patterns obtained by IF with MAb 13F1 are presented on the right. The sequences of isolates C31, E5, and RJ2 were identical to the sequence of isolate C7 (16). Isolate C33 had the same sequence as isolate C5 (16).