Typing of Candida glabrata in clinical isolates by comparative sequence analysis of the cytochrome c oxidase subunit 2 gene distinguishes two clusters of strains associated with geographical sequence polymorphisms

Abstract

We tested whether comparative sequence analysis of the mitochondrion-encoded cytochrome c oxidase subunit 2 gene (COX2) could be used to distinguish intraspecific variants of Candida glabrata. Mitochondrial genes are suitable for investigation of close phylogenetic relationships because they evolve much faster than nuclear genes, which in general exhibit very limited intraspecific variation. For this survey we used 11 clinical isolates of C. glabrata from three different geographical locations in Brazil, 10 isolates from one location in the United States, 1 American Type Culture Collection strain as an internal control, and the published sequence of strain CBS 138. The complete coding region of COX2 was amplified from total cellular DNA, and both strands were sequenced twice for each strain. These sequences were aligned with published sequences from other fungi, and the numbers of substitutions and phylogenetic relationships were determined. Typing of these strains was done by using 17 substitutions, with 8 being nonsynonymous and 9 being synonymous. Also, cDNAs made from purified mitochondrial polyadenylated RNA were sequenced to confirm that our sequences correspond to the expressed copies and not nuclear pseudogenes and that a frameshift mutation exists in the 3' end of the coding region (position 673) relative to the Saccharomyces cerevisiae sequence and the previously published C. glabrata sequence. We estimated the average evolutionary rate of COX2 to be 11.4% sequence divergence/10(8) years and that phylogenetic relationships of yeasts based on these sequences are consistent with rRNA sequence data. Our analysis of COX2 sequences enables typing of C. glabrata strains based on 13 haplotypes and suggests that positions 51 and 519 indicate a geographical polymorphism that discriminates strains isolated in the United States and strains isolated in Brazil. This provides for the first time a means of typing of Candida strains that cause infections by use of direct sequence comparisons and the associated divergence estimates.

FIG. 1

PCR and RT-PCR of COX2 genes from total cellular DNA and mtRNA. (A) COX2 was amplified as a 756-bp product from total cellular DNAs of 10 strains of C. glabrata (lanes 1 to 10, respectively) but not from S. cerevisiae total cellular DNA (lane 11). C. glabrata DNA samples correspond to strains 05, 06, 12, 13, 36, 37, 38, 39, 47 and ATCC 90030, respectively (TABLE 1). (B) COX2 was amplified from purified mtRNA of C. glabrata 06 (Table 1) (lanes 5 and 7). The cDNAs were synthesized either from oligo(dT) primers (lane 4) or from the COR primer (lane 7). Negative controls consisted of mock RT reactions with oligo(dT) (lane 4) or COR primer (lane 6) lacking reverse transcriptase to demonstrate that the COX2 product was reverse transcriptase dependent. Amplification of actin cDNA from oligo(dT) was used to control for RNA integrity, RT efficiency, and contamination with nonmitochondrial mRNA (lane 3) with the corresponding mock RT reaction-negative control to exclude the possibility of DNA contamination (lane 2). Amplification of COX2 from total cellular DNA was used as a positive control for PCR (lane 1). The identities of the COX2 and actin cDNA amplicons were checked by sequencing. Lane M, molecular size markers (phage lambda DNA cut with HindIII for panel A and a 100-bp ladder [GIBCO BRL] for panel B).

FIG. 2

Alignment of COX2 sequences from C. glabrata clinical isolates and those from previously published strain CBS 138 and S. cerevisiae (S.c). Sequence names correspond to the names in Table 1 (C.g, C. glabrata). The cDNA sequence (second from top to bottom) was determined from strain ATCC 90030. The frameshift mutation (C insertion) is located at position 673. Boxed residues indicate differences from the consensus sequence.

FIG. 2

Alignment of COX2 sequences from C. glabrata clinical isolates and those from previously published strain CBS 138 and S. cerevisiae (S.c). Sequence names correspond to the names in Table 1 (C.g, C. glabrata). The cDNA sequence (second from top to bottom) was determined from strain ATCC 90030. The frameshift mutation (C insertion) is located at position 673. Boxed residues indicate differences from the consensus sequence.

FIG. 2

Alignment of COX2 sequences from C. glabrata clinical isolates and those from previously published strain CBS 138 and S. cerevisiae (S.c). Sequence names correspond to the names in Table 1 (C.g, C. glabrata). The cDNA sequence (second from top to bottom) was determined from strain ATCC 90030. The frameshift mutation (C insertion) is located at position 673. Boxed residues indicate differences from the consensus sequence.

FIG. 3

Summary of alignment of variant positions of C. glabrata COX2. Open boxes indicated the synonymous substitutions, and underscores indicate the nonsynonymous substitutions. The asterisks at the bottom indicate the substitutions at positions 51 and 519 that separate type 1 strains from type 2 strains.

FIG. 4

Correlation between percent COX2 sequence distances and taxonomic distances. Points in the plot indicate the average distance for the pairwise comparison for a given category, and error bars indicate the associated standard deviation.

FIG. 5

Interspecific and intraspecific phylogenetic analysis of COX2 genes of Candida glabrata and closely related taxa. (A) Maximum likelihood tree for several fungi, rooted for S. pombe and N. crassa, to compare the average intraspecific distances of C. glabrata strains from their closest relatives. (B) Maximum likelihood phylogeny of C. glabrata strains supporting the existence of two types with a bootstrap value of 89% in 500 replications. The same topologies for trees in panels A and B were observed when neighbor-joining algorithms with HKY distances were used. The scale bars below the trees indicate percent divergence, and the numbers above the tree nodes indicate the percentage of that particular branch cluster in 500 bootstrap replications. Species abbreviations are as follows: S-c, S. cerevisiae; S.e, S. exiguus; W.su, W. saturnus var. suaveolens; W.sa, W. saturnus var. makrii; D.b, D. bruxellensis; K.l, K. lactis; K.t, K. thermotolerans; P.j, P. jadinii; C.g, C. glabrata; B.ci, B. custersii; N.c, N. crassa; Sc.p, S. pombe.