Small chromosomes among Danish Candida glabrata isolates originated through different mechanisms

Abstract

We analyzed 192 strains of the pathogenic yeast Candida glabrata from patients, mainly suffering from systemic infection, at Danish hospitals during 1985-1999. Our analysis showed that these strains were closely related but exhibited large karyotype polymorphism. Nine strains contained small chromosomes, which were smaller than 0.5 Mb. Regarding the year, patient and hospital, these C. glabrata strains had independent origin and the analyzed small chromosomes were structurally not related to each other (i.e. they contained different sets of genes). We suggest that at least two mechanisms could participate in their origin: (i) through a segmental duplication which covered the centromeric region, or (ii) by a translocation event moving a larger chromosome arm to another chromosome that leaves the centromere part with the shorter arm. The first type of small chromosomes carrying duplicated genes exhibited mitotic instability, while the second type, which contained the corresponding genes in only one copy in the genome, was mitotically stable. Apparently, in patients C. glabrata chromosomes are frequently reshuffled resulting in new genetic configurations, including appearance of small chromosomes, and some of these resulting "mutant" strains can have increased fitness in a certain patient "environment".

Fig. 1

Phylogenetic relationships among pathogenic C. glabrata strains, based on seven different haplotypes, as deduced by Neighbor-joining method. The analysis is based on the D1/D2 domain of the 26 rDNA encoding locus. The numbers correspond to the museum numbers of the initial collection and can be found in Supplementary materials Table S1. Among the analyzed sequences (for accession numbers see Table S1), which had the 489 positions, 177 were identical with the C. glabrata type strain CBS 138. The strains belonging to the same haplotype are described in Supplementary materials Table 1. The bootstrap values are shown on some branches and the tree was not rooted. The scale bar in Neigbour-joining analysis corresponds to 0.001substitution per nucleotide site

Fig. 2

Phylogenetic relationship, as deduced by Neighbor-joining method, based on the IGS region located between the CDH1 and EPR6 genes. 35 (plus CBS 138) different haplotypes (representing isolate sequences which had the available 474 positions) were deduced. The strain numbers correspond to the museum numbers of the initial collection and can be found in Supplementary materials Table 1. The names of the strains with small chromosomes are followed by a capital letter pointing out which CBS 138 chromosome is related to the small chromosome. Among the analyzed strains several sequences belonged to the same haplotype. The appearance of each haplotype, in addition to the shown strain (and if different from 1), is written in the brackets following the strain/sequence designation. The strains belonging to each of these haplotypes can be found in Supplementary methods Table S1. The group 002574 (analyzed for their karyotypes in Fig. 3) is arrowed. The bootstrap values are shown on some branches and the tree is not rooted

Fig. 3

Electrophoretic karyotyping of 25 C. glabrata clinical isolates belonging to the same phylogenetic sub-group KA002574 which is arrowed in Fig. 2. Five groups of chromosomes (according to the CBS 138 nomenclature, see also Supplementary materials Fig. S1) are shown on the left, and the chromosome sizes on the right. The number of chromosome bands ranges from ten to thirteen but KA002870 (Y663) has fourteen chromosome bands because of its small chromosome (arrowed as a). The large chromosome group (K-L-M) shows a clear variation, from one band as in KA005064 to three bands, as in KA003250, or even four bands, as in KA005129. KA004709 and KA004773, arrowed as b and c, were isolated in 1997 from the same hospital but have clearly different karyotypes. In b we can see only ten bands but the third smallest chromosome (located in the C-D-E group) is likely a double band, while in c there are 12 bands

Fig. 4

Electrophoretic karyotyping of nine clinical isolates of C. glabrata with small chromosomes. S. cerevisiae S288C (Y1307) and CBS 138 were included as references to determine the size of the new chromosomes. Y624a is a daughter strain of KA000127 (Y624) which has lost its small chromosome but the position of the small chromosome, as it would be in Y624, is circled. Y624 and its small chromosome were described previously (Poláková et al. 2009). The sizes of small chromosomes determined by calculation of chromosomal migration on the gel were estimated to be between 280 and 420 kb (see also Table 2). Note, strains Y1640 and Y1641 are from the same patient taken at different time points and the two small chromosomes have a slightly different size

Fig. 5

Chromosome separations (a and c) and Southern blots (b and d) of C. glabrata clinical isolates with small chromosomes. CBS 138 was used as a reference. The gel a was transferred to membrane b, which was hybridized with the “Gel” gene probe originating from CBS 138 chromosome G. The Y1640 and Y1641 small chromosomes (arrowed) hybridized to the gel probe showing that they share the origin with chromosome G. Note that in these two strains only one signal was obtained. The chromosomes from gel c were transferred to membrane d and hybridized with the probe “Dcl” (originating from chromosome D). Note that in both Y1642 and Y1644 there were two bands, the original chromosome and the small chromosome, hybridizing to the probe

Fig. 6

Chromosomal stability of two C. glabrata clinical isolates with small chromosomes grown in liquid YPD for 70 generations. a Karyotypes of the parental strain Y1644 (lane 1) and eight randomly selected progenies (lanes 2–9), the position of the small chromosome is indicated by a black arrow. Chromosome D rearrangement in one daughter lineage is arrowed in white. b Karyotypes of the parental strain Y1645 (lane 1) and ten (lanes 2–11) randomly selected progenies after 70 generations. The small chromosome is arrowed