Gross karyotypic and phenotypic alterations among different progenies of the Candida glabrata CBS138/ATCC2001 reference strain

Abstract

Genomic plasticity is a mechanism for adaptation to environmental cues such as host responses and antifungal drug pressure in many fungi including the human pathogenic yeast Candida glabrata. In this study we evaluated the phenotypic and genotypic stability of the world-wide used C. glabrata reference strain CBS138/ATCC2001 under laboratory conditions. A set of ten lineages of this wild type strain and genetically modified progenies were obtained from different scientific laboratories, and analyzed for genotypic and phenotypic alterations. Even though the derivates were indistinguishable by multi locus sequence typing, different phenotypic groups that correlated with specific karyotypic changes were observed. In addition, modifications in the adherence capacity to plastic surface emerged that were shown to correlate with quantitative changes in adhesin gene expression rather than subtelomeric gene loss or differences in the number of macrosatellite repeats within adhesin genes. These results confirm the genomic plasticity of C. glabrata and show that chromosomal aberrations and functional adaptations may occur not only during infection and under antimicrobial therapy, but also under laboratory conditions without extreme selective pressures. These alterations can significantly affect phenotypic properties such as cell surface attributes including adhesion and the cell wall carbohydrate composition and therefore, if unnoticed, may adulterate the outcome of genetic studies.

Figure 1. Electrophoretic karyotyping.

PFGE analysis of CBS138-derived gene deletion progenies and ten different CBS138 isolates (A) CBS138-derived gene deletion progenies of C. glabrata, which are based on the trp^- ura^- and his^- trp^- ura^- auxotrophic strains that were produced by Kitada et al. . All these strains carry the ChrK aberration (boxed, K*). (B) Variants of the parental CBS138 strain obtained from various laboratories. The isolates fall into three distinct karyotypic patterns, including the unaltered CBS138 reference karyotype (karyotype I), the karyotype observed in the Kitada-lineage (boxed, karyotype III), but also one additional, previously unobserved karyotype (arrows pointing to altered chromosomes, karyotype II).

Figure 2. Karyotypes of different CBS138 derivates correlate with cell wall phenotypes.

(A) Zymolyase-induced cell lysis is CBS138-karyotype dependent. Cell lysis rates by zymolyase activity are significantly (p = 0.009, indicated by asterisk) increased in karyotype I isolates as compared to karyotypes II and III. (B) Measurements of relative glucan and chitin levels showed that glucan content was CBS138-karyotype dependent. FACS measurements of CR-stained (dark bars) or WGA-stained (white bars) cell wall carbohydrates showed that karyotype I isolates had significantly higher content of alkali-insoluble glucan (CR-staining) than both karyotype II and III isolates (p_{(I vs. II)} = 0.0016; p_{(I vs. III)}<0.0001). Within karyoptype I isolates, CBS138/1 showed significantly higher CR-staining compared to the other members of this group and significant differences of all individual strains in comparison to the reference strain CBS138/1 are indicated by asterisks (p<0.05). Content of acid-insoluble chitin (WGA-staining) was not significantly altered between the groups.

Figure 3. Cell surface characteristics.

(A) Qualitative determination of adherence to polystyrene. (B) Quantitative determination of hydrophobicity (white bars), adherence to polystyrene (grey bars) and adherence to silicone (striped bars). All values are normalized against the values of isolate CBS138/1. Statistically significant differences were observed for karyotype II measurements of hydrophobicity and polystyrene adherence as compared to karyotype I and III (p_{(II vs. I)} = 0.0015; p_{(II vs. III)} = 0.0030 and p_{(II vs. I)}<0.0001; p_{(II vs. III)} = 0.0008, respectively), and for karyotype III for silicone adhesion (p_{(III vs. I)} = 0.0001; p_{(III vs. II)}<0.0001). The significant differences of CBS 138/6 (karyotpe II) in comparison to the reference strain CBS138/1(karyotype I) are indicated by asterisks (p<0.05).

Figure 4. Genomic analysis of gene presence and repeat length of selected adhesin genes in CBS138 derivates does not reveal alterations.

(A) The presence of selected adhesin genes in the genomes of the different isolates was analyzed by PCR. All genes tested were present in all strains. (B) PCR analysis of the repeats-containing C-terminal regions of AWP2 and AWP5. No differences were detected.

Figure 5. CBS138 derivates show differences in adhesin gene expression.

Gene expression analysis of adhesin genes in selected strains, representing the three karyotypes (CBS138/5 (karyotype I), 6 (karyotype II), and 7 (karyotype III)), revealed that adhesins are differentially expressed between the strains (ANOVA). T-tests showed significant reduced adhesin gene expression for CBS138/6 compared to CBS138/5 and CBS138/7 for EPA3, EPA6, EPA7, EPA22, AWP2, AWP4, AWP5 and AWP7 (indicated by asterisks (p<0.05)).