Frequency of <i>SSR1</i> genotypes in disease-causing isolates.

<p>Allele combinations are labeled with repeat numbers in allele 1 (Region A + Region B) followed by repeat numbers in allele 2 (Region A + Region B); alleles are sorted so that allele 1 is the smaller allele (when alleles are of the same size the allele with the larger region A precedes the allele with the smaller region A). Genotypes for 49 disease-causing clade 1 isolates and 46 other disease-causing isolates are shown.</p

Highly mutable tandem DNA repeats generate a cell wall protein variant more frequent in disease-causing <i>Candida albicans</i> isolates than in commensal isolates

<div><p>During adaptation to host environments, many microorganisms alter their cell surface. One mechanism for doing so is variation in the number of amino acid repeats in cell surface proteins encoded by hypermutable DNA tandem repeats. In the yeast <i>Candida albicans</i>, an opportunistic human pathogen, the gene <i>SSR1</i> encodes a GPI-anchored cell wall protein with a structural role. It contains two regions consisting of tandem repeats, almost exclusively encoding the amino acid pair Ser-Ala. As expected, the repeat regions make <i>SSR1</i> highly mutable. New <i>SSR1</i> alleles arose with a frequency of 1.11×10⁻⁴ per cell division in serially propagated cells. We also observed a large number (25) of <i>SSR1</i> alleles with different repeat lengths in a survey of 131 isolates from a global strain collection. <i>C</i>. <i>albicans</i> is diploid, and combinations of these allele generated 41 different <i>SSR1</i> genotypes. In both repeat regions, nonsynonymous mutations were largely restricted to one particular repeat unit. Two very similar allele combinations were largely restricted to one clade, clade 1. Each combination was present in ~30% of 49 infection-causing clade 1 strains, but one was rare (2%), the other absent in 46 infection-causing strains representing the remainder of the species (<i>P</i> < 0.00018 and 0.00004; Fisher’s exact test). These results indicate that both repeat regions are under selection and that amino acid repeat length polymorphisms generate Ssr1 protein variants most suitable for specific genetic backgrounds. One of these two allele combinations was 5.51 times more frequent, the other 1.75 times less frequent in 49 clade 1 strains that caused disease than in 36 commensal clade 1 strains (<i>P</i> = 0.0105; Chi² test). This indicates that insertion and deletion of repeats not only generates clade-optimized <i>Ssr1p</i> variants, but may also assist in short-term adaptation when <i>C</i>. <i>albicans</i> makes the transition from commensal to pathogen.</p></div

<i>SSR1</i> allele region structures.

<p>Predicted amino acid sequences of repeat regions of 23 different <i>SSR1</i> alleles and the repeat region from the <i>C</i>. <i>dubliniensis</i> CD36 ortholog. Different shadings of repeat units indicate different versions of DNA and amino acid repeats, with colored units representing nonsynonymous mutations, leading to amino acid pairs other than SER/ALA. Alleles of stains CLB49, YASM 42, OD9014, Var1.4vag (both alleles), RIHO30, HUN64, Au11, CH42, Var1.5vag, OTG6, Au19, RIHO16, and HUN91 were sequenced as part of this study, while the other alleles were from the <i>C</i>. <i>albicans</i> genome database (<a href="http://www.candidagenome.org/" target="_blank">http://www.candidagenome.org/</a>) or were identified by BLAST searches.</p

Frequency of genotypes in 36 commensal and 49 disease-causing clade 1 isolates.

<p>Allele combinations are labeled with repeat numbers in allele 1 (Region A + Region B) followed by repeat numbers in allele 2 (Region A + Region B) alleles are sorted so that allele 1 is the smaller allele (when alleles are of the same size the allele with the smaller region A precedes the allele with the larger region A).</p

Primers used and PCR conditions.

<p>Primers used and PCR conditions.</p

<i>SSR1</i> ORF as present in the SC5314 genome.

<p>Locations of the two repeat regions and a likely CFEM domain identified by us are indicated. Grey arrows indicate primers used (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180246#pone.0180246.t001" target="_blank">Table 1</a>).</p

Genotyping of repeat region B showing mutant alleles after 300 generations.

<p>(A) Size of one of the original repeat region B amplicons in strain RIHO30 (31 repeats) as displayed by the software Peak Scanner. (B),(C) Repeat region B amplicons, increased in size by 6 bp (by 1 repeat unit, to 32 repeats), from 2 different colonies after serial propagation of the strain for 300 generations.</p

Verification of <i>PNG2</i> expression by Northern hybridization.

<p>Messenger RNA (5 µg per lane) prepared from exponential phase cultures of SC5314 and HUN68 grown in YPD was hybridized under high-stringency conditions with a probe corresponding to the repeat region.</p

Frequencies of repeat regions of different lengths and frequencies of repeat region combinations.

<p>Frequencies (B) among GPG clinical isolates are indicated by black bars, frequencies among other clinical isolates by grey bars. Lengths (A) are expressed as numbers of 4 amino acid repeat units. One borderline GPG strain and one borderline non-GPG strain (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009614#pone-0009614-t001" target="_blank">Table 1</a>) were included in the analysis.</p

Alterations of repeat regions after 300 generations of serial transfer of strains RIHO10 and W53.

<p>PCR products amplified from genomic DNA using primers MC0repf and MC0repr were resolved on acrylamide gels. For each strain the middle lane shows PCR products from the clone with an altered allele (ALT), flanked by two lanes of PCR products from clones which retained alleles of the original lengths. Figures on the right show the numbers of repeats, with altered allele marked by an asterisk. Numbers of repeats were verified by sequencing (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009614#pone-0009614-g005" target="_blank">Fig. 5</a>).</p