Genome Comparisons of Candida glabrata Serial Clinical Isolates Reveal Patterns of Genetic Variation in Infecting Clonal Populations

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Overall, we detected small differences between the two genomes of a pair, with 0.037 SNPs/Kb between CMRL3/CMRL4 as the lowest value, and 0.047 SNPs/Kb between DSY562/DSY565 as the highest value.

“Overall, we detected small differences between the two genomes of a pair, with 0.037 SNPs/Kb between CMRL3/CMRL4 as the lowest value, and 0.047 SNPs/Kb between DSY562/DSY565 as the highest value (Figure 1A).”

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For clonal strains isolated from the same patients at different days, there was a positive trend, but no significant correlation, between the number of private SNPs and time-span between isolations (r = 0.46, p-value = 0.14).

“For clonal strains isolated from the same patients at different days, we observed a positive trend, but no significant correlation, between the number of private SNPs and time-span between isolations (r = 0.46, p-value = 0.14) (Figure 1B).”

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Most (161) genes with non-synonymous mutations in matched pairs also present mis-sense variants in comparisons of non-clonal strains from the same clade, while only 23 were exclusive to comparisons among clonal isolates.

“These comparisons revealed that most (161) genes with non-synonymous mutations also present mis-sense variants in comparisons of non-clonal strains from the same clade, while only 23 were exclusive to comparisons among clonal isolates.”

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With the exception of the pair NRZ-2016-057/NRZ-2016-058, the rest of the clonal strains present additionally two non-synonymous mutations (S76P and T143P) in PDR1.

“Also, with the exception of the pair NRZ-2016-057/NRZ-2016-058, the rest of the clonal strains present additionally two non-synonymous mutations (S76P and T143P).”

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CANGA2A and CANGA2B presented deletions in the MATalpha cassette (CAGL0B00242g and CAGL0B01243g) and a duplication in MATa (CAGL0E00341g).

“Among all analyzed samples, CANGA2A and CANGA2B presented deletions in the MATalpha cassette (CAGL0B00242g and CAGL0B01243g) and a duplication in MATa (CAGL0E00341g), which suggest these strains present an abnormal configuration of mating type MATa (triple a), as previously shown for other strains (Carreté et al., 2018).”

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Genetic distance was also high between strain pairs isolated the same day but from different body sites and sometimes similar to those isolated from the same site at different days.

“Moreover, the genetic distance was also high between strain pairs isolated the same day but from different body sites and sometimes similar to those isolated from the same site at different days (Figure 1B).”

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We detected 49 private exonic INDELs that differ between pairs of isolates, which globally affected 36 genes, of which 17 encoded cell wall associated proteins.

“Overall, we detect 49 private exonic INDELs that differ between pairs of isolates, which globally affected 36 genes, of which 17 encoded cell wall associated proteins (Supplementary Table S2).”

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MCA analysis provided consistent results with previously published clades, and suggested that all these new strains could be ascribed to several of the previously described clades.

“MCA analysis provided consistent results with previously published clades, and suggested that all these new strains could be ascribed to several of the previously described clades (Carreté et al., 2018): namely, pairs B1012M/B1012S and EB101M/BO101S belong to clade I; SAT strains to clade II; DSY562/DSY565 to clade III; P35_2/P35_3, CMRL1/CMRL2, and CANGA3A/CANGA3B strains belong to clade VI; NRZ-”

• cites Carreté et al., 2018 sourceNot checked yet

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CMRL4 presents one private mutation G143V in FEN1.

“Additionally, CMRL4 presents one private mutation in G143V (Figure 3).”

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DSY562 and DSY565 share an additional non-synonymous mutation A942T, and four other MSH2 non-synonymous mutations are distributed in three other strain pairs.

“We additionally found another non-synonymous mutation shared by these two strains (A942T), and four other MSH2 non-synonymous mutations distributed in three other strain pairs (Supplementary Figure S3).”

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Most tested conditions resulted in similar growth of the three clonal isolates, with the exception of oxidative (hydrogen peroxide) and acidic (pH2) stress conditions.

“Most tested conditions resulted in a similar growth of the three clonal isolates, with the exception of oxidative (hydrogen peroxide) and acidic (pH2) stress conditions (Figure 4A and Supplementary Table S6).”

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In our dataset, three different non-synonymous mutations were shared in three different pairs of isolates: V62I in CMRL1/CMRL2, E142K in NRZ-2016-057/NRZ-2016-058, and M155T in SAT strains.

“In our dataset, we observed three different non-synonymous mutations shared in three different pair of isolates: V62I in CMRL1/CMRL2, E142K in NRZ-2016-057/NRZ-2016-058, and M155T in SAT strains.”

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Non-synonymous mutations in genes potentially related to drug resistance were common, sometimes, but not always, correlating with experimentally determined lower susceptibilities to drugs.

“We found that non-synonymous mutations in genes potentially related to drug resistance were common, sometimes, but not always, correlating with experimentally determined lower susceptibilities to drugs.”

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SAT03BC and SAT02PL showed a faster growth rate as compared to the other isolates in the presence of hydrogen peroxide or low pH (pH = 2), respectively.

“SAT03BC and SAT02PL showed a faster growth rate as compared to the other isolates in the presence of hydrogen peroxide or low pH (pH = 2), respectively.”

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Genetically diverse C. glabrata lineages can recombine, leading to genetic admixture.

“Another ability of C. glabrata that has been confirmed recently, using comparison of whole genome sequences, is that genetically diverse lineages can recombine, leading to genetic admixture (Dodgson et al., 2005; Carreté et al., 2018).”

• cites Dodgson et al., 2005 sourceNot checked yet

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• cites Carreté et al., 2018 sourceNot checked yet

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In C. albicans genome studies, serial isolates allowed the identification of alterations that may have contributed to drug resistance over the treatment course, including segmental aneuploidies, loss of heterozygosity, and gene-level alterations.

“In genome studies of the other major yeast pathogen, C. albicans, serial isolates allowed the identification of several alterations that may have contributed to drug resistance over the treatment course, including segmental aneuploidies (Selmecki et al., 2006), loss of heterozygosity (LOH) in large parts of the chromosomes (Dunkel et al., 2008), and alterations at the gene level.”

• cites Selmecki et al., 2006 sourceNot checked yet

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• cites Dunkel et al., 2008 sourceNot checked yet

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C. glabrata is a haploid organism and thus, with the exception of aneuploidies, loss of heterozygosity does not play a role.

“These include the fact that C. glabrata is a haploid organism and thus, with the exception of aneuploidies, LOH does not play a role.”

• cites Gabaldón and Carreté, 2016Not checked yet

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There exists significant variation within C. albicans samples, suggesting that selection to become more resistant to drugs can shape the genetic variation of infecting populations.

“Despite the usually clonal nature of clinical isolates obtained from the same patient, it is known that there exist significant variation within C. albicans samples, suggesting that selection to become more resistant to drugs can shape the genetic variation of infecting populations (Ford et al., 2015; Hirakawa et al., 2015).”

• cites Ford et al., 2015 sourceNot checked yet

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• cites Hirakawa et al., 2015 sourceNot checked yet

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C. glabrata presents different infection strategies and antifungal properties as compared to C. albicans.

“In addition, C. glabrata presents different infection strategies and antifungal properties as compared to C. albicans.”

• cites Gabaldón and Carreté, 2016Not checked yet

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Genetic variation and the presence of non-synonymous mutations and copy number variations accumulated within the host.

“Genetic variation and the presence of non-synonymous mutations and copy number variations accumulated within the host, suggest that clonal populations entail a non-negligible level of genetic variation that may reflect selection processes that occur within the human body.”

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The C. glabrata genome harbors several members of a specific gene family encoding epithelial adhesin proteins (EPA genes), which are considered a key factor in its ability to infect humans.

“The C. glabrata genome harbors several members of a specific gene family encoding epithelial adhesin proteins (EPA genes), which are considered a key factor in its ability to infect humans (Cormack and Falkow, 1999; Roetzer et al., 2011; Gabaldón et al., 2013; Vale-Silva et al., 2017).”

• cites Cormack and Falkow, 1999Not checked yet

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• cites Roetzer et al., 2011 sourceNot checked yet

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• cites Gabaldón et al., 2013 sourceNot checked yet

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These C. albicans studies could not differentiate between mutations that occurred during the course of infection or selection over pre-existing standing variation.

“However, these studies could not differentiate between mutations that occurred during the course of infection or selection over pre-existing standing variation.”

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C. glabrata isolates can be ascribed to at least seven genetically distinct clades that lack a geographical structure.

“In a recent study, we have shown that C. glabrata isolates can be ascribed to at least seven genetically distinct clades that lack a geographical structure (Carreté et al., 2018).”

• cites Carreté et al., 2018 sourceNot checked yet

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The aberrant mating type configuration of CANGA2A/CANGA2B was not reported in a previous analysis of these strains.

“The aberrant mating type configuration of CANGA2A/CANGA2B was not reported in a previous analysis of these strains (Håvelsrud and Gaustad, 2017).”

• cites Håvelsrud and Gaustad, 2017Not checked yet

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FKS1 and FKS2 mutations can confer resistance to echinocandin drugs in C. glabrata.

“it is known that FKS1 and FKS2 mutations can confer resistance to echinocandin drugs in C. glabrata (Katiyar et al., 2012; Arendrup and Perlin, 2014).”

• cites Katiyar et al., 2012 sourceNot checked yet

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• cites Arendrup and Perlin, 2014Not checked yet

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C. glabrata is likely able to undergo mating and introgression from differentiated strains.

“Importantly, this study also revealed that C. glabrata is likely able to undergo mating and introgression from differentiated strains (Carreté et al., 2018).”

• cites Carreté et al., 2018 sourceNot checked yet

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L280F in PDR1 is known to mediate azole resistance in C. glabrata.

“which is known to mediate azole resistance in C. glabrata (Ferrari et al., 2009).”

• cites Ferrari et al., 2009 sourceNot checked yet

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The finding that two deletions appear only in the first isolate corroborates that many of the differences between strains may represent standing variation, as gene deletions cannot be reversed.

“Two of the deletions appear only in the first isolate, which corroborates the previously mentioned finding that many of the differences between strains may represent standing variation, as gene deletions cannot be reversed.”

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Genomes from a total of 25 isolates corresponding to twelve different patients were analyzed in a common analytical framework to avoid analytical biases.

“Including the SAT strains, genomes from a total of 25 isolates corresponding to twelve different patients (Table 1) were analyzed in a common analytical framework to avoid analytical biases.”

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Closely related strains belonging to the same clade can have large phenotypic differences.

“Recent studies have shown that closely related strains belonging to the same clade can have large phenotypic differences (Carreté et al., 2018).”

• cites Carreté et al., 2018 sourceNot checked yet

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The three SAT strains were sequenced using Illumina paired-end technology to an average coverage ranging from 463 to 575×.

“The three strains were sequenced using Illumina paired-end technology to an average coverage ranging from 463 to 575×.”

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Most recent analyses of C. glabrata isolates obtained from the same patient revealed very little genetic variation, supporting the idea that a single clonal population colonizes different body sites and jointly contributes to infection.

“Most of these analyses revealed very little genetic variation, supporting the idea that a single clonal population colonizes different body sites and jointly contributes to infection.”

• cites Singh-Babak et al., 2012 sourceNot checked yet

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• cites Biswas et al., 2017 sourceNot checked yet

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• cites Håvelsrud and Gaustad, 2017Not checked yet

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The coordinates S629P and S663P belong to hot spot regions in FKS1 and FKS2 in which mutations can confer elevated MICs.

“These coordinates belong to hot spot regions in these two genes in which mutations can confer elevated MICs (Arendrup and Perlin, 2014).”

• cites Arendrup and Perlin, 2014Not checked yet

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C. glabrata can undergo genomic re-arrangements as an adaptive survival mechanism.

“Previous studies found that C. glabrata can undergo genomic re-arrangements as an adaptive survival mechanism (Shin et al., 2007; Muller et al., 2009; Poláková et al., 2009).”

• cites Shin et al., 2007 sourceNot checked yet

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• cites Muller et al., 2009 sourceNot checked yet

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• cites Poláková et al., 2009 sourceNot checked yet

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Previously a MATa to MATalpha switching event has been described for the P35_2/P35_3 pair.

“Previously a MATa to MATalpha switching event has been described already for the P35_2/P35_3 pair (Lin et al., 2007; Carreté et al., 2018).”

• cites Lin et al., 2007 sourceNot checked yet

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• cites Carreté et al., 2018 sourceNot checked yet

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Assuming a per base mutation rate similar to that of haploid yeasts (0.33 10-9 per base per generation), each generation one would expect 0.0041 mutations in the genome.

“If we were to assume a per base mutation rate similar to that of haploid yeasts (estimated at 0.33 10-9 per base per generation) (Lynch et al., 2008), each generation one would expect 0.0041 mutations in the genome (0.33 10-9 × 12,3 106 bases in the genome = 4.1 10-3).”

• cites Lynch et al., 2008 sourceNot checked yet

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• AssumptionThe authors state this as an assumption: The per base mutation rate of C. glabrata is similar to that of haploid yeasts, estimated at 0.33 10-9 per base per generation.

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The genetic structure of C. glabrata populations suggests that humans may not be the natural niche for this yeast.

“Moreover, the genetic structure of C. glabrata populations suggest that human may not be the natural niche for this yeast (Carreté et al., 2018).”

• cites Carreté et al., 2018 sourceNot checked yet

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Assuming a doubling time close to 1 h in S. cerevisiae, the strains are separated by 374-474 days, or diverged 187-237 days ago.

“Further assuming a doubling time close to 1 h in S. cerevisiae, these would suggest that these strains are separated by 374–474 days (or that they diverged 187–237 days ago).”

• AssumptionThe authors state this as an assumption: The doubling time of C. glabrata is close to 1 h, as in S. cerevisiae.

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Aberrant mating type switching events resulting in unorthodox configurations of the mating type loci are common among C. glabrata clinical isolates.

“Aberrant mating type switching events resulting in unorthodox configurations of the mating type loci have been shown to be common among C. glabrata clinical isolates (Brockert et al., 2003; Carreté et al., 2018)”

• cites Brockert et al., 2003 sourceNot checked yet

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• cites Carreté et al., 2018 sourceNot checked yet

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DSY562 and DSY565 both carry a V239L mutation in MSH2 and may display a faster evolutionary rate.

“In a previous analysis it was reported that DSY562 and DSY565 both carry a V239L mutation in MSH2 and that these strains may display a faster evolutionary rate (Vale-Silva et al., 2017).”

• cites Vale-Silva et al., 2017 sourceNot checked yet

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Two or three isolates per patient represent a very poor sampling of the infecting population, and the results should be confirmed with larger datasets.

“Admittedly, two or three isolates per patient represent a very poor sampling of the infecting population and our results should be confirmed with larger datasets.”

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SIR3 and SIR4 from several clinical isolates present a larger number of non-synonymous polymorphisms as compared to genes encoding other silencing proteins, and some such polymorphisms relate to different levels of silencing of target genes.

“A recent study (Leiva-Peláez et al., 2018) has found that SIR3 and SIR4 from several clinical isolates present a larger number of non-synonymous polymorphisms as compared to genes encoding other silencing proteins, and that some such polymorphisms relate to different levels of silencing of target genes.”

• cites Leiva-Peláez et al., 2018 sourceNot checked yet

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Mutations in FEN1 have been related to acquisition of resistance to echinocandins in C. glabrata, with some mutations leading to reduced susceptibility to caspofungin but not to micafungin.

“Mutations in the FEN1 gene have been related to acquisition of resistance to echinocandins in C. glabrata, with some mutations leading to reduced susceptibility to caspofungin but not to micafungin (Healey et al., 2012).”

• cites Healey et al., 2012 sourceNot checked yet

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SIR4 is a protein involved in subtelomeric silencing and regulation of biofilm formation.

“a protein involved in subtelomeric silencing and regulation of biofilm formation (Iraqui et al., 2004).”

• cites Iraqui et al., 2004 sourceNot checked yet

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With the current sampling and the lack of knowledge on the mutation rate and generation time of C. glabrata, it is impossible to know whether the high genetic variability predated the infectious phase and represents genetic variability existing in a commensal population, or whether most observed genetic differences appeared during the course of infection.

“With the current sampling and the lack of knowledge on the mutation rate and generation time of C. glabrata it is impossible to know whether this high genetic variability predated the infectious phase and represents genetic variability existing in a commensal population, or, alternatively, whether most of the observed genetic differences appeared during the course of infection.”

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A large standing variation would be compatible with the infecting strains originating from an established commensal population rather than from a recent infection with few infecting cells.

“Similarly, a large standing variation would also be compatible with the infecting strains originating from an established commensal population rather than from a recent infection with few infecting cells.”

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Genes encoding cell-wall proteins may be more mutagenic, given their subtelomeric localization and the presence of repeats.

“Alternatively, genes encoding cell-wall proteins may be more mutagenic, given their subtelomeric localization and the presence of repeats.”

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Mutations in MSH2, a mismatch repair gene, can influence the capacity to acquire resistance to drugs.

“Additionally, it has been suggested that mutations in MSH2, a mismatch repair gene, can influence the capacity to acquire resistance to drugs (Healey et al., 2016).”

• cites Healey et al., 2016 sourceNot checked yet

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These estimates seem to suggest that the divergence of the strains is larger than the period between isolations, reinforcing the conclusion that many mutations observed between any pair of serial isolates represent standing variation.

“Nevertheless these estimates seem to suggest that the divergence of the strains is larger than the period between isolations, reinforcing our conclusions that many of the mutations observed between any pair of serial isolates represent standing variation.”