A spontaneous mutation in DNA polymerase POL3 during in vitro passaging causes a hypermutator phenotype in Cryptococcus species

Abstract

Passaging of microbes in vitro can lead to the selection of microevolved derivatives with differing properties to their original parent strains. One well characterised instance is the phenotypic differences observed between the series of strains derived from the type strain of the human pathogenic fungus Cryptococcus neoformans. A second case was reported in the close relative Cryptococcus deneoformans, in which a well-studied isolate ATCC 24067 (52D) altered its phenotypic characteristics after in vitro passaging in different laboratories. One of these derivatives, ATCC 24067A, has decreased virulence and also exhibits a hypermutator phenotype, in which the mutation rate is increased compared to wild type. In this study, the molecular basis behind the changes in the lineage of ATCC 24067 was determined by next-generation sequencing of the parent and passaged strain genomes. This analysis resulted in the identification of a point mutation that causes a D270G amino acid substitution within the exonuclease proofreading domain of the DNA polymerase delta subunit encoded by POL3. Complementation with POL3 confirmed that this mutation is responsible for the hypermutator phenotype of this strain. Regeneration of the mutation in C. neoformans, to eliminate the additional mutations present in the ATCC 24067A genetic background, demonstrated that the hypermutator phenotype of the pol3^D270G mutant causes rapid microevolution in vitro but does not result in decreased virulence. These findings indicate that mutator strains can emerge in these pathogenic fungi without conferring a fitness cost, but the subsequent rapid accumulation of mutations can be deleterious.

1. Introduction

The relative success of a species is determined by it’s ability to adapt rapidly to changes in environmental conditions. In microbes, the small proportion of the microbial population possessing phenotypes which facilitate growth are selected for and these cells become predominant in the population in a short time frame. The phenotypic traits are generated by mutations in the microbe’s DNA sequence arising from unrepaired errors occurring during DNA replication or from environmental damage to DNA. This process is termed either adaptive evolution, when the mutations are rapidly acquired in response to the environment, or microevolution, to reflect the small scale nature of the evolutionary changes which occur.

Cryptococcosis, caused by a complex of seven species (C. neoformans, C. deneoformans and five in the C. gattii species complex), is a significant fungal disease worldwide, with high incidence and morbidity. In 2014, an estimated 181,100 deaths globally were attributed to cryptococcosis [1]. Cryptococcus species undergo microevolution both in vitro during laboratory culturing and in vivo during the course of disease [2-7]. Constant passaging in vitro can lead to the selection of microevolved derivatives that exhibit phenotypic differences with their original parental strains. One example of this is the series of strains derived from the Cryptococcus neoformans type strain, H99 (C. neoformans var. grubii) [8]. This strain was originally isolated in 1978. Since its isolation, laboratory passaging and distribution to numerous research groups has resulted in the establishment of a number of distinct lineages that differ in genotype and phenotype as a result of selection for adaptation to laboratory conditions [7,8]. Variants of this strain differ in melanisation, mating capacity, environmental stress response, antifungal drug resistance, urease production and virulence [7,8].

While the variation in the sub-strains derived from strain H99 are mostly single nucleotides that likely reflect errors that have occurred during the normal DNA replication process, recent discoveries have found that a subset of C. neoformans strains acquire mutations at a high rate. In particular, some clinical isolates are hypermutators and carry mutations in the DNA mismatch repair pathway, especially the MSH2 gene [6,9]. Hypermutators carrying mutations in msh2 have also been found within one of the three clonal expansions of the Cryptococcus deuterogattii outbreak within the Pacific Northwest region of the United States and Canada [10,11]. How widespread this phenomenon is in the Cryptococcus genus is currently unclear.

Passaging of one of the best characterized Cryptococcus deneoformans (C. neoformans var. neoformans) strains, ATCC 24067 (also known as 52D) has also resulted in microevolution. A comparative study of ATCC 24067 isolates obtained from six different research laboratories revealed laboratory passaging generated isolates with different phenotypic characteristics and one passaged derivative, ATCC 24067A, became attenuated for virulence [12]. This derivative was used in a number of studies, particularly as it was able to undergo phenotypic switching (reviewed in [13]). However, this derivative was later shown to exhibit a hypermutator phenotype – a higher than expected mutation rate resulting in an increased frequency of phenotypic switching and spontaneous resistance to the chemical FK506 [14]. ATCC 24067A exhibited increased sensitivity to oxidative stress agents and increased resistance to ethidium bromide, leading to the hypothesis that this strain carries a mutation in a DNA repair gene [14].

In this study we aimed to identify the underlying basis for the change in mutation rate in ATCC 24067A and investigated if this was the cause of decreased virulence in this isolate. Whole genome sequence analysis revealed a mutation in POL3 that causes a D270G amino acid substitution in the exonuclease proofreading domain of the DNA polymerase delta subunit and complementation confirmed that this mutation is responsible for the hypermutator phenotype of ATCC 24067A. This mutation is likely to be a significant contributing factor to the rapid generation of microevolved derivatives from this strain. Regeneration of the mutation in a C. neoformans genetic background, to eliminate additional mutations that are present in the C. deneoformans ATCC 24067A background, shows the hypermutator phenotype is responsible for rapid microevolution in vitro but not for the additional phenotypes observed in ATCC 24067A, including decreased virulence.

2. Materials and methods

2.1. Plasmid construction

POL3 was amplified from genomic DNA isolated from strain ATCC 24067 (C. deneoformans) with the primers KB183 and KB184 (Table S2) and cloned into TOPO pCR2.1 (Invitrogen, Life Technologies, Grand Island, NY) to generate plasmid KBG040. The pol3^D270G mutation was introduced into KBG040 by inverse PCR with the primers KB181 and KB182 (Table S2), generating KBG051. Complementation constructs for ATCC 24067A were generated by cloning the XbaI fragments from KBG040 and KBG051 into the XbaI site of pPZP-NEO11 generating KBG047 (POL3⁺) and KBG049 (pol3^D270G), respectively.

The deletion construct, KBG056, was generated with the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) using XhoI-digested pCR2.1 TOPO and PCR generated with the primers KB243 and KB244 to amplify 5´ POL3, KB245 and KB246 to amplify the nourseothricin acetyltransferase (NAT) resistance cassette and KB247 and KB248 to amplify 3´ POL3 (Table S2).

POL3 was also amplified from genomic DNA isolated from strain KN99α (C. neoformans) using the primers KB219 and KB220 (Table S2) and cloned into TOPO pCR2.1 to generate plasmid KBG053. The pol3^D270G mutation was introduced into KBG053 by inverse PCR with the primers KB181 and KB212 (Table S2), generating KBG054. The pol3^D270G::NAT construct (KBG059) for homologous integration into strain KN99α at POL3 was made using the NEBuilder HiFi DNA Assembly Cloning Kit with BamHI-digested KBG054 with PCR products generated with the primers KB239 and KB240 to amplify 5´ POL3 and KB241 and KB242 to amplify NAT (Table S2). A complementation construct for the KBCN0074 strain was generated by cloning the PCR generated with the primers KB253 and KB254 into BamHI-digested vector pPZP-NEO11 (KBG012) using the NEBuilder HiFi DNA Assembly Cloning Kit.

2.2. Strains and growth conditions

Strains used in this study are listed in Table 1. Cryptococcus wild type strains used were C. neoformans KN99α/a [15], and C. deneoformans ATCC 24067 and ATCC 24067A. ATCC 24067A is derived from laboratory passage of ATCC 24067. Once generated, all strains were immediately stored on glycerol at −70 °C. The strains were struck off glycerol just prior to each experiment and propagating in the laboratory was kept to a minimum. A single similar-sized colony was used for inoculation. Cryptococcus strains were cultured in yeast extract-peptone dextrose (YPD) ± 2 % agar medium and stored as glycerol stocks at −70 °C.

Table 1.

Strains used in this study.

Strain Name	Strain obtained/derived from	Genotype
KN99α	[15]	Wild type
KN99a	[15]	Wild type
ATCC 24067	[51]	Wild type
ATCC 24067A	[12]	Wild type
KBCN0041	ATCC 24067A	[POL3+ NEO]
KBCN0043	ATCC 24067A	[POL3D270G NEO]
KBCN0074	KN99α	pol3D270G NATα
KBCN0076	KN99α	pol3D270G NATα
KBCN0131	KBCN0074 x KN99a	pol3D270G NATa
KBCN0077	KN99α	pol3D270G NAT [[POL3 NEO]
AISVCN195	[6]	msh2Δ::NAT
AI187	[17]	MATa/MATα ade2/ADE2 ura5/URA5
KBCN0048	AI187	MATa/MATα ade2/ADE2 ura5/URA5 po13Δ::NAT/POL3
KBCN0126	KBCN0131 x AISVCN195	pol3D270G NAT msh2Δ::NAT
KBCN0127	KBCN0131 x AISVCN195	pol3D270G NAT msh2Δ::NAT
KBCN0128	KBCN0131 x AISVCN195	pol3D270G NAT msh2Δ::NAT
KBCN0129	KBCN0131 x AISVCN195	pol3D270G NAT msh2Δ::NAT

The strains derived from sporulation of the POL3/pol3 heterozygote and those from in vitro microevolution are not listed.

To test complementation of strain ATCC 26067A with POL3, ATCC 26067A was transformed by Agrobacterium-mediated delivery of T-DNAs from plasmids KBG047 and KBG049 with selection on YPD medium containing cefotaxime (Sigma) (200 μg/mL) and Geneticin [G418] (Gibco Life Technologies) (100 μL/100 mL), generating strains KBCN0041 and KBCN0043.

A genetic segregation analysis strategy as previously described [16] was used to test if the pol3Δ allele is lethal in C. neoformans. One copy of POL3 was deleted by homologous recombination in the diploid strain AI187 [MATa/MATα ade2/ADE2 ura5/URA5; [17]] using the PCR amplified deletion cassette from KBG056 (amplified with KB243 and KB248). A heterozygous mutant strain (KBCN0048) was identified by PCR and confirmed by Southern blot analysis. KBCN0048 was inoculated on Murashige & Skoog medium and incubated in the dark at 22 °C to induce meiosis and the production of basidiospores. Basidiospores were micromanipulated onto YPD medium, and allowed to germinate at 30 °C. Colonies were phenotyped for genetic markers (URA5 and MAT) on YNB + adenine and YNB + uracil media and by PCRs specific for each mating type (Table S2).

To regenerate the pol3^D270G mutation in the KN99α background (KBCN0074), the PCR amplified deletion cassette from KBG059 (amplified with KB239 and KB220) was precipitated onto gold beads and strain KN99α transformed using a PDS/He-1000 biolistic apparatus [Bio-Rad, Hercules, CA [18]] and plated onto YPD with nourseothricin (Jena Bioscience) (100 μg/ml). Gene replacement was confirmed by PCR and Southern blot analysis and the introduction of the D270G mutation confirmed by sequencing over the region with primers KB190 and KB259. KBCN0074 was complemented with the plasmid KBG064 by Agrobacterium-mediated transformation and selection on cefotaxime (200 μg/mL) and Geneticin [G418] (50 μL/100 mL), generating strain KBCN0077.

The KBCN0074 strain (pol3^D270G) was crossed to KN99a to generate KBCN0131 (Table 1). This strain was crossed to AISVCN195 (msh2Δ) and meiotic progeny obtained. The 21 progeny obtained from this cross were genotyped by growth on nourseothricin and screening for the pol3^D270G and msh2Δ mutations by PCR. 6 wildtype, 6 pol3^D270G, 5 msh2Δ and 4 pol3^D270G msh2Δ progeny were obtained. The genotypes of the pol3^D270G msh2Δ progeny were confirmed by PCR, Southern blot analysis and sequencing over the pol3^D270G region with primers KB190 and KB259.

Differential growth rate was assessed in three independent experiments by comparison of the average growth rate after 2 days to wild type (2 mL of liquid YPD was inoculated with 1 × 10⁵ cells and the number of cells was measured per mL after 2 days incubation). Viability was measured in three independent experiments by plating 1 × 10² cells onto YPD and incubating for 4 days at 30 °C and counting the resultant number of colonies. Colony diameter was measured in two independent experiments, by plating 50 cfus of an overnight YPD culture onto YPD plates and incubating for 4 days at 28 °C and diameter (mm) measured in ImageJ for 50 colonies per strain: means and standard errors of the mean were calculated in GraphPad Prism4 version 4.0c.

Fluconazole E tests were performed on yeast nitrogen base agar plates pH 7.0 as per the manufacturer’s instructions and [19]. To assess the frequency of colony formation on fluconazole, 1 × 10⁵ cells of each strain from an overnight YPD culture was used to inoculate 3 separate YPD cultures. After growth for 48 h in a roller drum at room temperature, 1 × 10⁵ cells were plated onto YPD + 72 μg/mL fluconazole (16 × MIC) and plates incubated at 30 °C for 7 days. Frequencies were calculated as number of colonies on the drug plate divided by the total CFU plated. Frequency averages and standard errors of the mean were calculated using Prism 8.0.2. Two tailed Student t tests and one-way ANOVA were performed to determine statistical significance.

2.3. Sequence analysis to identify mutations

Next-generation (Ion torrent) sequencing was performed by the Australian Genome Research Facility (AGRF). The ATCC24067 and ATCC24067A sequences had a 211 bp mean length, with 3.3 and 1.6 million reads, respectively (~37× and ~18× genome coverage). Sequencing reads from each strain were aligned in Geneious version 8.1.7 [20] onto the genome sequence of strain JEC21 [21]. Variants between the ATCC24067 strains and JEC21 were detected using the criteria of a minimum coverage of six reads, minimum similarity of 80 %, and the maximum P value of 1 e-6. Polymorphisms unique to ATCC 24067A were identified by a visual inspection across each chromosome. Sequencing reads were deposited to the GenBank SRA database as accession SRP131707.

Genomic DNA was extracted from 20 5-FOA and 20 5-FU resistant mutants isolated during the wild type and pol3^D270G fluctuation analysis (see below), using a CTAB buffer (100 mM Tris–HCl pH 7.5, 0.7 M NaCl, 10 mM EDTA, 1 % β-mercaptoethanol and 1 % CTAB [alkyl-trimethyl ammonium bromide, Sigma]). The URA5 gene was PCR amplified from 5-FOA resistant mutants using the primers KB187 and KB188 (Table S2). FUR1 was PCR amplified from 5-FU resistant mutants using the primers KB207 and KB208. The PCR products were sequenced with the primers used for amplification and an additional internal primer for FUR1, KB209 (Table S2). Sanger sequencing was performed at the Australian Genome Research Facility (AGRF). Two tailed Student t tests and one-way ANOVA were performed to determine statistical significance.

2.4. Mutation rate analysis

Spontaneous resistance to 5-fluoroorotic acid (5-FOA) and 5-fluorouracil (5-FU) was used to measure mutation rate by fluctuation analysis. For each strain, 1 × 10⁵ cells from an overnight YPD culture was used to inoculate 20 separate YPD cultures. 1 × 10⁵ cells from the overnight YPD culture used as the inoculum was also plated onto YNB +0.05 mg/mL uracil +1 mg/mL 5-FOA medium to check for the presence of pre-existing mutations in the culture. No pre-existing mutations were detected in any of the strains. After growth of the 20 separate YPD cultures for 48 h in a roller drum at room temperature, 1 × 10⁷ cells of each culture were plated onto YNB +0.05 mg/mL uracil +1 mg/mL 5-FOA or YPD +0.25 mg/mL 5-FU medium. The numbers of spontaneously arising colonies were counted from the 20 independent cultures. The mutation rate plates were examined under dissection microscope and no mutant microcolonies could be detected. The bz method was used to estimate the number of mutations, from the observed values of mutants, across the parallel cultures [22,23]. Mutation rates per cell division corrected by plating efficiency and the 95 % upper and lower confidence limits are shown in Table S3. Plating efficiency did not vary between replica platings.

2.5. Testing for sensitivity or tolerance to stress agents

Strains were cultured overnight in YPD medium, ten-fold serially diluted, and plated onto YPD ± stress agents at 28 °C for 2 days: 0.06 % ethidium bromide solution (EtBr) (Sigma Aldrich, E1510), 0.04 % methyl methanesulfonate (MMS) (Sigma Aldrich, 129925), 0.25 mM menadione (Sigma Aldrich, M5625), 5 mM hydrogen peroxide solution (H₂O₂) (Sigma Aldrich, 216763) and 0.4 mM Luperox TBH70X tert-butyl hydroperoxide solution (tBOOH) (Sigma Aldrich, 458139). One set of plates was exposed to UV light (120 Jm⁻²) and one set was incubated at 37 °C. Strains were also plated on l-DOPA medium to assess melanization.

2.6. Phenotypic characterisation of microevolving strains

Wild type and the pol3^D270G mutant were streaked for single colonies on YPD or YPD + nourseothricin (100 μg/ml) plates and incubated at 28 °C. Single colonies were re-streaked every 7 days for 2 months (approximately 650 generations). Four independent strains were generated for wild type and the pol3^D270G mutant (Table S2). Strains were then cultured overnight in YPD medium, ten-fold serially diluted, and plated onto YPD ± stress agents: 0.25 mM menadione, 5 mM hydrogen peroxide solution (H₂O₂) and 0.4 mM Luperox TBH70X tert-butyl hydroperoxide solution (tBOOH). Strains were also plated on l-DOPA medium to assess melanization. Plates were incubated at 28 °C for 2 days, and one set of plates was incubated at 37 °C.

2.7. Virulence studies in vivo and fungal burden in infected organs

Yeast strains were grown at 30 °C overnight, and cultures were washed twice with PBS and resuspended at a final concentration of 2 × 10⁶ cells/ml. Groups of female A/Jcr mice (Jackson Laboratory) were infected intranasally with 1 × 10⁵ yeast cells of each strain. Over the course of experiments, animals that appeared moribund or in pain were sacrificed by CO₂ inhalation. Survival data from the murine experiments were statistically analysed between paired groups by using the log rank test in Prism program 4.0 (GraphPad Software, San Diego, CA). P values of < 0.01 were considered significant.

To compare fungal burdens, infected lungs and brains at the endpoint of the experiment were isolated and homogenized in PBS buffer using a homogenizer. Resuspensions were diluted, and 100 μl of each dilution were spread on YPD medium. Fungal colonies were counted after 3 days of incubation at 30 °C. Statistical analyses used the two-tailed Student’s t test. P values of < 0.05 were considered statistically significant. Ethics statement: This animal study was performed according to the guidelines of NIH and Institutional Animal Care and Use Committee (IACUC). The animal models and procedures used have been approved by the IACUC at Rutgers University (PROT0999901066).

3. Results

3.1. The microevolved laboratory strain ATCC 24067A has a mutation in the exonuclease proofreading domain of the DNA Polymerase delta subunit encoded by POL3

A microevolved laboratory strain, ATCC 24067A, generated by the passaging of one of the best characterised and intensively studied strains of C. deneoformans, ATCC 24067, was previously shown to exhibit an elevated mutation rate and decreased virulence [12,14](Supp. Fig. 1). To determine the molecular cause of these phenotypes the genomes of ATCC 24067 and ATCC 24067A were obtained by next-generation sequencing. We examined the genome sequencing information for strain ATCC 24067A to identify DNA mutations that could account for the altered phenotypes in this passaged strain. There were a total of 129 single nucleotide changes, as 72 transitions and 51 transversions and six deletions, between strains ATCC 24067 and the passaged ATCC 24067A (Table SI). Many of the mutations were intragenic, with predictions of altered protein sequences for at least 57 genes. Most of the ~7,000 genes in the C. neoformans and C. deneoformans genome remain to have functions linked to them. Nonetheless, mutations were found in six genes, ADE13, HLH2, RAD1, SNF102, TRP3, XRN1, which, based on the reported phenotypes of the deletion strains, are likely to account for the alterations in growth, fertility, pathogenicity, melanin production and stress resistance observed in the ATCC 24067A strain [24-30]. One SNP was located in the coding region of CNAG_02563 (A1132G), an uncharacterized gene in Cryptococcus species predicted to encode the DNA polymerase delta subunit, orthologous to S. cerevisiae POL3. The SNP results in an amino acid substitution (D270G) in the exonuclease proofreading domain (Fig. 1A). Mutation of that same amino acid residue in S. cerevisiae leads to a 86 times higher mutation rate [31].

Fig. 1. Introduction of POL3 to strain ATCC 24067A complements the hypermutator phenotype.

A. Protein alignment of the Pol3 exonuclease domain from S. cerevisiae (Sc), C. neoformans (Cn) and C. deneoformans (Cd) showing the aspartate (D) residue mutated in ATCC 24067A (red arrow).

B&C. Quantitative assessment of mutation rate (mutation rate per cell division corrected by plating efficiency (μcorr)) in the ATCC 24067, ATCC 24067A, ATCC 24067A [POL3⁺] and ATCC 24067A [pol3^D270G] strains using fluctuation analysis and the bz method and spontaneous resistance to 5-FU (B) and 5-FOA (C). The error bars represent the 95 % upper and lower confidence limits. Introduction of POL3, but not pol3^D270G, into strain ATCC 24067A results in a mutation rate almost equivalent to wild type.

To assess if the pol3^D270G mutation is responsible for the elevated mutation rate in ATCC 24067A, a wild type copy of POL3 and a pol3^D270G allele were introduced into ATCC 24067A at an ectopic location using Agrobacterium tumefaciens mediated transformation and selection with G-418. Mutation rate was qualitatively assessed by observing the generation of spontaneously resistant 5-fluorouracil (5-FU) and 5-fluoroorotic acid (5-FOA) colonies. 5-FU inhibits the growth of wild type strains by inhibiting the activity of thymidylate synthetase, which affects pyrimidine synthesis and leads to an imbalance of intracellular dNTP pools. 5-FOA inhibits the growth of wild type strains as 5-FOA is converted by orotine-5´-monophosphate decarboxylase to toxic 5-flurouracil. Introduction of the wild type copy of POL3, but not an extra copy of pol3^D270G, resulted in a mutation rate almost equivalent to ATCC 24067, suggesting it is the pol3^D270G mutation that is causing the increased mutation rate in ATCC 24067A. The increased mutation rate was quantitatively assessed using fluctuation analysis and resistance to 5-FU and 5-FOA [22,23]. The ATCC 24067A strain shows a mutation rate significantly higher than ATCC 24067 (Fig. 1B and C). Introduction of wild type POL3 into ATCC 24067A restored the mutation rate to close to that of ATCC 24067 (Fig. 1B and C). There is a significant difference between ATCC 24067 and ATCC 24067A transformed with wild type POL3⁺. The residual mutator phenotype may be a result of other mutations in this genetic background elevating the mutation rate or the pol3^D270G allele having a semi-dominant effect, as has been observed in S. cerevisiae pol3-01 [36]. In contrast, introduction of another copy of pol3^D270G into ATCC 24067A did not alter the mutation rate compared to ATCC 24067A (Fig. 1B).

3.2. POL3 is a gene essential for viability in C. neoformans

In S. cerevisiae and S. pombe deletion of POL3 is lethal. To assess if deletion of POL3 is also lethal in C. neoformans, one copy of POL3 was deleted in the diploid strain AI187 by replacing the open reading frame via homologous recombination, with a cassette conferring nourseothricin resistance (Fig. 2A-C). The heterozygote strain pol3Δ::NAT/POL3 was induced to undergo meiosis and sporulation. 18 haploid progeny were isolated and PCR screening showed all were wild type for POL3, rather than the expected 1:1 ratio of wild type vs. mutant. Consistent with this, none of the progeny were resistant to nourseothricin, i.e. carried the pol3Δ::NAT allele (Fig. 2D). Phenotypes controlled by two markers that are on separate chromosomes in strain AI187 (i.e. segregation of MATa/MATα; ura5/URA5) were tested, showing independent segregation of the mating type (MAT) and URA5 loci (Fig. 2D). These results indicate that the deletion of POL3 in C. neoformans is lethal.

Fig. 2. Deletion of POL3 is lethal in C. neoformans.

A. Schematic diagram of POL3 in the diploid strain AI187 (POL3 POL3) and the pol3Δ::NAT/POL3 heterozygote strain KBCN0048 (POL3 pol3Δ) showing the POL3 coding sequence in dark blue, 5´ and 3´ homologous flanking sequences present in the deletion construct in light blue, the NAT selectable marker in red. Screening primers for specific amplification of the 5´ region (green) and 3´ region (pink) in diploid strains with one copy of POL3 successfully replaced are indicated. Primers to determine if only one copy of POL3 is deleted in the diploid are indicated in orange. B. PCR amplification in the diploid strain AI187 (POL3 POL3) and the pol3Δ::NAT/POL3 heterozygote strain KBCN0048 (POL3 pol3Δ) using primers indicated in A. C. Southern blot analysis of the wild type (POL3), diploid strain AI187 (POL3 POL3) and pol3Δ::NAT/POL3 heterozygote strain KBCN0048 (POL3 pol3Δ) confirming the deletion of one copy of POL3. D. The 18 meiotic haploid progeny isolated from the pol3Δ::NAT/POL3 heterozygote strain KBCN0048 plated on YPD, YPD + nourseothricin, YNB + adenine or with mating type indicated as alpha (dark grey) or a (light grey). The position of the AI187 diploid (D) and pol3Δ::NAT/POL3 heterozygote (Δ) controls are indicated in the mating type panel. None of the meiotic progeny are resistant to nourseothricin. Growth on YNB + adenine and mating type shows independent segregation of the URA5 and mating type (MAT) loci.

3.3. The pol3^D270G mutant possesses an elevated mutation rate

Strain ATCC 24067A possesses additional phenotypic differences to ATCC 24067 which could be due to the pol3^D270G allele or to other mutations that have accumulated during strain propagation. To examine the phenotype of pol3^D270G without these background mutations the pol3^D270G mutation was regenerated in strain KN99α (C. neoformans) (Fig. 3). KN99α was chosen so a direct comparison could be made between the pol3^D270G mutant and the mismatch repair (MMR) mutants such as the msh2Δ, which were previously generated in this genetic background [6]. Pol3 is 97.5 % identical and 98.8 % positive in C. neoformans and C. deneoformans and 98.3 % identical and 100 % positive across the exonuclease domain (Fig. 1A). A complemented strain was generated by re-introduction of the wild type gene at an ectopic location using Agrobacterium tumefaciens mediated transformation and resistance to G-418. Mutation rate was assessed by observing the generation of spontaneously resistant 5-fluorouracil (5-FU) colonies in wild type, pol3^D270G and pol3^D270G POL3⁺ strains. Similar to the ATCC 24067A strain, the pol3^D270G mutant exhibited an elevated mutation rate compared to wild type and the pol3^D270G POL3⁺ strain. The increased mutation rate was quantified using fluctuation analysis on 5-FU (Fig. 4A) and 5-FOA (Fig. 4B) [22,23]. The pol3^D270G mutant possesses a mutation rate significantly higher than wild type and the complemented strain (Fig. 4A and B).

Fig. 3. The generation of a pol3^D270G mutant in C. neoformans.

A. Schematic diagram of POL3 in wild type (WT) and (B) in the pol3^D270G mutant in C. neoformans (pol3^D270G) showing the POL3 coding sequence in dark blue, 5´ homologous flanking sequences present in the integration construct in light blue, the NAT selectable marker in red. Screening primers for specific amplification of the 5´ region (green), integration of the NAT construct (pink) and sequencing of the region of POL3 to confirm the introduction of the mutation (orange) are indicated. C. PCR of the wild type (WT) and pol3^D270G mutant KBCN0074 (pol3^D270G) with primer combinations 255/259 and 255/256 showing integration of the construct at POL3 in thepol3^D270G mutant. D. Southern blot analysis of the wild type (WT), two independently isolated pol3^D270G mutants (KBCN0074 and KBCN0076) mating type α (pol3^D270G α), the pol3^D270G mutant of mating type a (pol3^D270G a) obtained from crossing KBCN0074 to KN99a (strain KBCN0131) and the AISVCN195 (msh2Δ) strain, probed with POL3, showing successful integration of the pol3^D270G construct at POL3.

Fig. 4. The pol3^D270G mutation elevates mutation rate.

Quantitative assessment of mutation rate (mutation rate per cell division corrected by plating efficiency (μcorr)) in the wild type (WT), pol3^D270G and pol3^D270G [POL3⁺] strains using fluctuation analysis and the bz method based on the spontaneous resistance to 5-FU (A) and 5-FOA (B). The error bars represent the 95 % upper and lower confidence limits.

3.4. The pol3^D270G mutation increases transition and transversion mutations

The most common causes of resistance to 5-FOA and 5-FU in C. neoformans are mutations of the URA5 and FUR1 genes [6,14,32]. URA5 and FUR1 were sequenced from 20 5-FOA resistant and 20 5-FU resistant strains isolated from the wild type and pol3^D270G mutants, derived from the independent parallel cultures used in the fluctuation analysis, in order to determine the types of mutations generated in the absence of DNA polymerase proofreading. URA5 from wild type 5-FOA resistant strains contained a variety of mutations including small and large insertions or deletions, transversions and transitions (Fig. 5A and C). The pol3^D270G mutant displayed a shift in the mutation profile of URA5 (Fig. 5A and C). In contrast to wild type, URA5 from pol3^D270G mutant 5-FOA resistant strains possessed a smaller number of insertion and deletion mutations and a statistically significant increase in the number of transition mutations, from 30 % in wild type to 65 % in the pol3^D270G mutants (Fig. 5C).

Fig. 5. The pol3^D270G mutant shows an increased proportion of transition and transversion mutations.

A. Schematic representation of the URA5 gene indicating the location and type of spontaneous mutations generated in 20 5-FOA resistant isolates derived each from the wild type (WT) and pol3^D270G strains. Insertions are indicated with black arrows (large arrowhead depicts a large insertion), large deletions with black bars (Δ), transitions as solid lines and transversions as dashed lines.

B. Schematic representation of the FUR1 gene indicating the location and type of spontaneous mutations generated in the 20 wild type (WT) and 20 pol3^D270G 5-FU resistant isolates. Insertions are indicated with black arrows, large deletions with black bars (Δ), small deletions with grey arrows, deletions in homopolymeric tracts as red arrows, transitions as solid lines and transversions as dashed lines. The three homopolymeric tracts in FUR1 are indicated as red boxes.

C. Percentage of URA5 insertions (black), deletions (dark grey), transversions (white) and transitions (light grey) in the wild type (WT) and pol3^D270G 5-FOA resistant isolates. The pol3^D270G mutant shows an increased proportion of single bp transition mutations. Asterisks indicate statistical significance using a two-tailed student’s t test; ** p < 0.005.

D. Graph showing the percentage of FUR1 with no mutations (white), large deletions (grey spots), small deletions (checkered grey), small insertions (hatched grey), deletions in homopolymeric tracts (light grey), transitions (dark grey) and transversions (black) in the wild type (WT) and pol3^D270G 5-FU resistant isolates. Compared to wild type, the pol3^D270G mutant shows an increased proportion of transversions and transitions and no deletions in homopolymeric tracts. Asterisks indicate statistical significance using a two-tailed student’s t test; ** p < 0.005, *** p < 0.0005.

Unlike URA5, FUR1 contains a number of homopolymeric tracts; (C)₇ at +81-87, (A)₆ at +460-465 and (T)₁₄ at +941-954. Not all 5-FU resistant strains contained mutations in FUR1, 3/20 genes from wild type and 3/20 from the pol3^D270G mutant did not contain a mutation in FUR1. The FUR1 gene sequenced from 17/20 wild type 5-FU resistant strains contained a variety of mutations including large and small deletions and insertions, transitions and a large number (60 %) of deletions in the (A)₆ homopolymeric tract (Fig. 5B and D). The pol3^D270G mutant displayed a shift in the mutation profile of FUR1 compared to wild type (Fig. 5D). In contrast to wild type, FUR1 from 17/20 of the pol3^D270G 5-FU resistant strains possessed no deletions in the (A)₆ homopolymeric tract and an increase in the number of transitions and transversions (Fig. 5D). This shift in mutational profile was shown to be statistically significant using two-tailed t tests and two way ANOVA.

3.5. The newly-generated C. neoformans pol3^D270G mutant does not possess the additional phenotypes observed in the C. deneoformans ATCC 24067A strain

In addition to exhibiting an increased mutation rate, strain ATCC 24067A also shows additional phenotypes that could be attributed to an absence of repair of DNA replication errors, such as sensitivity or tolerance to DNA damaging agents [14]. The S. cerevisiae pol3-01 mutant displays increased sensitivity to the DNA damaging agent methyl methanesulfonate (MMS) [33]. To explore this further, the wild type and pol3^D270G mutant were cultured on a selection of DNA damaging chemicals or treated with UV light. The pol3^D270G mutant showed no difference in sensitivity compared to wild type on DNA damaging agents that affect DNA replication; ethidium bromide (EtBr), methyl methanesulfonate (MMS), cadmium sulfate (CdSO₄) and ultraviolet light (UV) (Fig. 6A), or on chemicals which generate reactive oxygen species (ROS); menadione, hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (tBOOH) (Fig. 6B). This suggests that the increased sensitivity to oxidative stress and resistance to ethidium bromide exhibited by the ATCC 24067A strain [14] is attributed to other SNPs in the genome and not to the pol3^D270G mutation. Strain ATCC 24067A also exhibits reduced melanization and colony size compared to ATCC 24067 [12]. However, melanization (Fig. 6B) and colony diameter (wild type 2.48 ± 0.05 mm; pol3^D270G 2.26 ± 0.05 mm) of the wild type and pol3^D270G mutant were equivalent.

Fig. 6. Loss of DNA polymerase proofreading leads to rapid microevolution of new phenotypes.

A & B. The wild type (WT) and pol3^D270G strains were cultured overnight in YPD medium, ten-fold serially diluted and plated at 28 °C for 2 days onto (A) YPD ± DNA stress agents: 0.06 % ethidium bromide solution (EtBr), 0.04 % methyl methanesulfonate (MMS), 0.01 mM cadmium sulfate (CdSO₄) and one set of plates was exposed to UV light (120 Jm⁻²) or onto (B) oxidative stress agents: 0.25 mM menadione, 5 mM hydrogen peroxide solution (H₂O₂) and 0.4 mM tert-butyl hydroperoxide solution (tBOOH). Strains were also incubated at 37 °C or plated on l-DOPA medium to assess melanization (B).

C. Growth of 4 strains of wild type (WT) and pol3^D270G derived from independent passaging for approximately 650 generations with population bottlenecks every 90 generations. The passaged strains were cultured overnight in YPD medium, ten-fold serially diluted, and plated onto YPD ± stress agents at 28 °C for 2 days: 0.25 mM menadione, 5 mM hydrogen peroxide solution (H₂O₂) and 0.4 mM tert-butyl hydroperoxide solution (tBOOH). Strains were also incubated at 37 °C or plated on l-DOPA medium to assess melanization. Unlike wild type, the pol3^D270G passaged strains display phenotypic differences compared to the original strain (B) and each other (C).

3.6. The hypermutator phenotype of pol3^D270G does not result in a reduction in virulence

Compared to the parental C. deneoformans ATCC 24067 strain, microevolved derivative ATCC 24067A is attenuated for virulence [12]. To determine if the pol3^D270G mutation contributes to decreased virulence, the C. neoformans pol3^D270G mutant was assessed in a conventional murine inhalation virulence assay. The virulence, as measured by mouse survival over time and fungal load in the lung or brain, of the pol3^D270G mutant did not significantly differ from the wild type (Fig. 7).

Fig. 7. The pol3^D270G mutation does not result in an attenuation of virulence.

A. Percentage survival of wild type and pol3^D270G mutant in a murine inhalation virulence assay. B. Colony forming units measured from brain and lung tissue of mice when sacrificed.

3.7. A lack of DNA polymerase delta proofreading leads to rapid microevolution

To determine if the increased mutation rate observed in the pol3^D270G mutant results in an increased rate of microevolution and phenotypic change, the wild type and pol3^D270G mutant were passaged as four independent cultures for approximately 650 generations. The cultures were passaged through population bottlenecks every 90 generations to allow non-lethal mutations to accumulate as if they are neutral by mitigating the effect of selection. The resultant strains were assessed for phenotypes that are associated with the ability to grow in vivo including growth at 37 °C, resistance to oxidative stress (paraquat and H₂O₂), alterations in the cell wall (congo red and calcofluor white stress) and melanization. No phenotypic differences were observed between the original wild type strain (Fig. 6B) and the 4 passaged wild type strains at 37 °C or on menadione, H₂O₂ and tBOOH (Fig. 6C). In contrast, the phenotypes of the passaged pol3^D270G mutant differed dramatically under these conditions from the original mutant (Fig. 6B) and from each other (Fig. 6C), suggesting that the rate of microevolution in these strains is higher than in wild type. Likewise, no differences in melanization were observed between the original and passaged wild type strains (Fig. 6B and C). In contrast, melanization differed dramatically in the passaged pol3^D270G mutant strains compared to the original mutant (Fig. 6B), with two strains exhibiting a decrease and one strain showing an increase in melanization (Fig. 6C).

3.8. The rapid microevolution of the pol3^D270G mutant leads to an increase in the emergence of spontaneous resistance to antifungal agents

Disrupting mismatch repair in C. neoformans results in an increase in spontaneously-arising fluconazole resistant colonies [6]. To investigate if this also occurs in the pol3^D270G mutant, which also displays rapid microevolution, the fluconazole minimum inhibitory concentration (MIC) was determined using E tests. The pol3^D270G mutant had a MIC equivalent to wild type (wild type 4.7 ± 0.7 μg/mL; pol3^D270G 5.3 ± 0.7 μg/mL). A small number of spontaneously arising fluconazole resistant colonies in the zone of clearing was observed compared to wild type (Fig. 8A). To quantify this increase in spontaneously-arising fluconazole resistant colonies, the colony frequency was calculated on 72 μg/mL (16 x MIC). The msh2Δ and pol3^D270G mutants showed a statistically significant increase in the frequency of fluconazole resistant colonies (Fig. 8B). The msh2Δ mutant displayed a higher number of spontaneously arising fluconazole resistant colonies than the pol3^D270G mutant (Fig. 8B).

Fig. 8. Themsh2Δand pol3^D270G mutations result in an increase in the spontaneous emergence of resistance to fluconazole.

A. Fluconazole minimum inhibitory concentrations (MIC) of the wild type (WT), pol3^D270G strains and msh2Δ strains determined by E tests, and the emergence of resistance seen in the halo of the msh2Δ and pol3^D270G mutants.

B. Quantification of the number of spontaneously resistant colonies arising on 72 μg/mL fluconazole (16 × MIC) media. Asterisks indicate statistical significance using a two-tailed student’s t test; * p < 0.05.

3.9. pol3^D270G msh2Δ double mutants exhibit reduced viability and growth

DNA replication errors are repaired by the action of two sequential systems, DNA polymerase 3´–5´ exonuclease activity and the mismatch repair (MMR) system. Deletion of the MMR component encoded by MSH2 results in an increase in mutation rate equivalent to that observed in the pol3^D270G mutant (Fig. 4B and C). To investigate if these mutations have an additive effect on mutation rate or are synthetically lethal, pol3^D270G msh2Δ double mutants were generated by crossing the pol3^D270G mutant with a msh2Δ mutant and obtaining meiotic progeny. Four pol3^D270G msh2Δ double mutants were obtained. In comparison to the wild type, pol3^D270G and msh2Δ strains, the pol3^D270G msh2Δ strains exhibited dramatically reduced growth rate and produced smaller colonies which varied greatly in size (Fig. 9A and B). Two of the strains showed dramatically reduced viability, most likely due to the accumulation of a detrimental mutation (Fig. 9C). Two of the strains showed variable viability (Fig. 9C), presumably from detrimental or advantageous mutations occurring rapidly within the time-course of the viability assessment. The increased mutation rate was quantitatively assessed using fluctuation analysis across the 20 independent parallel cultures on 5-FOA and the bz-rates method to account for the differential growth rate between wild type and the mutant cells (and plating efficiency) (Fig. 9D) [23]. The pol3^D270G msh2Δ mutants possess a mutation rate either higher or within the same range as the pol3^D270G and msh2Δ single mutants (Fig. 9D).

Fig. 9. The pol3^D270G msh2Δ double mutants exhibit reduced growth and viability.

A. Colonies of the wild type (WT), pol3^D270G, msh2Δ and pol3^D270G msh2Δ strains on YPD medium after 4 days. B. Differential growth rate of the pol3^D270G, msh2Δ and pol3^D270G msh2Δ strains compared to wild type (WT). C. Viability of the wild type (WT), pol3^D270G, msh2Δ and pol3^D270G msh2Δ strains. D. Quantitative assessment of mutation rate (mutation rate per cell division corrected by plating efficiency (μcorr)) using fluctuation analysis and the bz method based on the spontaneous resistance to 5-FOA. The error bars represent the 95 % upper and lower confidence limits.

4. Discussion

Hypermutation is a newly described property found in some isolates of C. neoformans, with to date this being attributed to mutations in a specific gene, MSH2, for mismatch repair (MMR) [6,9,11]. The process remains poorly understood, particularly from the perspectives of the impact of hypermutation on virulence. Here, the underlying basis for the change in mutation rate in the microevolved C. deneoformans derivative ATCC 24067A was identified as a mutation in the exonuclease proofreading domain of the DNA polymerase delta subunit encoded by POL3. DNA polymerase 3´–5´exonuclease activity acts prior to the mismatch repair (MMR) system to sequentially repair DNA replication errors. The ATCC 24067A derivative, and the comparable mutant in the C. neoformans genetic background, exhibited a hypermutator phenotype with an ~200 fold increase in mutation rate compared to wild type. This mutation rate is equivalent to that exhibited by the C. neoformans mismatch repair (MMR) mutants msh2Δ, mlh1Δ and pms1Δ [6]. Both the pol3^D270G mutant and MMR mutants are viable as they exhibit a mutation rate that falls below the error threshold for haploid cells [34]. The error threshold is the point at which inactivating mutations in essential genes leads to extinction. In the case of haploid organisms, this is estimated to be one inactivating mutation per essential gene per cell division, which corresponds to ~1000 fold increase in the wild type mutation rate [34,35]. Mutations which give rise to a mutation rate > 1000 fold are lethal, for example loss of exonuclease activity in the Escherichia coli DNA polymerase III epsilon subunit which results in a 1,000-10,000 fold increase in mutation rate which is equivalent to 1.3 inactivating mutation per essential gene per cell division [35].

The increased proportion of single nucleotide mutations observed both in the pol3^D270G and MMR mutants of C. neoformans [6] suggest Pol3 and MMR are repairing single nucleotide mismatches occurring during replication, as expected. Although DNA polymerase exonuclease activity and MMR act sequentially to correct errors arising during DNA replication to increase replication fidelity in a multiplicative effect, the mutational profiles differ between mutant strains, also highlighting specific roles of each repair component [36]. The pol3^D270G mutant possesses an increased proportion of transitions and transversions, whereas, MMR mutants display an increased proportion of transitions and mutations in homopolymeric tracts [6]. This suggests Pol3 exonuclease activity, and not MMR, repairs most mismatches resulting in transversions and conversely implies that mismatches in homopolymeric tracts occurring from DNA polymerase slippage are repaired predominantly by MMR, and not Pol3. Both mechanisms are used to repair mismatches resulting in transition mutations. The mutational profiles of the pol3 and msh2Δ mutants also differ in S. cerevisiae [36-38]. The S. cerevisiae pol3-01 and C. neoformans pol3^D270G mutants generate similar mutational profiles; mainly single bp mutations which are predominately A/G transitions [36]. The pol3^D270G msh2Δ double mutants in C. neoformans were found to be viable but displayed reduced growth and viability. The decreased viability is most likely observed due to mutations occurring in essential genes. As this phenotype is not observed in either of the single mutant strains, it suggests that the pol3 and msh2Δ mutations are having a synergistic effect and the combination of these mutations is resulting in a > 200 fold increase in mutation rate. The S. cerevisiae msh2Δ mutant was shown to have a 8 fold increase in mutation rate using fluctuation analysis based on resistance to canavanine and a 215 fold increase in mutation rate using whole genome sequencing [37]. The po13-01 msh2Δ double mutant is lethal in S. cerevisiae, where error-induced extinction occurs within 6–7 mitotic divisions due to a 10,000 fold increase in mutation rate [34-36,39]. The C. neoformans poi3^D270G msh2Δ double mutant is likely existing on the edge of extinction and colonies observed in the fluctuation analysis arise from suppressor mutations which are occurring to suppress the high mutation rate and enable growth.

Similar to what we previously observed with MMR mutants [6], a lack of Pol3 proofreading leads to rapid microevolution in vitro. Similar to the MMR mutants, this rapid microevolution leads to an increase in spontaneous resistance to fluconazole. However the emergence of resistance is at a much lower rate than the msh2Δ mutant. This may suggest that resistance is more commonly a result a mutation in a gene or genes – currently unidentified – containing a homopolymeric tract. The rapid microevolution of the pol3^D270G mutant does likely account for some of the additional phenotypes observed in the ATCC 24067A strain compared to the original parent strain, however whether these mutations occurred before or after the pol3^D270G mutation occurred cannot be determined. Interestingly, the pol3^D270G mutant in C. neoformans, which eliminated the additional mutations present in the ATCC 24067A C. deneoformans genetic background, did not exhibit decreased virulence in a murine inhalation infection model. This suggests that the additional mutations that have accumulated in ATCC 24067A, such as those resulting in phenotypic changes known to alter growth, fertility, pathogenicity, melanin production and stress resistance such as in genes ADE13, HLH2, RAD1, SNF102, TRP3, XRN1, are likely responsible for the attenuation of virulence in this ATCC 24067 derivative [24-30]. A similar situation has been observed in C. deuterogattii msh2 mutants exhibiting reduced virulence [11]. One of the three clonal expansions of the C. deuterogattii outbreak within the Pacific Northwest region of the United States and Canada contains three closely related isolates that are less virulent, are hypermutators and carry mutations in msh2 [10,11]. Billmyre et al. showed that the msh2 mutations in these isolates are not directly responsible for the decrease in virulence, but, similar to ATCC 24067A, are the likely result of the accumulation of mutations in critical pathways affecting virulence [11]. It is noteworthy that both the pol3^D270G mutant and msh2Δ mutant do not have an immediate effect on virulence in a standard murine inhalation model, as previously a large scale screen of 1200 defined deletion mutants of C. neoformans showed that MMR mutants (msh2Δ, pms1Δ and mlh1Δ) exhibit increased proliferation in a pooled infection experiment [40]. These results suggest that hypermutators are better able to adapt to the mouse lung. However, this increase in proliferation may only be observed in conditions where mutants are in a competitive environment, when a small number of cells are inoculated effectively creating a population bottleneck, or when by chance a mutation occurs which provides a selective advantage in the host.

High mutation rates are favoured in changing environmental conditions as they enhance the probability of a mutation occurring that provides a selective advantage and allows microevolution to occur. Nevertheless, most mutations will be deleterious and hypermutators will gradually lose fitness as they accumulate mutations and will ultimately become extinct after serially passaging through population bottlenecks. In bacteria this conundrum is overcome by making the hypermutator phenotype transient by returning to wild type mismatch repair using horizontal gene transfer. In fungi, this may be achieved using sexual recombination or the accumulation of anti-hypermutator mutations. Despite this, it is clear that a hypermutator phenotype can be utilized by pathogenic microorganisms to enable rapid adaptation to the challenges faced both in vitro and in vivo. The bacterial pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica have a large proportion of hypermutators with defects in MMR in their populations [41-44]. Recent studies in fungi also support the hypothesis that clinical populations of fungal pathogens may contain a large proportion of hypermutators [6,9,45-48]. We identified two C. neoformans clinical isolates that are hypermutators and have mutations in the MSH2 gene [6] and another study on the recurrence of meningitis due to C. neoformans identified one isolate with mutations in the RAD5 DNA repair gene and the MMR genes MSH2 and MSH5 [9]. In addition, C. glabrata clinical isolates possessing non-synonymous variation in MSH2 have now been detected in clinical populations in many parts of the world with varying prevalence [45-48]. However, the proportion of hypermutators in C. glabrata clinical populations is likely to be an overestimation [49]. These studies identified non-synonymous mutations in MSH2 but lacked an assessment of accurate mutation rate using fluctuation analysis [45-48]. A recent study by Shor et al. (2019) used a new method for comparing mutation rates across strains by employing the green fluorescent protein (GFP) as the marker and measurement of mutations within populations using fluorescent assisted cell sorting (FACS) [50]. This study reported no difference in mutation rates between two different naturally-occurring alleles of MSH2 [50]. This suggests that some of the non-synonymous mutations in MSH2 present in clinical isolates do not result in a mutator phenotype [49,50]. It is therefore of interest to investigate the frequency of non-synonymous mutations in MSH2 within large populations of fungal pathogens and how many of these mutations result in true hypermutators, in addition to, elucidating the implications this has on microevolution during disease progression.

The key finding from this study is that there are independent ways in which pathogenic fungi can develop a hypermutator phenotype. The mechanism discovered here, of a base pair substitution that changes a single amino acid, is likely a highly uncommon event within Cryptococcus populations relative to the mutations in the MSH2 gene previously reported. The D270 G change in Pol3 causes a loss of just part of this protein’s function, whereas the most likely outcome of a mutation in the gene is complete loss of function and a lethal phenotype. In contrast, Cryptococcus strains can gain mutations in many ways within MSH2 (or potentially other MMR pathway genes) without impacting fitness.