Recombinant Candida glabrata DNA mismatch repair protein MSH3 (MSH3), partial

What is the role of MSH3 in Candida glabrata DNA repair?

MSH3 is a key component of the post-replicative DNA mismatch repair system (MMR) in C. glabrata. It functions by heterodimerizing with MSH2 to form the MutSβ complex, which specifically recognizes and binds to DNA containing large insertion-deletion loops (IDLs) . This initial recognition step triggers downstream repair processes involving additional MMR components. Unlike many other Candida species, C. glabrata is haploid, making single mutations in DNA repair genes like MSH3 sufficient to cause a mutator phenotype that can accelerate the emergence of resistance-conferring mutations .

How does MSH3 function differ from other mismatch repair proteins?

MSH3 has specialized functions that distinguish it from other MMR proteins:

This division of labor allows for efficient repair of different types of DNA damage. MSH3's specific role in recognizing larger IDLs makes it particularly important for maintaining genomic stability in regions prone to replication slippage .

What genetic interactions have been identified with MSH3 in C. glabrata?

While much research has focused on MSH2 mutations, MSH3 operates within a complex network of DNA repair pathways. Studies have shown that mutations in MMR genes contribute to C. glabrata's ability to develop resistance to multiple drug classes . The genetic interaction between MSH3 and MSH2 is particularly critical, as they form the functional MutSβ complex. Additionally, MMR pathway defects appear to interact with homologous recombination (HR) pathways, as evidenced by research showing that "mutants of MMR and DSBR (double-strand break repair), but not BER (base excision repair) or NER (nucleotide excision repair), lead to increased appearance of fluconazole-resistant colonies" .

What expression systems are optimal for producing recombinant C. glabrata MSH3?

Based on the literature, several expression systems have been used for producing recombinant C. glabrata MSH3:

For most research applications, E. coli expression systems represent a good starting point, with progression to more complex systems if functionality issues arise .

How can MSH3 activity be accurately measured in vitro?

Several complementary approaches can be employed to measure the activity of recombinant MSH3:

• DNA binding assays: Electrophoretic mobility shift assays (EMSA) or fluorescence anisotropy to assess binding to DNA containing insertion-deletion loops

• ATPase activity measurements: Quantifying ATP hydrolysis rates in response to binding mismatched DNA

• Mismatch repair reconstitution: In vitro reconstitution of repair using purified components, measuring repair efficiency on substrates with defined IDLs

• Protein-protein interaction assays: Pull-down assays, analytical ultracentrifugation, or size-exclusion chromatography to verify MSH3-MSH2 complex formation

When conducting these assays, it's essential to include wild-type protein as a positive control and known inactive mutants as negative controls to validate the experimental system.

What critical controls should be included when studying MSH3 function?

A comprehensive set of controls is essential for valid experimental interpretation:

• Protein controls:

  • Wild-type MSH3 as positive control

  • Catalytically inactive MSH3 mutant as negative control

  • MSH3 alone versus MSH2-MSH3 complex

  • Concentration gradients

• Substrate controls:

  • DNA with various sizes of insertion-deletion loops

  • Perfectly matched DNA as negative control

  • Base-base mismatches (preferential MSH2-MSH6 substrates)

• Reaction condition controls:

  • ATP versus non-hydrolyzable ATP analogs

  • Divalent cation dependency tests

  • Time course experiments

These controls ensure that experimental results accurately reflect MSH3's biological functions and not experimental artifacts.

How do defects in MSH3 contribute to antifungal resistance?

Defects in MSH3 likely contribute to antifungal resistance through several mechanisms:

• Elevated mutation rate: Compromised MutSβ function leads to accumulation of certain types of mutations, particularly in regions with repetitive sequences.

• Accelerated acquisition of resistance mutations: Studies have shown that MMR defects lead to "elevated propensity to breakthrough antifungal treatment" , with MSH3 defects contributing to this phenomenon by increasing the likelihood of acquiring resistance-conferring mutations.

• Genomic plasticity enhancement: The genomic instability resulting from MSH3 defects could facilitate chromosomal rearrangements, which are known to occur in C. glabrata and contribute to its adaptability .

Research indicates that "mutants of MMR and DSBR, but not BER or NER, lead to increased appearance of fluconazole-resistant colonies" , supporting the role of MSH3, as part of the MMR pathway, in resistance development.

What is the relationship between MSH3 and MSH2 in drug resistance?

The relationship between MSH3 and MSH2 in C. glabrata drug resistance is complex:

How do researchers determine if clinical resistance is related to MSH3 defects?

Researchers employ several approaches to link clinical resistance to MSH3 defects:

• Genomic sequencing: Direct sequencing of MSH3 genes from clinical isolates to identify potential mutations.

• Mutation rate assessment: Quantifying mutation rates in clinical isolates to identify hypermutator phenotypes characteristic of MMR defects.

• Complementation studies: Introducing wild-type MSH3 into clinical isolates to see if normal MMR function and drug susceptibility can be restored.

• Functional assays: Measuring the ability of extracts from clinical isolates to perform MMR on defined substrates.

• Multi-gene analysis: Examining the combined status of MSH3, MSH2, and other repair genes for a more comprehensive assessment, as "additional mechanisms of DNA repair... may influence the mutagenic potential of C. glabrata strains" .

How is MSH3 involved in C. glabrata's response to DNA-damaging agents?

Research on C. glabrata's response to DNA damage provides insights into MSH3's potential roles:

• DNA damage signaling: While not directly addressed in the search results for MSH3, the MMR system in other organisms is known to play a role in DNA damage signaling and checkpoint activation.

• Interaction with other repair pathways: Studies have shown that when C. glabrata is exposed to methyl methanesulfonate (MMS), "damaged DNA was repaired by the homologous recombination (HR) pathway" . This suggests potential crosstalk between MMR (including MSH3) and HR pathways.

• Potential regulation under stress: The search results indicate that exposure to DNA-damaging agents can trigger transcriptional responses, such as "MMS exposure triggered a significant downregulation of histone H4 transcript and protein levels" . Similar regulations might affect MSH3 expression under stress conditions.

• Strain-dependent variation: C. glabrata has "a complex population structure" , suggesting different strains might show distinct MSH3-dependent responses to DNA damage.

How could MSH3 be exploited as a target for novel antifungal strategies?

MSH3 offers several potential avenues for novel antifungal development:

• Restoring MMR function: Compounds that restore functional MMR activity in strains with defective MSH3 could potentially slow the acquisition of resistance.

• Synthetic lethality approaches: Identifying pathways that become essential when MSH3 is defective could reveal novel drug targets.

• Targeting MMR-deficient strains: Developing compounds that specifically target cells with MMR defects could selectively eliminate drug-resistant subpopulations.

• Combination therapies: Using MSH3-targeting compounds in combination with traditional antifungals might prevent the emergence of resistance during treatment.

These approaches represent more sophisticated strategies than traditional antifungals, potentially targeting C. glabrata's evolutionary capacity rather than simply its viability.

What role does MSH3 play in C. glabrata's genomic plasticity?

C. glabrata shows high levels of genomic plasticity, with chromosomal rearrangements being common . MSH3 likely influences this plasticity in several ways:

• Maintenance of repetitive sequences: MSH3, as part of MutSβ, helps maintain the stability of repetitive sequences, which are often hotspots for recombination.

• Prevention of inappropriate recombination: Functional MMR systems normally suppress recombination between divergent sequences, suggesting MSH3 defects might increase recombination rates.

• Interaction with DNA repair pathways: The search results indicate complex interactions between repair pathways, with one study finding that exposure to DNA-damaging agents affected the "CgRad52, a DNA strand exchange-promoter protein of the HR system" . This suggests MSH3 may interact with HR factors to influence genomic stability.

• Population-level genomic diversity: The search results show evidence of "recombination, inbreeding and clonal expansion within and between hospitals, including evidence for nosocomial transmission among coronavirus disease 2019 (COVID-19) patients" , highlighting how MMR defects might contribute to C. glabrata's genomic diversity at the population level.

How do MSH3 mutations correlate with treatment outcomes in C. glabrata infections?

While the search results don't provide direct data on MSH3 mutations and clinical outcomes, several inferences can be made:

• Potential for rapid resistance development: Clinical isolates with MSH3 defects would likely develop resistance more rapidly during treatment, potentially leading to treatment failure.

• Multi-drug resistance concerns: The search results indicate that MMR defects contribute to "multidrug resistance in Candida glabrata" , suggesting MSH3 mutations could correlate with poor outcomes across multiple drug classes.

• Fitness trade-offs: Research suggests "complete loss of MMR function (msh2Δ) impacts fitness of C. glabrata in the host" , indicating a complex relationship between MMR defects and virulence that could affect clinical outcomes.

• Population-specific patterns: The geographic differences in MMR gene mutations suggest potential population-specific correlations with treatment outcomes.

How can understanding MSH3 function improve diagnostic approaches for resistant C. glabrata?

Knowledge of MSH3 function could enhance diagnostic approaches through:

• Molecular diagnostics: Developing rapid tests to identify MSH3 mutations or dysfunction in clinical isolates could help predict resistance potential before conventional susceptibility testing.

• Mutator phenotype identification: Methods to identify hypermutator phenotypes associated with MMR defects could flag high-risk isolates.

• Comprehensive MMR profiling: Testing the status of multiple MMR genes (MSH2, MSH3, MSH6, etc.) could provide a more complete picture of resistance risk.

• Biomarker development: Identifying downstream consequences of MSH3 dysfunction that could serve as biomarkers for resistant infections.

• Strain typing enhancement: The search results indicate that "both MSH2 genotypes and chromosomal patterns cluster consistently into specific strain types" , suggesting MSH3 status could contribute to improved strain typing approaches.

What is the evidence for MSH3 mutations affecting virulence in C. glabrata?

The relationship between MSH3 mutations and virulence appears complex:

• Host adaptation: MSH3 defects may accelerate adaptation to the host environment, potentially affecting virulence.

• Fitness considerations: Research shows that "complete loss of MMR function (msh2Δ) impacts fitness of C. glabrata in the host" , suggesting a potential fitness cost to severe MMR defects.

• Strain-dependent effects: The search results indicate that C. glabrata has "a complex population structure where genomic variants arise, perhaps during the process of adaptation to environmental changes, and persist over time" , suggesting strain-specific interactions between MMR status and virulence.

• Host interaction factors: C. glabrata's "ability to replicate in macrophages and survive a high level of oxidative stress contributes to its virulence in the mammalian host" , and MMR defects might influence these interactions.

Additional research specifically examining the relationship between MSH3 status and virulence factors would help clarify these potential connections.