Recombinant Treponema denticola MutS2 protein (mutS2), partial

What is the structural organization of bacterial MutS2 proteins and how does it differ from MutS1?

MutS2 proteins represent a distinct branch of the MutS protein family with unique structural features. Unlike MutS1 proteins, which are primarily involved in DNA mismatch repair (MMR), MutS2 proteins have evolved specialized functions in regulating homologous recombination.

Based on detailed analyses of MutS2 proteins, they typically possess:

• A conserved middle domain with approximately 27% identity (41% similarity) to MutS1 proteins

• A conserved ATP binding motif (TGXNXXGK) essential for ATPase activity

• A unique C-terminal domain of approximately 100 amino acids known as the smr (small mutS-related) domain

• Notably, MutS2 proteins lack the N-terminal domain responsible for mismatch recognition in MutS1 proteins

The absence of the mismatch-binding domain suggests that MutS2 proteins do not participate in traditional mismatch repair pathways. Instead, their structure indicates specialized functions in other DNA metabolism processes, particularly in the regulation of homologous recombination.

What biochemical activities have been identified for bacterial MutS2 proteins?

Research on bacterial MutS2 proteins, particularly from H. pylori, has identified several key biochemical activities:

• ATPase activity: MutS2 proteins possess an intrinsic ATP hydrolyzing activity

• DNA substrate preferences: MutS2 ATPase activity is specifically stimulated by certain DNA structures, particularly:

  • Four-way junction (FWJ) DNA structures resembling Holliday junctions

  • D-loop structures representing recombination intermediates

• DNA binding activity: MutS2 proteins preferentially bind to recombination intermediate structures rather than to DNA mismatches

These biochemical properties align with the observed role of MutS2 in regulating homologous recombination rather than participating in mismatch repair pathways.

How does MutS2 function differ from MutS1 in bacterial DNA metabolism?

Experimental evidence clearly demonstrates that MutS2 serves different functions than MutS1 in bacterial DNA metabolism:

Studies in H. pylori demonstrate that mutS2 deletion strains show:

• No significant change in spontaneous mutation rates to rifampicin resistance

• 5-21 fold increases in homologous recombination frequencies depending on the genomic locus

• 25-fold increase in the incorporation of point mutations by recombination

These findings confirm that MutS2 functions primarily as a recombination regulator rather than as a component of mismatch repair.

What methods are recommended for expressing and purifying recombinant bacterial MutS2 proteins?

For effective expression and purification of recombinant MutS2 proteins, researchers have successfully employed the following methodology:

• Cloning strategy:

  • Clone the mutS2 gene in frame with a histidine tag (typically at C-terminus)

  • Use an expression vector suitable for bacterial expression

• Expression conditions:

  • Express in E. coli using standard induction protocols

  • Optimize temperature and induction conditions to enhance solubility

• Purification approach:

  • Affinity chromatography using metal chelate resins for His-tagged proteins

  • Further purification using ion exchange or size exclusion chromatography if needed

• Quality assessment:

  • SDS-PAGE to verify protein purity and expected molecular weight (~86.7 kDa for H. pylori MutS2)

  • Western blotting with anti-His antibodies to confirm identity

  • Functional assays (ATPase activity) to verify proper folding

This approach has yielded near-homogeneous MutS2 protein suitable for biochemical and functional studies.

What experimental evidence demonstrates MutS2's role in suppressing homologous recombination?

The role of MutS2 in suppressing homologous recombination is supported by multiple lines of experimental evidence:

These findings provide strong evidence that MutS2 functions as a general inhibitor of recombination processes, affecting both homologous (perfectly matched) and homeologous (partially divergent) recombination pathways.

How can isogenic mutants of mutS2 be generated in T. denticola?

Based on established methodologies for creating defined mutants in T. denticola, researchers can employ the following approach to generate mutS2 mutants:

• Construction of targeting vector:

  • Amplify DNA fragments flanking the mutS2 gene (typically 500-1000 bp each)

  • Clone these fragments into a plasmid vector flanking a selectable marker

  • Common selectable markers for T. denticola include:

    • ermB (erythromycin resistance)

    • aphA2 (kanamycin resistance)

• Transformation procedure:

  • Linearize the targeting construct

  • Introduce the DNA into T. denticola by electroporation

  • Plate on selective media containing the appropriate antibiotic

• Mutant verification:

  • PCR analysis to confirm correct integration

  • DNA sequencing to verify the mutation

  • Western blotting to confirm absence of the MutS2 protein

  • Functional assays to characterize the mutant phenotype

This approach has been successfully used to generate defined isogenic mutants in T. denticola for other genes, including components of the dentilisin protease complex .

What potential interactions might exist between MutS2 and virulence factors in T. denticola?

While direct evidence of interactions between MutS2 and T. denticola virulence factors is not currently available, research on other T. denticola proteins suggests potential relationships:

• Possible interaction with the dentilisin protease complex:

  • The dentilisin complex (PrtP, PrcA, PrcB) is a major virulence factor in T. denticola

  • Mutations in components of this complex can affect expression of other proteins

  • For example, disruption of prtP affects expression of Msp, a major surface protein

• Potential regulation by two-component systems:

  • The AtcSR two-component system regulates multiple virulence factors in T. denticola

  • Deletion of atcS results in significant transcriptome changes affecting:

    • Genes encoding motility proteins

    • The dentilisin protease complex

    • These changes correlate with reduced virulence in infection models

• Indirect effects through genomic stability:

  • If T. denticola MutS2 functions similarly to H. pylori MutS2, it may influence genomic stability

  • Changes in recombination rates could affect:

    • Acquisition of genetic material

    • Expression of virulence factors

    • Adaptation to the host environment

Research approaches to explore these potential interactions could include co-immunoprecipitation studies, transcriptomic and proteomic analyses of mutS2 mutants, and phenotypic characterization of mutants in virulence assays.

How might environmental factors in periodontal disease affect MutS2 expression and function?

The periodontal pocket represents a dynamic environment with multiple stressors that may influence MutS2 expression and function:

• Potential regulatory mechanisms:

  • Two-component systems like AtcSR respond to environmental signals and regulate gene expression

  • RNA sequencing of an atcS deletion mutant revealed significant changes in the T. denticola transcriptome

  • This suggests environmental sensing mechanisms influence multiple cellular processes

• Impact of host-derived factors:

  • T. denticola interacts with host cells, including gingival epithelial cells

  • These interactions involve virulence factors like the Msp protein

  • Environmental signals during these interactions might influence MutS2 expression or activity

• Methodological approaches to investigate environmental effects:

  • qRT-PCR to measure mutS2 expression under different environmental conditions

  • Recombination assays using reporter systems to measure MutS2 activity

  • Proteomics to identify post-translational modifications of MutS2 in response to stress

  • Chromatin immunoprecipitation to identify potential regulators of mutS2 expression

Understanding how environmental factors influence MutS2 could provide insights into T. denticola adaptation during periodontal disease progression.

What methodologies can assess the impact of MutS2 on T. denticola virulence?

To investigate the potential role of MutS2 in T. denticola virulence, researchers could employ several experimental approaches:

• In vitro cell interaction assays:

  • Compare wild-type and mutS2 mutant strains for:

    • Attachment to gingival epithelial cells

    • Invasion of host cells

    • Induction of cytokine responses

    • Similar approaches have been used to characterize atcS mutants

• Murine periodontitis model:

  • Oral infection with wild-type and mutS2 mutant strains

  • Assessment of alveolar bone loss using micro-CT imaging

  • Histological analysis of tissue destruction

  • Quantification of inflammatory markers

  • This approach demonstrated attenuated virulence of atcS-deficient T. denticola

• Polymicrobial biofilm models:

  • Co-culture with other periodontal pathogens

  • Analysis of biofilm formation and structure

  • Assessment of competitive fitness within biofilms

  • Evaluation of virulence factor expression

• Transcriptomic and proteomic analyses:

  • RNA sequencing to identify genes differentially expressed in mutS2 mutants

  • Proteomic analysis to characterize protein expression changes

  • Similar approaches with atcS mutants revealed effects on motility and dentilisin expression

These methodological approaches would provide comprehensive insights into the potential role of MutS2 in T. denticola virulence and pathogenicity.

What are the key knowledge gaps in understanding T. denticola MutS2?

Despite advances in understanding bacterial MutS2 proteins, several significant knowledge gaps remain regarding T. denticola MutS2:

• The precise DNA binding preferences and specificity of T. denticola MutS2

• The regulatory mechanisms controlling mutS2 expression during infection

• Potential interactions between MutS2 and established virulence factors such as dentilisin and Msp

• The impact of MutS2 on T. denticola genomic stability and adaptation

• Whether MutS2 regulates acquisition of genetic material from other oral bacteria

Addressing these knowledge gaps would enhance our understanding of T. denticola pathogenicity and potentially identify new targets for therapeutic intervention in periodontal disease.

What future research directions would advance understanding of T. denticola MutS2?

Future research on T. denticola MutS2 could productively focus on:

• Structural studies of the protein to define domain architecture and DNA binding interfaces

• Generation and phenotypic characterization of defined mutS2 mutants in T. denticola

• Investigation of potential regulatory links between MutS2 and two-component systems like AtcSR

• Comparative genomic analyses to identify genes affected by altered recombination rates in mutS2 mutants

• Development of recombination reporter systems to quantify MutS2 activity under various conditions