Recombinant Porphyromonas gingivalis Undecaprenyl pyrophosphate synthase (uppS)

What is Porphyromonas gingivalis and why is it significant in research?

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium commonly found in human subgingival plaque. It serves as a major etiologic agent for periodontitis and has been associated with multiple systemic pathologies beyond oral health. Periodontitis affects approximately 42.2% of American adults aged 30 years or older, with 7.8% experiencing severe forms of the disease. Untreated periodontitis leads to impaired oral function, eventual tooth loss, and reduced quality of life. Additionally, it has been linked to systemic disorders including cardiovascular disease, diabetes, Alzheimer's disease, respiratory tract infections, and adverse pregnancy outcomes.

The significance of P. gingivalis extends beyond periodontal disease; it is considered a keystone pathogen of the dysbiotic oral microbiome, capable of disrupting host-microbe homeostasis. This bacterium employs sophisticated virulence mechanisms including the type-IX secretion system (T9SS) to shuttle proteins across the outer membrane for pathogenesis.

What is Undecaprenyl pyrophosphate synthase (UppS) and what role does it play in bacterial physiology?

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme that catalyzes consecutive condensation reactions involving farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP). During these reactions, new cis-double bonds are formed, generating undecaprenyl pyrophosphate—a crucial lipid carrier for peptidoglycan synthesis in bacterial cell walls.

How do different P. gingivalis strains vary in their expression and characteristics?

P. gingivalis strains demonstrate significant diversity in virulence factors, which directly impacts their pathogenicity. Current research has identified numerous strains, each possessing different virulence profiles. Standard microbiome approaches using 16S or shotgun sequencing have traditionally been unable to differentiate between these strains.

The avirulent strain ATCC33277/381 has been identified as the most abundant strain across various sample types. In contrast, the W83/W50 strain shows significant enrichment in periodontitis cases, with approximately 13% of periodontitis patients harboring this strain. The W83 strain is commonly used as a wild-type reference strain in laboratory research.

Recent advances in strain identification techniques, particularly those targeting the intergenic spacer region (ISR) which varies between P. gingivalis strains, have improved our ability to distinguish between different strains. This approach employs a two-step PCR to amplify the P. gingivalis ISR region specifically, followed by Illumina sequencing and strain-specific mapping.

What are the optimal conditions for recombinant expression of P. gingivalis UppS?

When expressing recombinant P. gingivalis UppS, researchers should consider several factors that influence protein yield and activity. Based on comparable studies with other bacterial proteins, optimal expression typically involves:

• Expression System Selection: Escherichia coli expression systems are commonly employed for recombinant bacterial protein production. For UppS, BL21(DE3) or Rosetta strains may provide adequate expression levels while accommodating potential codon usage differences between E. coli and P. gingivalis.

• Vector Design: Incorporating an N-terminal hexahistidine tag facilitates purification while minimally affecting enzyme function. This approach has been successfully applied to related bacterial proteins, as demonstrated in research with PorU from P. gingivalis.

• Induction Parameters: IPTG concentrations between 0.1-0.5 mM and induction at lower temperatures (16-25°C) for extended periods (overnight) often improve the solubility of bacterial enzymes.

• Growth Media Supplementation: For proteins requiring metal cofactors like UppS, which depends on Mg²⁺, supplementation of expression media with appropriate concentrations of magnesium salts (approximately 1 mM) may enhance proper folding and activity.

When designing expression constructs, researchers should note that recombinant proteins from P. gingivalis may require careful optimization due to the organism's unique genetic characteristics and protein folding requirements.

What purification strategies are most effective for obtaining highly pure and active P. gingivalis UppS?

A multi-step purification protocol is recommended for obtaining highly pure and functionally active P. gingivalis UppS:

• Initial Capture: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins provides efficient initial purification. Buffer conditions should include:

  • 50 mM Tris-HCl or HEPES (pH 7.5-8.0)

  • 300-500 mM NaCl

  • 1 mM MgCl₂

  • 5-10% glycerol for stability

  • Gradient elution with imidazole (20-250 mM)

• Intermediate Purification: Ion exchange chromatography (IEX) can further separate UppS from contaminants with different charge characteristics. Based on the theoretical pI of UppS, either anion or cation exchange can be selected.

• Polishing Step: Size-exclusion chromatography (SEC) provides final purification and allows determination of the oligomeric state of the enzyme. This is particularly important given that related proteins like PorU from P. gingivalis transition between monomeric and dimeric states, which may affect activity.

• Quality Assessment: Purified enzyme should be assessed by:

  • SDS-PAGE for purity (>95%)

  • Western blotting for identity confirmation

  • Activity assays measuring condensation of FPP with IPP

  • Mass spectrometry for molecular weight confirmation

Throughout purification, it is critical to maintain appropriate Mg²⁺ concentrations (approximately 1 mM) in all buffers, as this cofactor is essential for UppS activity. Higher concentrations (>50 mM) should be avoided as they significantly reduce enzyme activity.

How can researchers confirm the proper folding and activity of recombinant P. gingivalis UppS?

Confirmation of proper folding and activity of recombinant P. gingivalis UppS requires a multi-faceted approach:

• Structural Analysis:

  • Circular Dichroism (CD) spectroscopy to assess secondary structure content

  • Thermal shift assays to evaluate protein stability

  • Dynamic Light Scattering (DLS) to confirm homogeneity and absence of aggregation

  • Native PAGE to examine oligomeric state, particularly important as enzyme activity may depend on proper oligomerization states

• Functional Assays:

  • Enzymatic activity measurement using either radiometric assays with ¹⁴C-labeled IPP or HPLC-based methods

  • Kinetic parameter determination (Km, Vmax) using varying concentrations of FPP and IPP substrates

  • Metal dependency tests, particularly examining the effect of Mg²⁺ concentration on activity

• Ligand Binding Studies:

  • Isothermal Titration Calorimetry (ITC) to measure binding affinities for substrates

  • Fluorescence-based assays to monitor substrate interaction

  • Differential Scanning Fluorimetry (DSF) in the presence and absence of substrates and cofactors

The following results would indicate properly folded and active UppS:

• CD spectrum consistent with the expected secondary structure composition

• Enzymatic activity showing dependence on Mg²⁺ with optimal activity at ~1 mM

• Substrate binding constants in the expected micromolar range

• Thermal stability enhancement in the presence of substrates or cofactors

What are the key structural features of P. gingivalis UppS and how do they compare to UppS from other bacterial species?

While specific structural data for P. gingivalis UppS is not extensively documented in the provided search results, key structural features can be inferred from UppS enzymes characterized in other bacterial species like E. coli:

The enzyme likely contains:

• An active site coordinating Mg²⁺, which is essential for catalytic activity

• Binding sites for both FPP and IPP substrates

• Conserved catalytic residues similar to those in E. coli UppS, including aspartate residues involved in Mg²⁺ coordination

In E. coli UppS, Mg²⁺ is coordinated by the pyrophosphate of farnesyl thiopyrophosphate (an FPP analogue), the carboxylate of Asp26, and three water molecules. Mutation of Asp26 to other charged amino acids significantly decreases UppS activity, indicating its critical role in enzyme function.

Other conserved residues in bacterial UppS enzymes include His43, Ser71, Asn74, and Arg77, which likely serve as general acid/base and pyrophosphate carriers. These residues are expected to be conserved in P. gingivalis UppS as well, though confirmation through sequence alignment and structural characterization would be necessary.

How does the catalytic mechanism of UppS function, and what experimental approaches can elucidate reaction intermediates?

The catalytic mechanism of UppS involves several key steps in the conversion of FPP and multiple IPP molecules to undecaprenyl pyrophosphate:

• Initiation: Mg²⁺-facilitated ionization of the pyrophosphate group from FPP

• Condensation: Nucleophilic attack by IPP on the resulting carbocation

• Chain Elongation: Iterative addition of IPP units

• Termination: After eight condensation reactions

The role of Asp26 (in E. coli UppS) appears to be assisting the migration of Mg²⁺ from IPP to FPP, initiating the condensation reaction through ionization of the pyrophosphate group from FPP.

To elucidate reaction intermediates, researchers can employ:

• Rapid Kinetics Approaches:

  • Stopped-flow spectroscopy with fluorescent substrate analogues

  • Quenched-flow techniques coupled with mass spectrometry

• Structural Biology Methods:

  • X-ray crystallography of enzyme-substrate complexes at various stages of the reaction using substrate analogues or transition state mimics

  • Cryo-electron microscopy (cryo-EM) for capturing dynamic states

• Spectroscopic Techniques:

  • NMR studies with isotopically labeled substrates

  • Infrared spectroscopy to monitor bond formation/breakage

• Computational Approaches:

  • Molecular dynamics simulations

  • Quantum mechanics/molecular mechanics (QM/MM) calculations

A systematic mutagenesis approach targeting key residues can provide additional insights into the roles of specific amino acids in the catalytic mechanism, similar to studies performed with E. coli UppS where mutations of Asp26 to other charged amino acids resulted in significant decreases in enzyme activity.

What molecular interactions govern substrate specificity in P. gingivalis UppS, and how can these be experimentally manipulated?

The substrate specificity of UppS is determined by molecular interactions between the enzyme and its substrates (FPP and IPP). While specific details for P. gingivalis UppS are not explicitly provided in the search results, insights can be drawn from studies on related bacterial UppS enzymes:

Key determinants of substrate specificity likely include:

• Binding Pocket Architecture: The size and shape of the substrate binding pocket determine accommodation of FPP and subsequent IPP molecules

• Key Residue Interactions: Specific amino acids that form hydrogen bonds, ionic interactions, or hydrophobic contacts with substrates

• Metal Coordination: The interaction of Mg²⁺ with substrate pyrophosphate groups and catalytic residues

• Conformational Changes: Protein dynamics that may regulate substrate access and product release

To experimentally manipulate these interactions, researchers can employ:

• Structure-Guided Mutagenesis:

  • Alter residues lining the substrate binding pocket

  • Modify residues involved in Mg²⁺ coordination

  • Target residues at the entrance to the active site to affect substrate access

• Substrate Analogue Studies:

  • Test modified FPP or IPP analogues with altered chain lengths or functional groups

  • Incorporate fluorescent or photoaffinity labels for binding studies

• Directed Evolution Approaches:

  • Develop high-throughput screening methods to identify UppS variants with altered substrate preferences

  • Apply error-prone PCR or DNA shuffling to generate diversity

• Computational Design:

  • Use molecular docking to predict interactions with modified substrates

  • Apply molecular dynamics to explore binding energy landscapes

Experimental validation of substrate specificity manipulations would require enzymatic assays comparing activity with natural and modified substrates, binding affinity measurements, and ideally, structural characterization of enzyme-substrate complexes.

How does UppS inhibition affect P. gingivalis cell wall biosynthesis and bacterial viability?

UppS inhibition would significantly impact P. gingivalis cell wall biosynthesis and viability through several mechanisms:

• Disruption of Peptidoglycan Synthesis: By inhibiting UppS, the production of undecaprenyl pyrophosphate would be impaired, limiting the availability of this essential lipid carrier required for peptidoglycan synthesis. This would directly compromise cell wall integrity.

• Impact on Bacterial Growth: With compromised cell wall synthesis, P. gingivalis would likely exhibit growth defects, morphological abnormalities, and increased susceptibility to osmotic stress.

• Downstream Effects on Virulence Factors: P. gingivalis employs the type-IX secretion system (T9SS) to shuttle virulence proteins across the outer membrane. Disruptions in cell wall integrity could potentially affect the assembly and function of this secretion system, thereby reducing bacterial virulence.

• Altered Strain Dynamics: Different P. gingivalis strains (such as the virulent W83/W50 versus the avirulent ATCC33277/381) might exhibit varying sensitivities to UppS inhibition based on potential differences in cell wall composition or UppS expression levels.

Experimental approaches to study these effects include:

• Growth inhibition assays with UppS inhibitors

• Microscopy to observe morphological changes

• Cell wall composition analysis following UppS inhibition

• Transcriptomic analysis to identify compensatory responses

• Virulence factor secretion assays to assess T9SS functionality

What is the relationship between P. gingivalis UppS and the type-IX secretion system (T9SS) in virulence expression?

While direct evidence linking P. gingivalis UppS and the T9SS is not explicitly detailed in the search results, a relationship can be inferred based on known bacterial physiology:

• Cell Wall Integrity and T9SS Function: The T9SS is a complex machinery that spans the cell envelope, including the cell wall. UppS, by providing the lipid carrier necessary for peptidoglycan synthesis, maintains cell wall integrity, which is likely prerequisite for proper T9SS assembly and function.

• Protein Translocation: The T9SS is responsible for shuttling virulence proteins, including those with C-terminal domains (CTDs) serving as secretion signals, across the outer membrane. These proteins undergo CTD cleavage and lipopolysaccharide attachment by PorU, a bifunctional signal peptidase and sortase.

• Impact on Virulence Factor Delivery: UppS inhibition could indirectly impair virulence factor delivery by compromising the cellular architecture required for T9SS operation. In P. gingivalis, this would affect the secretion of gingipains and other virulence factors that contribute to periodontitis.

To experimentally investigate this relationship, researchers could:

• Develop conditional UppS mutants to examine effects on T9SS component localization

• Assess T9SS cargo secretion under conditions of partial UppS inhibition

• Examine co-expression patterns of UppS and T9SS components during infection

• Investigate potential protein-protein interactions between cell wall synthesis machinery and T9SS components

How does strain variation in P. gingivalis affect UppS function and potential as a therapeutic target?

Strain variation in P. gingivalis could significantly impact UppS function and its potential as a therapeutic target:

• Strain-Specific UppS Variations: Different P. gingivalis strains may exhibit sequence variations in UppS that could affect enzyme kinetics, substrate binding, or susceptibility to inhibitors. The virulent W83/W50 strain versus the avirulent ATCC33277/381 strain may display different UppS characteristics.

• Expression Level Differences: Virulent strains might differentially regulate UppS expression, potentially as part of their pathogenic strategy. This could influence the efficacy of UppS-targeted interventions across different strains.

• Associated Virulence Factors: The research shows that different P. gingivalis strains possess distinct virulence factors. The W83/W50 strain, which is significantly enriched in periodontitis, may display a different relationship between UppS function and virulence factor expression compared to the more abundant but avirulent ATCC33277/381 strain.

• Therapeutic Target Validation: For UppS to be a viable therapeutic target, researchers must establish that inhibition would be effective against clinically relevant strains. The finding that 13% of periodontitis patients harbor the W83/W50 strain suggests this would be an important strain to validate for UppS-targeting strategies.

Methodological approaches to investigate strain variations include:

• Comparative genomics and protein sequence analysis of UppS across strains

• Recombinant expression and biochemical characterization of UppS from different strains

• Inhibitor screening against UppS from multiple strains

• In vitro and in vivo efficacy testing of UppS inhibitors against a panel of clinically relevant strains

A comprehensive strain typing approach, such as the ISR-based method described for P. gingivalis, would be valuable for correlating UppS characteristics with strain virulence profiles.

What are the common challenges in working with recombinant P. gingivalis proteins and how can they be addressed?

Researchers working with recombinant P. gingivalis proteins face several challenges that require specific strategies to overcome:

• Expression Difficulties:

  • Challenge: P. gingivalis proteins often express poorly in common hosts like E. coli due to codon usage differences.

  • Solution: Use specialized strains like Rosetta that supply rare tRNAs, or optimize codons in the synthetic gene construct.

• Protein Solubility Issues:

  • Challenge: Many recombinant proteins form inclusion bodies.

  • Solution: Lower induction temperature (16-18°C), reduce inducer concentration, co-express with chaperones, or use solubility tags like SUMO or MBP.

• Protein Stability Problems:

  • Challenge: Proteins may be unstable outside their native environment.

  • Solution: Include stabilizing additives (glycerol, specific ions), optimize buffer conditions, and maintain appropriate cofactor concentrations (e.g., 1 mM Mg²⁺ for UppS).

• Oligomerization State Variation:

  • Challenge: Proteins like PorU from P. gingivalis transition between monomeric and dimeric states, which may affect activity.

  • Solution: Carefully monitor oligomeric state by size exclusion chromatography or native PAGE, and determine which state correlates with optimal activity.

• Post-translational Modifications:

  • Challenge: P. gingivalis proteins may require specific modifications absent in recombinant systems.

  • Solution: Consider using eukaryotic expression systems or in vitro modification approaches when necessary.

• Functional Assay Development:

  • Challenge: Establishing reliable activity assays for enzymes like UppS.

  • Solution: Adapt established assays from related organisms, considering optimal cofactor concentrations (e.g., [Mg²⁺] = 1 mM for UppS activity).

How should researchers design mutation studies to investigate structure-function relationships in P. gingivalis UppS?

A systematic approach to mutation studies for investigating structure-function relationships in P. gingivalis UppS should include:

• Target Selection Strategy:

  • Conserved Residues: Identify residues conserved across bacterial UppS enzymes (similar to Asp26, His43, Ser71, Asn74, and Arg77 in E. coli UppS).

  • Structural Elements: Target residues in predicted binding pockets, catalytic sites, and oligomerization interfaces.

  • Unique Features: Focus on residues unique to P. gingivalis UppS that might confer strain-specific properties.

• Mutation Design Principles:

  • Conservative Substitutions: Replace residues with similar amino acids to probe subtle effects.

  • Charge Reversals: Convert acidic to basic residues (or vice versa) to drastically alter electrostatic properties.

  • Size Alterations: Substitute residues with significantly larger or smaller amino acids to investigate spatial requirements.

  • Alanine Scanning: Systematically replace residues with alanine to remove side chain functions while maintaining backbone structure.

• Validation Approaches:

  • Expression and Purification: Confirm that mutations don't disrupt protein folding or stability.

  • Structural Analysis: Use CD spectroscopy or thermal shift assays to verify structural integrity.

  • Activity Assays: Measure kinetic parameters (Km, kcat) for each mutant.

  • Metal Binding Studies: For residues involved in Mg²⁺ coordination, assess metal binding through ITC or activity at varying metal concentrations.

• Advanced Characterization:

  • Crystallography: Obtain structures of key mutants to visualize structural changes.

  • Substrate Analog Studies: Test mutants with modified substrates to probe binding pocket alterations.

  • Molecular Dynamics: Compare simulations of wild-type and mutant enzymes to understand dynamic effects.

Similar to studies with E. coli UppS, researchers should pay particular attention to residues involved in metal coordination, as the activity of UppS is highly dependent on proper Mg²⁺ binding and concentration.

What biosafety considerations should researchers be aware of when working with recombinant P. gingivalis components?

Researchers working with recombinant P. gingivalis components must adhere to specific biosafety guidelines:

• Regulatory Compliance:

  • Principal Investigators conducting research with recombinant DNA must register their research with the Institutional Biosafety and Chemical Safety Committee (IBCC) and ensure compliance with NIH Guidelines for rDNA research.

  • Any significant violation of NIH Guidelines must be reported to the Biosafety Officer.

• Risk Assessment Factors:

  • P. gingivalis is associated with periodontitis and potentially systemic diseases including cardiovascular disease, diabetes, and Alzheimer's disease.

  • While working with recombinant components rather than viable organisms reduces risk, the potential biological activity of certain components (especially virulence factors) requires careful consideration.

• Laboratory Containment Measures:

  • Work with recombinant P. gingivalis components typically requires Biosafety Level 2 (BSL-2) practices and facilities.

  • Use of certified biosafety cabinets for procedures that may generate aerosols.

  • Proper personal protective equipment including lab coats, gloves, and eye protection.

• Waste Management:

  • Decontamination of all waste containing recombinant materials before disposal.

  • Use of appropriate disinfectants effective against biological materials.

  • Proper labeling and disposal of sharps and contaminated materials.

• Emergency Response Procedures:

  • Development of clear protocols for spills or exposures.

  • Immediate reporting of incidents to laboratory supervisors and biosafety officers.

  • Medical follow-up for any potential exposures.

• Personnel Training:

  • Comprehensive training on biosafety procedures specific to recombinant P. gingivalis work.

  • Documentation of training and regular refresher courses.

  • Restriction of laboratory access to trained personnel only.

What are promising approaches for developing selective inhibitors of P. gingivalis UppS?

Several promising approaches exist for developing selective inhibitors of P. gingivalis UppS:

• Structure-Based Drug Design:

  • Based on crystal structures of bacterial UppS (such as those from E. coli), researchers can develop homology models of P. gingivalis UppS to identify unique structural features that might be exploited for selective inhibition.

  • Virtual screening against these models can identify compounds predicted to bind selectively to P. gingivalis UppS over human counterparts or UppS from commensal bacteria.

• Substrate Analogue Development:

  • Design of non-hydrolyzable analogues of FPP or IPP that competitively inhibit UppS activity.

  • Incorporation of features that exploit differences in substrate binding pockets between P. gingivalis UppS and related enzymes.

• Allosteric Inhibitor Screening:

  • Identification of compounds that bind outside the active site but alter enzyme conformation or dynamics.

  • These might target regions involved in oligomerization or unique regulatory elements.

• Metal Chelation Strategies:

  • Development of compounds that interfere with Mg²⁺ coordination specifically in the context of P. gingivalis UppS.

  • Design of inhibitors that exploit the enzyme's sensitivity to metal ion concentrations.

• Strain-Specific Targeting:

  • Investigation of potential differences in UppS between virulent strains (like W83/W50) and avirulent strains to develop inhibitors particularly effective against pathogenic variants.

• Combination Approaches:

  • Development of dual-targeting inhibitors that simultaneously affect UppS and other essential enzymes in P. gingivalis cell wall biosynthesis.

  • Design of prodrugs activated specifically in the P. gingivalis cellular environment.

These approaches would require rigorous validation through enzymatic assays, binding studies, crystallography, and ultimately testing in bacterial culture and animal models of periodontitis.

How might research on P. gingivalis UppS contribute to understanding the link between periodontitis and systemic diseases?

Research on P. gingivalis UppS could significantly contribute to understanding the connection between periodontitis and systemic diseases through several avenues:

• Cell Wall Integrity and Bacterial Translocation:

  • UppS inhibition affects cell wall biosynthesis, potentially altering bacterial cell surface properties and the ability to translocate from periodontal pockets to systemic circulation.

  • This could help elucidate mechanisms by which P. gingivalis contributes to systemic diseases like cardiovascular disease, diabetes, and Alzheimer's disease.

• Virulence Factor Expression and Secretion:

  • By understanding how UppS activity influences cell wall structure and consequently T9SS function, researchers can better comprehend how P. gingivalis expresses and secretes virulence factors that contribute to both local periodontitis and systemic pathologies.

• Strain-Specific Pathogenicity:

  • Investigation of potential variations in UppS across different P. gingivalis strains may reveal correlations between specific strains and their propensity to contribute to particular systemic diseases.

  • This is particularly relevant given that the virulent W83/W50 strain is significantly enriched in periodontitis patients.

• Biomarker Development:

  • Identification of specific byproducts or immune responses related to P. gingivalis UppS activity could lead to biomarkers that predict risk of systemic complications in periodontitis patients.

• Therapeutic Intervention Studies:

  • UppS-targeted interventions could serve as tools to investigate whether selective inhibition of P. gingivalis in the oral cavity reduces risk or severity of associated systemic diseases.

  • This would provide causative evidence for the role of this specific bacterium in systemic pathologies.

• Immune Response Characterization:

  • Understanding how P. gingivalis cell wall components, dependent on UppS activity, interact with host immunity could reveal mechanisms for systemic inflammation that links periodontitis to conditions like cardiovascular disease.

What emerging technologies might advance our understanding of P. gingivalis UppS in bacterial communities and host interactions?

Several emerging technologies hold promise for advancing our understanding of P. gingivalis UppS in bacterial communities and host interactions:

• Single-Cell Technologies:

  • Single-cell RNA sequencing to examine heterogeneous expression of UppS within P. gingivalis populations during colonization and infection.

  • Single-cell proteomics to correlate UppS expression with virulence factor production at the individual cell level.

• Advanced Imaging Techniques:

  • Super-resolution microscopy to visualize UppS localization within bacterial cells in multispecies biofilms.

  • Live-cell imaging with fluorescent UppS activity probes to monitor enzyme function during host cell interactions.

  • Correlative light and electron microscopy to examine structural changes in cell walls following UppS manipulation.

• Microfluidic Systems:

  • Organ-on-a-chip technologies modeling the periodontal pocket to study P. gingivalis behavior under controlled conditions.

  • Gradient-generating devices to examine bacterial responses to UppS inhibitors in complex communities.

• CRISPR-Based Technologies:

  • CRISPR interference systems for conditional knockdown of UppS to study dynamic effects on bacterial physiology.

  • CRISPR-based precise genome editing to introduce specific UppS variants into P. gingivalis strains.

• Strain-Specific Analysis Tools:

  • Further development of strain identification methods, like the intergenic spacer region (ISR) sequencing approach, to correlate specific P. gingivalis strains with UppS characteristics and disease outcomes.

• Systems Biology Approaches:

  • Multi-omics integration combining transcriptomics, proteomics, and metabolomics to understand UppS within the broader context of bacterial physiology.

  • Network analysis to identify interactions between UppS-dependent pathways and host response networks.

• In Situ Techniques:

  • Proximity labeling methods to identify proteins interacting with UppS in native conditions.

  • FISH-based approaches to simultaneously detect strain types and gene expression in clinical samples.

These technologies would enable researchers to move beyond static, in vitro studies of UppS to understand its dynamic role in the complex environment of the periodontal pocket and its potential implications for both oral and systemic health.