Recombinant Parabacteroides distasonis ATP-dependent zinc metalloprotease FtsH (ftsH)

What is the molecular architecture of P. distasonis FtsH and how does it compare to other bacterial FtsH proteins?

P. distasonis FtsH, like other bacterial FtsH proteins, is an ATP-dependent integral membrane metalloprotease. The molecular architecture consists of two distinctive rings: the protease domains form a flat hexagonal ring with an all-helical fold, while the AAA domains create a toroid structure that covers the protease ring . This hexameric structure is critical for its function, though interestingly, there can be a symmetry mismatch between the AAA and protease moieties during the catalytic cycle. This is similar to the symmetry breakdown observed in other enzyme complexes like the T7 gene 4 ring helicase, where symmetry reduction has been interpreted as facilitating sequential nucleotide hydrolysis .

When comparing P. distasonis FtsH to its E. coli counterpart, the structural foundations remain conserved, particularly in the catalytic domains. Of note, contrary to previous assumptions, the active site of FtsH classifies it as an Asp-zincin, with the third zinc ligand being a conserved aspartic acid residue (Asp-500 in the model system) rather than the previously reported glutamic acid .

How does the catalytic mechanism of FtsH differ from other ATP-dependent proteases?

The catalytic mechanism of FtsH involves a distinct interplay between its ATPase activity and proteolytic function. Unlike other ATP-dependent proteases such as ClpXP, FtsH possesses relatively weak unfoldase activity and cannot efficiently degrade stable proteins . The energy from ATP hydrolysis is used primarily for extracting target proteins from the membrane and for substrate translocation into the proteolytic chamber.

FtsH's active site contains the characteristic HEXXH motif of zinc-dependent metalloproteases, where the two histidines coordinate with the zinc ion and the glutamate serves as a catalytic base . The critical third zinc ligand is an aspartic acid residue (Asp-500), whose mutation to alanine completely abolishes proteolytic activity, as confirmed by crystal structure analysis showing the loss of the zinc ion . The glutamic acid residue (Glu-486) previously thought to be the third ligand actually forms hydrogen bonds that stabilize the histidine side chains in the proper conformation for zinc coordination, explaining why its mutation still retains approximately 10% residual proteolytic activity .

What expression systems are most effective for producing functional recombinant P. distasonis FtsH?

• Membrane protein expression challenges: As FtsH is an integral membrane protein, expression systems that can properly insert the protein into membranes are crucial. Common approaches include:

  • Using C41/C43(DE3) E. coli strains specifically designed for membrane protein expression

  • Employing vectors with regulatable promoters (like pBAD) to control expression rate

  • Including fusion tags that enhance membrane targeting and solubility

• Truncated constructs: Consider expressing soluble FtsH constructs lacking the transmembrane domains but retaining ATPase and protease functionality. This approach was successful with other FtsH proteins, resulting in constructs that maintained caseinolytic and ATPase activities in biochemical assays .

• Codon optimization: Adjust codon usage to match E. coli preferences while accounting for rare codon clusters that might be important for proper folding.

When working with full-length FtsH, detergent screening is essential for solubilization and maintaining enzymatic activity throughout purification. Mild detergents like DDM (n-dodecyl β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) are typically more effective than harsh detergents like SDS or Triton X-100.

What are the optimal conditions for assessing the enzymatic activity of recombinant P. distasonis FtsH?

When assessing enzymatic activity of recombinant P. distasonis FtsH, researchers should consider the following optimal conditions:

Buffer composition:

• pH range: 7.5-8.0 (neutral to slightly basic)

• Salt concentration: 150-300 mM NaCl or KCl

• Divalent cations: 5-10 mM MgCl₂ (required for ATPase activity)

• Zinc ion: 10-50 μM ZnCl₂ (essential for proteolytic activity)

Activity assays:

• ATPase activity: Measure ATP hydrolysis using colorimetric phosphate detection (malachite green assay) or coupled enzyme assays (pyruvate kinase/lactate dehydrogenase system).

• Proteolytic activity: Casein is a standard substrate for initial activity screening . Use fluorogenic peptide substrates or FRET-based assays for more quantitative measurements.

• Combined assays: Assess ATP-dependent proteolysis by measuring substrate degradation in the presence and absence of ATP and non-hydrolyzable ATP analogs.

For mutational analysis, the D500A mutation that abolishes zinc binding should be included as a negative control for proteolytic activity .

How can researchers effectively analyze the oligomeric state of recombinant P. distasonis FtsH?

Analyzing the oligomeric state of recombinant P. distasonis FtsH requires specialized techniques due to its membrane protein nature and hexameric assembly. The following methods are recommended:

• Blue Native-PAGE (BN-PAGE): This technique preserves protein complexes during electrophoresis and can be coupled with second-dimension denaturing SDS-PAGE to assess complex composition.

• Two-dimensional clear-native/Phos-tag SDS-PAGE: This approach allows simultaneous analysis of oligomerization state and phosphorylation status, revealing how phosphorylation might affect complex formation .

• Size Exclusion Chromatography (SEC): When combined with Multi-Angle Light Scattering (SEC-MALS), this provides accurate molecular weight determination of membrane protein complexes in detergent micelles.

• Analytical Ultracentrifugation (AUC): Particularly sedimentation velocity experiments can distinguish between different oligomeric states in solution.

• Electron Microscopy (EM): Negative stain EM or cryo-EM can directly visualize the hexameric ring structure and potential symmetry mismatches between AAA and protease rings .

For detergent-solubilized complexes, researchers should assess stability across different detergent conditions. The hexameric assembly may be sensitive to detergent choice, protein concentration, and buffer composition. Additionally, crosslinking experiments using agents like glutaraldehyde or BS3 can capture transient interactions and help stabilize complexes prior to analysis.

What are the known substrates of P. distasonis FtsH and how does it recognize them?

The specific substrates of P. distasonis FtsH have not been comprehensively characterized, but based on knowledge of bacterial FtsH proteins, they likely include:

• Membrane protein quality control targets: Unfolded or damaged membrane proteins that compromise membrane integrity .

• Regulatory proteins: In E. coli, FtsH degrades regulatory soluble proteins such as σ32 (heat shock transcription factor) and λ-CII transcriptional activator . P. distasonis FtsH may target similar regulatory factors.

• Orphan membrane protein subunits: Like the uncomplexed SecY subunit of translocase or the a-subunit of F₀F₁-ATPase in E. coli .

Substrate recognition by FtsH likely involves:

• Exposed degradation tags (degrons)

• Hydrophobic patches in partially unfolded proteins

• Recognition of specific sequence motifs by adaptor proteins

Unlike more powerful ATP-dependent proteases like ClpXP, FtsH possesses only weak unfoldase activity and primarily targets proteins that are already partially unfolded or intrinsically unstable .

What experimental approaches can be used to identify novel substrates of P. distasonis FtsH?

Several cutting-edge experimental approaches can be employed to identify novel substrates of P. distasonis FtsH:

• Quantitative proteomics with stable isotope labeling:

  • Compare protein abundance in wild-type versus FtsH-depleted or inactive mutant strains

  • Pulse-chase experiments with SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to measure protein degradation rates

• Proximity-dependent labeling:

  • Engineer BioID or APEX2 fusions to FtsH to biotinylate proximal proteins

  • Identify biotinylated proteins by streptavidin pull-down and mass spectrometry

• Trapped proteolysis intermediates:

  • Generate substrate-trapping FtsH variants (e.g., Walker B mutant that binds but does not hydrolyze ATP)

  • Identify trapped substrates by co-immunoprecipitation and mass spectrometry

• In vitro degradation assays:

  • Screen candidate substrates using purified recombinant FtsH

  • Monitor degradation by SDS-PAGE, western blotting, or fluorescence-based methods

• Global stability profiling:

  • Apply thermal proteome profiling (TPP) or limited proteolysis coupled with mass spectrometry

  • Compare protein stability landscapes between wild-type and FtsH-deficient strains

Validating candidate substrates requires demonstrating direct degradation and biological relevance through genetic complementation studies.

How does P. distasonis FtsH contribute to gut colonization and interaction with the host immune system?

P. distasonis FtsH likely plays several critical roles in gut colonization and host immune interactions, though direct evidence is still emerging:

• Membrane protein quality control: FtsH's primary function in maintaining membrane integrity is essential for bacterial survival in the competitive and fluctuating gut environment . This includes degradation of misfolded or damaged membrane proteins that could compromise bacterial viability.

• Stress response regulation: As observed in E. coli, FtsH regulates stress response factors (like σ32) , which may help P. distasonis adapt to host-derived stressors including antimicrobial peptides, bile acids, and immune effectors.

• Processing of surface proteins: FtsH may process surface-exposed proteins that mediate adhesion to intestinal surfaces. Research has shown that P. distasonis exhibits variable adhesion and biofilm formation capacities that are affected by host stress hormones . The electrokinetic features of its surface, which influence interactions with host tissues, may be partly regulated by FtsH activity.

• Immune modulation: P. distasonis contains proteins with sequence mimicry to host factors, particularly the hprt4-18 sequence that mimics insulin B:9-23 epitope . Whether FtsH regulates the expression or processing of such immunomodulatory factors remains to be determined, but this could represent an important aspect of how P. distasonis influences host immunity.

Studies in NOD mice have shown that P. distasonis colonization enhanced diabetes onset in female mice, and children harboring the hprt4-18 sequence had higher rates of seroconversion . The role of FtsH in regulating these immunomodulatory effects deserves further investigation.

What evidence suggests that P. distasonis FtsH activity is modulated by host factors such as stress hormones?

While direct evidence for stress hormone modulation of P. distasonis FtsH activity is limited, several indirect observations suggest this possibility:

• Surface property alterations: Host stress hormones significantly impact P. distasonis adhesion and biofilm formation capacities in approximately 35% and 23% of experimental assays, respectively . These surface behaviors are likely dependent on properly functioning membrane proteins that are subject to FtsH quality control.

• Inter-strain variability: The 14 unrelated strains of P. distasonis demonstrated significant variability in their response to stress hormones , suggesting genetic differences that may include variations in stress-responsive regulatory systems like FtsH-mediated proteolysis.

• Electrokinetic surface property changes: Stress molecules affect the colloidal stability and surface charge density of P. distasonis cells . These properties are influenced by the composition and organization of the bacterial cell envelope, which is maintained in part by FtsH activity.

Future research directions should include:

• Direct measurement of FtsH activity in P. distasonis exposed to stress hormones

• Analysis of FtsH phosphorylation status under stress conditions

• Identification of stress-induced substrates specific to P. distasonis FtsH

• Comparative transcriptomics and proteomics of stress hormone-exposed bacteria

Understanding these interactions could provide insights into how host psychological state influences gut microbiome composition and function, with potential implications for microbiome-related diseases.

What is the relationship between P. distasonis FtsH and autoimmune disease pathogenesis?

The relationship between P. distasonis FtsH and autoimmune disease pathogenesis represents an emerging area of research with several intriguing connections:

• Molecular mimicry: P. distasonis contains an insB:9-23 mimic in its hprt protein (residues 4-18) that activates insB:9-23 specific T-cells . This molecular mimicry mechanism may contribute to the development of type 1 diabetes (T1D). While FtsH itself may not contain mimics, it could regulate the expression or processing of such immunogenic proteins.

• Experimental evidence in animal models: Colonization of P. distasonis in female NOD mice enhanced diabetes onset and induced severe insulitis in both specific pathogen-free (SPF) and germ-free (GF) NOD mice . This occurred without causing significant alterations in the gut microbiome composition, gut permeability, serum metabolome, or broad cytokine responses, suggesting a specific immune mechanism.

• Clinical correlations: Children harboring the hprt4-18 sequence in their gut microbiome showed a higher rate of seroconversion in the DIABIMMUNE study , indicating potential clinical relevance of this mechanism.

The specific role of FtsH in this process is not fully elucidated, but as a key regulator of membrane protein quality control, it may influence:

• Surface presentation of immunogenic epitopes

• Processing of proteins involved in host-microbe interactions

• Bacterial adaptation to the inflammatory environment

A particularly noteworthy finding is that P. distasonis lysate can induce insB:9-23 specific T cells , suggesting that bacterial components released upon cell lysis (which might include FtsH or its substrates) have immunomodulatory properties.

How does P. distasonis FtsH compare structurally and functionally to FtsH from model organisms like E. coli?

P. distasonis FtsH shares fundamental structural and functional features with E. coli FtsH while likely possessing adaptations specific to its niche as a gut commensal:

Structural similarities:

• Both are ATP-dependent integral membrane metalloproteases with N-terminal transmembrane domains .

• Both contain the characteristic HEXXH motif of zinc-dependent metalloproteases .

• Both form hexameric complexes with AAA+ and protease domains arranged in ring structures .

Key differences and adaptations:

• Sequence divergence: While maintaining core functional domains, sequence variations in substrate-binding regions may reflect different target preferences.

• Membrane environment: P. distasonis, as a Bacteroidetes member, has a distinctive membrane composition compared to Proteobacteria like E. coli, potentially affecting how FtsH integrates into and functions within the membrane.

• Regulatory context: The different ecological niches (gut commensal vs. facultative anaerobe) suggest different regulatory networks controlling FtsH expression and activity.

From an evolutionary perspective, the conservation of FtsH across diverse bacterial lineages underscores its essential function. The E. coli FtsH is the only ATP-dependent protease that is both essential and universally conserved in bacteria . This high degree of conservation suggests that P. distasonis FtsH likely maintains core cellular housekeeping functions while potentially acquiring specialized roles related to host adaptation.

The human mitochondrial FtsH homolog paraplegin shares approximately 40% sequence identity with E. coli FtsH , suggesting that insights from bacterial FtsH research, including P. distasonis, may have relevance to understanding human mitochondrial disorders like hereditary spastic paraplegia.

What insights can phylogenetic analysis of FtsH across Bacteroidetes provide about its evolution and specialization?

Phylogenetic analysis of FtsH across Bacteroidetes can reveal important insights about its evolution and specialization:

• Core conservation vs. variable regions: By aligning FtsH sequences from diverse Bacteroidetes, researchers can identify:

  • Universally conserved catalytic residues (including the HEXXH motif and Asp-500 zinc ligand)

  • Variable regions that may confer species-specific substrate preferences

  • Lineage-specific insertions or deletions that could reflect ecological adaptations

• Selection pressure analysis: Calculating the ratio of non-synonymous to synonymous substitutions (dN/dS) across different regions of the FtsH gene can reveal:

  • Domains under purifying selection (likely essential for core function)

  • Regions under positive selection (potentially involved in host-specific adaptations)

  • Co-evolving residues that maintain structural or functional relationships

• Horizontal gene transfer assessment: Analyzing GC content, codon usage bias, and gene synteny can determine if:

  • FtsH has been horizontally transferred between Bacteroidetes and other phyla

  • Gene duplication events have occurred, potentially leading to functional specialization

• Correlation with ecological niches: Comparing FtsH sequences from gut Bacteroidetes (like P. distasonis) with those from other environments can identify:

  • Gut-specific adaptations that might facilitate colonization or host interaction

  • Environment-specific variations that correspond to different stress responses

This phylogenetic framework can guide experimental approaches, such as domain swapping or site-directed mutagenesis, to functionally validate the significance of evolutionary patterns identified in silico.

How might the function of P. distasonis FtsH differ in various host species or gut environments?

The function of P. distasonis FtsH likely varies across host species and gut environments due to several adaptation mechanisms:

• Host-specific selective pressures:

  • Different mammalian hosts present varying gut pH, transit times, bile acid compositions, and immune defenses

  • These differences may select for FtsH variants with optimized activity under specific host conditions

  • Research in NOD mice has shown that P. distasonis colonization enhances diabetes onset , but this effect may differ in other host genetic backgrounds

• Dietary and metabolic adaptations:

  • Host diet significantly shapes gut environment and nutrient availability

  • FtsH may regulate the expression or turnover of metabolic enzymes differently depending on available carbon sources

  • P. distasonis strains from hosts with different diets may show functional variations in FtsH substrate specificity

• Microbiome context:

  • The surrounding microbial community influences competitive dynamics and cross-feeding relationships

  • FtsH may play different roles in monoassociated germ-free animals versus complex microbiomes

  • Studies showed that P. distasonis colonization had minimal impact on gut microbiome composition, altering only 28 ASVs (Amplicon Sequence Variants)

• Stress response variations:

  • Different host species present varied stress hormone profiles and immune factors

  • P. distasonis shows significant inter-strain variability in response to stress hormones

  • FtsH may regulate different stress responses depending on the specific host environment

Experimental approaches to explore these differences could include:

• Comparative genomics of P. distasonis isolates from different host species

• Gnotobiotic animal models with defined genetic backgrounds

• In vitro simulations of different gut environments to assess FtsH activity and substrate profiles

• Metaproteomic analysis of P. distasonis in diverse host contexts

What are the most reliable methods for assessing the impact of P. distasonis FtsH mutations on bacterial physiology?

Assessing the impact of P. distasonis FtsH mutations requires a comprehensive approach combining molecular, cellular, and physiological techniques:

• Genetic manipulation strategies:

  • Site-directed mutagenesis targeting key residues:

    • Catalytic site mutations (e.g., D500A that abolishes zinc binding)

    • Walker A/B motif mutations in the AAA+ domain to disrupt ATPase activity

    • Transmembrane domain alterations to affect membrane integration

  • Complementation systems using plasmid-based expression

  • Conditional expression systems for essential gene studies

• Growth and stress response analyses:

  • Growth curve analysis under various conditions

  • Stress tolerance assays (oxidative, osmotic, temperature stress)

  • Membrane integrity assessment using fluorescent dyes

  • Antibiotic susceptibility testing

• Molecular and cellular assessment:

  • Proteome stability analysis using pulse-chase labeling

  • Membrane protein turnover rates

  • Electron microscopy to assess cell morphology changes

  • Protein complex formation using Blue Native PAGE

• Host interaction models:

  • Adhesion to intestinal epithelial cell lines

  • Colonization efficiency in gnotobiotic animal models

  • Immune activation assessment

  • Competitive colonization with wild-type strains

The S212 residue is highlighted because mutagenesis studies showed that this potential phosphorylation site may play a role in FtsH stability in thylakoid membranes, which could have analogous importance in bacterial membranes .

What techniques can researchers use to study the phosphorylation status of P. distasonis FtsH and its impact on function?

Studying the phosphorylation status of P. distasonis FtsH requires specialized techniques that can detect and characterize this post-translational modification:

• Detection of phosphorylation:

  • Phos-tag SDS-PAGE: This technique incorporates Phos-tag molecules into polyacrylamide gels to specifically retard the migration of phosphorylated proteins, allowing separation of phosphorylated and non-phosphorylated forms .

  • Western blotting with phospho-specific antibodies: Develop or use commercially available antibodies that recognize phosphorylated serine, threonine, or tyrosine residues.

  • Mass spectrometry: Phosphopeptide enrichment (using TiO₂ or IMAC) followed by LC-MS/MS analysis can identify specific phosphorylation sites and their stoichiometry.

• Site identification and characterization:

  • Bioinformatic prediction: Use phosphorylation site prediction tools (e.g., NetPhos, PhosphoSitePlus) to identify potential phosphorylation sites.

  • Site-directed mutagenesis: Generate non-phosphorylatable mutants (S/T→A) and phosphomimetic mutants (S/T→D/E) of predicted sites.

  • Two-dimensional clear-native/Phos-tag SDS-PAGE: This combined approach allows simultaneous analysis of oligomerization state and phosphorylation status .

• Functional analyses:

  • In vitro phosphorylation/dephosphorylation: Identify kinases and phosphatases that act on FtsH using purified components.

  • Activity assays with phosphorylated/dephosphorylated protein: Compare ATPase and proteolytic activities under different phosphorylation states.

  • Membrane association studies: Determine if phosphorylation affects membrane integration or complex stability.

• Physiological relevance:

  • Phosphoproteomics under different growth conditions: Analyze changes in FtsH phosphorylation in response to stress, nutrient limitation, or host factors.

  • In vivo functionality of phosphorylation site mutants: Test complementation with wild-type, non-phosphorylatable, and phosphomimetic FtsH variants.

From prior research on plant FtsH, neither different light conditions nor the lack of two major thylakoid kinases (STN7 and STN8) resulted in clear differences in FtsH phosphorylation . This suggests that FtsH phosphorylation may be independent of light-dependent regulation observed for other photosynthesis-related proteins. By analogy, P. distasonis FtsH phosphorylation might also be regulated by mechanisms distinct from common stress response pathways.

How can researchers effectively assess interactions between P. distasonis FtsH and host factors in experimental settings?

Investigating interactions between P. distasonis FtsH and host factors requires multi-disciplinary approaches spanning microbiology, immunology, and molecular biology:

• In vitro interaction studies:

  • Direct binding assays: Use purified recombinant FtsH and host factors (e.g., stress hormones, antimicrobial peptides) to assess direct interactions via techniques like surface plasmon resonance (SPR).

  • Activity modulation: Measure FtsH enzymatic activity in the presence of host-derived molecules to identify potential regulators.

  • Structural studies: Employ X-ray crystallography or cryo-EM to visualize potential binding of host factors to FtsH.

• Cell culture models:

  • Co-culture systems: Grow P. distasonis with intestinal epithelial cell lines (e.g., Caco-2, HT-29) and assess changes in FtsH expression, localization, or activity.

  • Transepithelial electrical resistance (TEER): Measure epithelial barrier function in response to wild-type versus FtsH-mutant P. distasonis.

  • Immunomodulation: Assess changes in cytokine production by immune cells exposed to P. distasonis with wild-type or mutant FtsH.

• Ex vivo approaches:

  • Intestinal organoids: Culture organ-like structures derived from intestinal stem cells to study host-microbe interactions in a physiologically relevant system.

  • Intestinal explants: Short-term culture of intestinal tissue to assess immediate responses to bacterial exposure.

• In vivo models:

  • Gnotobiotic animals: Colonize germ-free mice with wild-type or FtsH-mutant P. distasonis to assess colonization dynamics and host responses.

  • Disease models: Use models of intestinal inflammation or metabolic disorders to determine how FtsH impacts disease progression.

  • Specific phenotype assessment: In NOD mice, analyze how FtsH mutations affect the previously observed enhancement of diabetes onset by P. distasonis .

• Multi-omics integration:

  • Transcriptomics: RNA-seq of both bacterial and host cells to identify reciprocal gene expression changes.

  • Proteomics: Identify changes in protein expression and post-translational modifications in both organisms.

  • Metabolomics: Assess metabolite profiles to determine if FtsH influences metabolic cross-talk between bacteria and host.

For stress hormone studies specifically, researchers should note that P. distasonis shows significant inter-strain variability in its response to stress hormones . Therefore, multiple isolates should be tested, and strain-specific effects should be carefully documented.

What are the most promising therapeutic applications of research on P. distasonis FtsH?

Research on P. distasonis FtsH offers several promising therapeutic avenues:

• Microbiome modulation for autoimmune disease:

  • Understanding how P. distasonis FtsH influences type 1 diabetes progression could lead to targeted microbiome interventions.

  • The finding that P. distasonis colonization enhances diabetes onset in NOD mice suggests that inhibiting specific FtsH functions might mitigate autoimmune responses.

  • Engineered P. distasonis strains with modified FtsH activity could serve as next-generation probiotics with reduced immunogenicity.

• Novel antimicrobial strategies:

  • As an essential bacterial protein, FtsH represents a potential target for narrow-spectrum antimicrobials.

  • Structural differences between bacterial and human mitochondrial FtsH homologs could be exploited to develop selective inhibitors.

  • Targeting FtsH could be particularly effective against organisms resistant to conventional antibiotics.

• Stress-responsive therapeutic delivery:

  • P. distasonis responds to host stress hormones , and this response might involve FtsH-mediated protein quality control.

  • Engineered bacteria with modified FtsH systems could potentially release therapeutic compounds specifically in response to stress signals.

  • This approach could enable targeted intervention during stress-exacerbated conditions like inflammatory bowel disease.

• Biomarkers for personalized medicine:

  • FtsH variants or activity levels in gut Bacteroidetes could serve as biomarkers for disease risk or treatment response.

  • The association between P. distasonis hprt4-18 sequence and seroconversion suggests that bacterial features might predict autoimmune disease development.

Future therapeutic development will require deeper understanding of:

• Structure-function relationships in P. distasonis FtsH

• Specific substrates and regulatory networks in different host contexts

• Mechanisms linking FtsH activity to host immune responses

• Individual variation in responses to P. distasonis colonization

What are the most significant unresolved questions regarding P. distasonis FtsH function and regulation?

Several critical questions about P. distasonis FtsH remain unresolved:

• Substrate specificity determinants:

  • What specific features allow P. distasonis FtsH to recognize its substrates?

  • Are there adaptor proteins that modulate substrate recognition, similar to other proteolytic systems?

  • Do substrates differ between commensal and pathogenic contexts?

• Regulatory mechanisms:

  • How is FtsH expression and activity regulated in response to environmental changes in the gut?

  • What is the significance of FtsH phosphorylation in P. distasonis physiology?

  • Do host-derived molecules directly modulate FtsH activity?

• Role in microbiome dynamics:

  • How does FtsH activity influence P. distasonis competitive fitness in the complex gut ecosystem?

  • Does FtsH process secreted factors that affect neighboring microbes?

  • How does FtsH activity change across different stages of colonization?

• Host-microbe interaction mechanisms:

  • How does FtsH contribute to the molecular mimicry observed with the hprt4-18 sequence ?

  • Does FtsH process surface proteins involved in gut adhesion and biofilm formation ?

  • What is the mechanism by which P. distasonis enhances diabetes onset in NOD mice , and is FtsH involved?

• Structural biology questions:

  • What is the complete structure of membrane-integrated P. distasonis FtsH?

  • How does the symmetry mismatch between ATPase and protease rings contribute to function?

  • Are there structural adaptations specific to P. distasonis FtsH compared to other bacterial homologs?

• Technological challenges:

  • How can we develop improved genetic tools for manipulating P. distasonis?

  • What high-throughput approaches might identify all FtsH substrates in vivo?

  • How can we monitor FtsH activity in real-time within the native gut environment?

Addressing these questions will require interdisciplinary approaches combining structural biology, proteomics, microbial genetics, immunology, and advanced animal models.

What emerging technologies and methodologies might advance our understanding of P. distasonis FtsH?

Several cutting-edge technologies and methodologies promise to significantly advance our understanding of P. distasonis FtsH:

• Structural biology innovations:

  • Cryo-electron microscopy (cryo-EM): Allows visualization of FtsH in its native membrane environment without crystallization, potentially revealing dynamic conformational states.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Can map protein dynamics and conformational changes during substrate processing.

  • Integrative structural biology: Combining X-ray crystallography, NMR, cryo-EM, and computational modeling to build complete structural models.

• Genetic manipulation advances:

  • CRISPR-Cas9 systems optimized for Bacteroidetes: Would enable precise genome editing of P. distasonis.

  • Inducible gene expression systems: For controlled expression of wild-type or mutant FtsH in vivo.

  • Single-cell tracking methods: To monitor FtsH activity in individual bacteria within complex communities.

• Proteomics innovations:

  • Proximity labeling proteomics: BioID or APEX2 fusions to identify FtsH interaction partners and substrates in vivo.

  • Top-down proteomics: Analysis of intact proteins to characterize post-translational modifications.

  • Targeted proteomics with parallel reaction monitoring (PRM): For quantitative analysis of low-abundance FtsH substrates.

• Microbiome research technologies:

  • Gnotobiotic models with humanized microbiomes: To study P. distasonis FtsH function in physiologically relevant settings.

  • Organ-on-chip technology: Microfluidic devices that mimic human intestinal physiology for controlled host-microbe studies.

  • In situ microbial phenotyping: Methods to characterize microbial function directly in the gut environment.

• Single-cell technologies:

  • Single-cell RNA-seq of host-associated bacteria: To capture heterogeneity in FtsH expression within bacterial populations.

  • Live-cell imaging with activity-based probes: To visualize FtsH activity in real-time.

  • Single-cell proteomics: Emerging methods to analyze protein content in individual bacterial cells.

• Computational approaches:

  • Molecular dynamics simulations: To model FtsH substrate processing and membrane interactions.

  • Machine learning for substrate prediction: Algorithms trained on known substrates to predict new targets.

  • Systems biology modeling: Integration of multi-omics data to understand FtsH within broader regulatory networks.

These technologies will be particularly powerful when applied in combination, offering complementary insights into FtsH structure, function, and physiological roles in host-microbe interactions.