Recombinant Treponema denticola GTPase Era (era)

What is the role of GTPase Era in Treponema denticola virulence?

GTPase Era likely functions as a critical regulator of T. denticola cellular processes similar to other bacterial GTPases. While specific Era functions in T. denticola require further characterization, related GTPases in this organism contribute to virulence through cytoskeletal manipulation and immune evasion. For example, T. denticola can activate small GTPases including RAC1, RhoA, and Ras in fibroblasts, contributing to actin reorganization . When investigating Era's potential role, researchers should examine both bacterial survival mechanisms and host-pathogen interactions using knockout mutants alongside wild-type controls to identify phenotypic differences in biofilm formation, stress response, and interaction with host cells.

How does T. denticola GTPase Era compare structurally to Era homologs in other bacterial species?

To conduct structural comparison, researchers should first obtain purified recombinant Era protein through established protein expression systems. Following crystallization, X-ray crystallography would reveal the protein's three-dimensional structure. Computational analysis comparing T. denticola Era with homologs from other species should focus on key functional domains, particularly the G-domain containing GTP-binding motifs and the KH domain involved in RNA binding. While specific Era structural data for T. denticola is limited, research on other bacterial GTPases from this organism shows unique structural features compared to counterparts in other species. For instance, the Major Outer Sheath Protein (Msp) selectively inhibits Rac1 activation in murine neutrophils , suggesting T. denticola has evolved specialized GTPase-related mechanisms.

What expression systems are most effective for producing recombinant T. denticola GTPase Era?

For optimal expression of recombinant T. denticola GTPase Era, E. coli-based systems represent the primary choice given their established protocols for bacterial protein expression. Based on successful expression of other T. denticola proteins, researchers should consider BL21(DE3) or Rosetta strains to address potential codon bias issues. The gamma-glutamyltransferase (GGT) gene from T. denticola was successfully transformed into E. coli, resulting in expression of a functional recombinant protein . A similar approach with the Era gene using a pET vector system with an N-terminal His-tag would facilitate purification. Researchers should optimize induction conditions (IPTG concentration, temperature, duration) through small-scale expression trials before scaling up, with typical conditions being 0.1-0.5 mM IPTG at 16-25°C for 16-20 hours to minimize inclusion body formation.

What methodological approaches can resolve contradictory data regarding GTPase Era's role in T. denticola virulence?

To address contradictory findings regarding Era's role in virulence, implement a multi-faceted experimental approach combining genetics, biochemistry, and host-pathogen interaction studies. First, generate Era mutants using complementary approaches: (1) conditional knockdowns if Era is essential, and (2) point mutations of key functional residues based on structural predictions. Characterize these mutants through multiple assays: growth kinetics under various stress conditions, proteomics to identify altered protein pathways, transcriptomics to detect compensation mechanisms, and multiple virulence assays including neutrophil interaction and actin manipulation studies . The contradictory data often results from using different experimental systems; therefore, perform all experiments in standardized conditions across multiple T. denticola strains. Incorporate appropriate controls including complementation strains to confirm phenotypes are specifically attributed to Era modification.

How can researchers accurately assess the GTPase activity of recombinant T. denticola Era and identify potential regulatory partners?

To accurately assess GTPase activity of recombinant T. denticola Era, implement a comprehensive biochemical characterization workflow. Begin with a malachite green phosphate assay to quantify GTP hydrolysis rates under various conditions (temperature, pH, divalent cations). Complement this with radioactive [γ-32P]GTP hydrolysis assays for higher sensitivity to determine kinetic parameters (Km, Vmax, kcat). To identify regulatory partners, employ pull-down assays using purified His-tagged Era as bait with T. denticola lysates, followed by mass spectrometry identification of interacting proteins. Cross-validate interactions using surface plasmon resonance (SPR) or microscale thermophoresis (MST) to determine binding affinities.

Additionally, investigate whether Era interacts with components of pathways already identified in T. denticola, such as the RASA4 pathway described for actin reorganization . Consider potential RNA interactions, as bacterial Era proteins often function in ribosome biogenesis, through RNA immunoprecipitation followed by sequencing (RIP-seq).

What challenges exist in differentiating the specific effects of Era GTPase versus other small GTPases in T. denticola pathogenicity models?

Differentiating Era-specific effects from other GTPases presents significant methodological challenges. Address this through a hierarchical experimental design beginning with selective inhibition approaches. Deploy small molecule inhibitors with validated specificity for Era versus other GTPases (like Rac1 or RhoA), confirming selectivity through in vitro biochemical assays. Create an Era dominant-negative mutant by introducing mutations in the conserved G1 or G3 motif, expression of which would specifically interfere with Era function.

Design cellular assays that can discriminate between phenotypes associated with different GTPases based on existing knowledge. For instance, since T. denticola inhibits neutrophil polarization and migration through Rac1 pathway suppression , compare Era mutants with Rac1 manipulation to identify unique versus overlapping phenotypes. Implement CRISPR interference (CRISPRi) for temporal control of Era expression, enabling observation of immediate versus compensatory effects. Finally, develop dual-reporter systems where Era and other GTPase activities are simultaneously monitored in real-time through FRET-based biosensors to directly compare their activation dynamics during infection processes.

How do post-translational modifications affect Era GTPase function in T. denticola, and what methodologies best characterize these modifications?

Post-translational modifications (PTMs) likely play a crucial role in regulating Era GTPase function in T. denticola. To characterize these modifications, implement a multi-analytical approach. First, perform mass spectrometry analysis of purified native Era from T. denticola using multiple proteolytic digestions and enrichment strategies for phosphorylation, methylation, and acetylation. Compare PTM profiles under different growth conditions (e.g., biofilm versus planktonic, different pH levels) to identify condition-specific modifications.

Develop site-specific antibodies against predicted PTM sites to monitor modification status in vivo. For functional characterization, create point mutants at identified PTM sites (phosphomimetic and non-phosphorylatable) and assess their impact on GTPase activity, protein-protein interactions, and virulence phenotypes. Since calcium-dependent signaling affects T. denticola interactions with host cells and activates pathways like RASA4 , investigate whether calcium-dependent modifications regulate Era activity using phosphatase and kinase inhibitors followed by activity assays.

What is the optimal experimental design to assess Era GTPase's contribution to T. denticola's evasion of host immune responses?

To assess Era GTPase's contribution to immune evasion, design experiments focusing on T. denticola's known interactions with neutrophils. Create conditional Era knockdown strains and strains expressing Era mutants with altered GTPase activity. Using these variants, perform neutrophil chemotaxis assays similar to those that demonstrated Msp's inhibitory effect on neutrophil polarization and Rac1 activation . Assess neutrophil migration toward chemoattractants like fMLP in the presence of different T. denticola variants, measuring both directional movement and cellular polarization through time-lapse microscopy.

Quantify activation of neutrophil small GTPases (Rac1, Rac2, cdc42, RhoA) using PBD pulldown assays following exposure to different bacterial strains . Complement these with real-time visualization using fluorescent probes like PAK-PBD-YFP to track GTPase activation patterns in living neutrophils. Additionally, measure neutrophil effector functions including phagocytosis efficiency, reactive oxygen species production, and NET formation to comprehensively assess Era's impact on neutrophil antibacterial activity.

What strategies can overcome expression and purification challenges for recombinant T. denticola Era protein?

Overcoming expression and purification challenges for recombinant T. denticola Era requires a systematic optimization approach. First, address codon bias by either using Rosetta strains or synthesizing a codon-optimized Era gene. Test multiple expression constructs with different fusion tags (His, GST, MBP) at both N and C termini, as MBP can particularly enhance solubility. Optimize induction conditions through a factorial design varying temperature (16°C, 25°C, 37°C), IPTG concentration (0.1-1.0 mM), and duration (4-24h).

For purification, implement a staged approach starting with affinity chromatography corresponding to the fusion tag, followed by ion exchange and size exclusion chromatography. Add stabilizing agents to all buffers (5-10% glycerol, 1-5 mM DTT or TCEP) to prevent aggregation. If protein remains insoluble, attempt on-column refolding protocols or explore alternative solubilization strategies such as SMALP (styrene maleic acid lipid particles) if Era associates with membranes. The successful expression approach used for T. denticola gamma-glutamyltransferase, which involved transformation into E. coli followed by chromatographic purification , provides a viable template for Era purification strategy.

How can researchers effectively measure the impact of Era GTPase on T. denticola's interaction with extracellular matrix components?

To measure Era GTPase's impact on interactions with extracellular matrix (ECM) components, establish quantitative binding assays using purified ECM proteins, particularly fibronectin, which is known to interact with T. denticola . Compare binding of wild-type bacteria versus Era-depleted or Era-mutant strains to immobilized fibronectin, laminin, and collagen using crystal violet staining or radiolabeled bacteria for quantification.

Develop more sophisticated assays using fluorescently labeled bacteria to visualize ECM interactions in real-time through confocal microscopy. Complement this with ELISA-based binding assays and surface plasmon resonance to determine binding kinetics. To assess functional consequences, measure the degradation of ECM components by different bacterial strains through zymography or by quantifying breakdown products with mass spectrometry.

Additionally, investigate whether Era regulates expression of known adhesins or proteases involved in ECM interactions, using qRT-PCR and Western blotting to measure expression levels. Consider developing 3D tissue models incorporating relevant ECM components to assess bacterial invasion and tissue destruction patterns in a physiologically relevant context, comparing Era-mutant strains to wild-type bacteria.

How should researchers interpret contradictory results regarding Era GTPase activity in different experimental systems?

When confronting contradictory results regarding Era GTPase activity across different experimental systems, implement a structured analytical framework. First, systematically catalog all experimental variables including bacterial strain differences, growth conditions, protein preparation methods, and assay conditions. Create a standardized experimental matrix that tests Era activity across these variables to identify condition-dependent effects.

Recognize that in vitro biochemical data may not translate directly to cellular environments due to missing cofactors or regulatory partners. Therefore, complement in vitro studies with cellular assays, considering that Era may function differently in bacterial versus host cell environments. Employ statistical approaches like multivariate analysis to identify which experimental factors most significantly influence Era activity.

If contradictions persist between recombinant and native Era activity, investigate potential post-translational modifications or binding partners present in the native context but absent in recombinant systems. Finally, consider that Era may have multiple distinct functions depending on cellular location or activation state, similar to how T. denticola influences multiple host signaling pathways including RASA4 upregulation and selective Rac1 inhibition .

What bioinformatic approaches best predict Era GTPase binding partners and cellular pathways in T. denticola?

For predicting Era GTPase binding partners and cellular pathways, implement a multi-layered bioinformatic strategy. Begin with sequence-based approaches including conservation analysis across bacterial species to identify conserved interaction motifs. Apply protein-protein interaction prediction algorithms such as PIPE (Protein-Protein Interaction Prediction Engine) and SPRINT specifically trained on bacterial datasets.

Conduct structural bioinformatics by generating a homology model of T. denticola Era based on crystal structures of Era from other bacteria, then use molecular docking simulations to predict interactions with candidate partners. Complement this with protein domain analysis to identify functional motifs that might participate in specific interactions.

For pathway prediction, perform gene neighborhood analysis, as functionally related genes often cluster in bacterial genomes. Apply guilt-by-association methods using existing transcriptomic data to identify genes with expression patterns correlated with Era. Additionally, leverage known GTPase pathways in T. denticola, such as those involving Rac1 inhibition and RASA4 upregulation , to identify potential regulatory networks where Era might participate.

Validate top computational predictions through targeted experimental approaches such as bacterial two-hybrid screening or co-immunoprecipitation followed by mass spectrometry.

What are the promising therapeutic applications of targeting T. denticola Era GTPase in periodontal disease treatment?

Targeting T. denticola Era GTPase offers several therapeutic avenues for periodontal disease treatment. Based on the essential nature of Era in bacterial physiology, small molecule inhibitors specific to bacterial Era could potentially disrupt T. denticola growth and virulence with minimal host toxicity. The development pipeline should begin with high-throughput screening of compound libraries against purified recombinant Era, followed by secondary assays to confirm specificity and activity in bacterial cultures.

Therapeutic approaches could focus on disrupting Era's role in T. denticola's immune evasion strategies, similar to how Msp inhibits neutrophil polarization and chemotaxis . Peptide-based inhibitors designed to interfere with Era's interaction with specific binding partners might selectively disrupt virulence without affecting commensal bacteria. Additionally, since GTPases like RASA4 are involved in T. denticola's manipulation of host actin dynamics , Era inhibitors could potentially prevent tissue invasion and bacterial persistence.

Future therapeutic development should also consider combination approaches targeting multiple T. denticola virulence mechanisms simultaneously, such as both Era GTPase and the proteolytic enzyme dentilisin, to address the complex pathogenesis of periodontal disease.

How might research on T. denticola Era GTPase inform understanding of other oral pathogens and polymicrobial infections?

Research on T. denticola Era GTPase has broader implications for understanding oral pathogen biology and polymicrobial infections. Comparative genomic and functional analyses of Era across oral pathogens can reveal conserved and species-specific mechanisms of virulence regulation, potentially identifying common targets for broad-spectrum therapeutic approaches.

Era's potential role in regulating T. denticola interactions with other bacteria should be investigated through co-culture systems, particularly with known periodontal pathogens like Porphyromonas gingivalis. Since T. denticola can influence other bacteria through enzymes like GGT (which increases glutathione metabolism in P. gingivalis ), Era might similarly affect polymicrobial community dynamics.

Methodologically, establishing protocols for Era characterization in T. denticola could provide templates for studying this essential GTPase in more difficult-to-culture oral spirochetes. Additionally, understanding how Era regulates T. denticola's response to host defenses could reveal mechanisms by which polymicrobial communities collectively resist immune clearance in periodontal pockets.