Recombinant Treponema denticola UPF0178 protein TDE_2151 (TDE_2151)

What is the basic structure and sequence of TDE_2151?

TDE_2151 is a 155-amino acid protein with a molecular weight of 17.4 kDa from Treponema denticola strain ATCC 35405 / CIP 103919 / DSM 14222. The complete amino acid sequence is: MKIWVDADSCPVRIRQITAKAGERLKLPVIFAANREIPVPKGASMVVTENTEQAADLYITENSVEGDLAITRDIPLAKLLVDKGLYVINDRGTIFTRDNINTYLSARNFMYELQANGLAPEKTNSFGKKEIQKFSNLLDSLLAKALKQRHLDSRF . The protein belongs to the UPF0178 family, which consists of proteins with unknown function that share sequence similarity . Preliminary structural analysis can be performed using computational tools such as SWISS-MODEL or Phyre2 for homology modeling, followed by verification through circular dichroism spectroscopy or X-ray crystallography.

How can I express recombinant TDE_2151 in laboratory settings?

Expression of recombinant TDE_2151 can be achieved using established protocols for T. denticola proteins. For heterologous expression, E. coli systems (BL21, Rosetta, or SHuffle strains) are recommended with optimization of temperature, IPTG concentration, and incubation time . For native-like expression, consider using the T. denticola shuttle plasmid system described by researchers working with other T. denticola proteins . When designing expression constructs, codon optimization may significantly improve protein yields, as demonstrated with other T. denticola proteins . Expression vectors should include appropriate fusion tags (His6, GST, or MBP) to facilitate purification while maintaining protein folding and solubility.

What are the recommended methods for purifying recombinant TDE_2151?

Purification of recombinant TDE_2151 can be achieved through a multi-step process beginning with affinity chromatography (Ni-NTA for His-tagged protein), followed by ion-exchange chromatography and size-exclusion chromatography for higher purity . Based on its predicted molecular weight of 17.4 kDa, a simulated SDS-PAGE profile can guide identification of the protein during purification steps . For removal of endotoxins, which is critical for functional studies involving host cells, Triton X-114 phase separation or polymyxin B columns are recommended. Protein purity should be assessed via SDS-PAGE analysis and mass spectrometry, while proper folding can be verified through circular dichroism spectroscopy.

How can I determine the cellular localization of TDE_2151 in T. denticola?

Determining the cellular localization of TDE_2151 requires a multi-faceted approach. Begin with computational prediction tools like PSORT and SignalP to analyze sequence features indicative of subcellular targeting . For experimental verification, generate anti-TDE_2151 antibodies and perform immunofluorescence microscopy on fixed T. denticola cells, comparing with known markers for different cellular compartments . Cell fractionation studies separating outer membrane, periplasmic, inner membrane, and cytoplasmic fractions followed by Western blot analysis can provide biochemical evidence of localization. For in vivo tracking, consider creating a TDE_2151-fluorescent protein fusion (such as with Bs2) using the genetic tools developed for T. denticola . Expression of the fluorescent fusion protein can be driven by native promoters (like P-msp) for physiologically relevant expression levels .

What approaches can be used to identify potential binding partners or substrates of TDE_2151?

Identifying binding partners of TDE_2151 requires multiple complementary approaches. Co-immunoprecipitation using anti-TDE_2151 antibodies followed by mass spectrometry can identify protein-protein interactions in T. denticola lysates . Pull-down assays with purified recombinant TDE_2151 can be used to identify interactions with host proteins, potentially revealing roles in host-pathogen interactions similar to other T. denticola proteins like FhbB . Yeast two-hybrid screening can identify binary interactions, while bacterial two-hybrid systems may be more suitable for membrane-proximal interactions. For quantitative binding studies, surface plasmon resonance or isothermal titration calorimetry can characterize the kinetics and thermodynamics of identified interactions. Crosslinking mass spectrometry can provide insight into transient interactions and structural arrangement of interaction interfaces.

How can I develop a knockout or knockdown system for TDE_2151 to study its function?

Creating genetic tools to manipulate TDE_2151 expression requires consideration of T. denticola's genetic characteristics. For gene knockout, homologous recombination-based approaches using the ermB cassette (encoding 23S rRNA methylase) as a selectable marker have been established for T. denticola . When designing the knockout construct, consider the potential polar effects on neighboring genes. For inducible knockdown, consider adapting CRISPR interference (CRISPRi) systems with a catalytically inactive Cas9 targeted to the TDE_2151 promoter region . When transforming T. denticola, pay careful attention to DNA methylation status and restriction-modification systems, as these significantly affect transformation efficiency . To confirm successful genetic manipulation, use qPCR for transcript analysis and Western blotting for protein expression, with complementation assays to verify phenotypes.

What computational methods can predict the potential function of TDE_2151?

Computational prediction of TDE_2151 function should employ a multi-tiered approach. Begin with sequence-based analyses including BLAST, HHpred, and InterProScan to identify conserved domains and distant homologs that might suggest function . Structure prediction using AlphaFold2 followed by structural comparison with functionally characterized proteins using DALI can reveal functional similarities not evident from sequence alone. Gene neighborhood analysis examining conservation of genomic context across different Treponema species can provide functional clues through guilt-by-association logic. Examination of gene expression patterns under different conditions using existing transcriptomic data from T. denticola can identify co-regulated genes that may have related functions . Protein-protein interaction networks can be computationally predicted using STRING database and similar tools to place TDE_2151 in a functional context.

How can I assess if TDE_2151 contributes to T. denticola's virulence?

Assessing TDE_2151's contribution to virulence requires a combination of genetic manipulation and functional assays. Create isogenic mutant strains through targeted gene disruption as described previously, and compare them with wild-type and complemented strains in a range of assays . Adhesion assays using human gingival epithelial cells or fibroblasts can determine if TDE_2151 affects bacterial attachment . Invasion assays using cell monolayers can assess the ability of mutant strains to penetrate host tissues . Cytotoxicity assays measuring LDH release from host cells can identify if TDE_2151 contributes to T. denticola's cytopathic effects . Biofilm formation capacity can be assessed using crystal violet staining and confocal microscopy. For immune evasion assessment, complement resistance assays similar to those used for FhbB characterization may be informative if TDE_2151 has similar functions .

What biochemical assays are appropriate for characterizing TDE_2151's enzymatic activity?

Although the function of UPF0178 family proteins remains unknown, several approaches can systematically investigate potential enzymatic activities. Begin with broad substrate screening using commercial enzyme activity kits that test for common activities (hydrolase, transferase, oxidoreductase) . Based on T. denticola's pathogenic lifestyle and the UPF0178 family characteristics, test for specific activities including proteolysis against various protein substrates, especially host extracellular matrix proteins relevant to periodontal disease . Phosphatase/kinase activity assays using various phosphorylated substrates can identify potential signaling roles. Nucleic acid binding or modifying activities can be tested through gel shift assays and nuclease activity tests. Lipid interaction studies using liposome binding assays may reveal membrane-associated functions. For all assays, include appropriate positive and negative controls, and verify activity using site-directed mutagenesis of predicted catalytic residues.

How do different promoter systems affect TDE_2151 expression in T. denticola?

Promoter selection significantly impacts recombinant protein expression in T. denticola. Based on comparative studies of promoter strength in T. denticola, the msp promoter (P-msp) demonstrates strong activity and could be used for high-level expression of TDE_2151 . The tap1 promoter shows moderate activity in T. denticola but is inactive in E. coli, making it useful for T. denticola-specific expression . Quantitative comparison of these promoters shows that P-msp drives approximately 4-fold higher expression than other promoters tested . For titratable expression, consider adapting inducible systems from related organisms. When designing expression constructs, the plasmid copy number must be considered—shuttle plasmids like pCF728 exist at approximately 24-27 copies per cell in T. denticola, which would result in significantly higher expression than chromosomal integration .

Table 1: Relative Promoter Strengths in T. denticola (Based on similar experiments with other T. denticola proteins)

What challenges might arise when expressing TDE_2151 and how can they be addressed?

Expression of recombinant TDE_2151 may face several challenges that require systematic troubleshooting. Protein solubility issues can be addressed by optimizing growth conditions (lower temperature, reduced inducer concentration) or using solubility-enhancing fusion partners like MBP or SUMO . Codon optimization for the expression host can significantly improve yields as shown for other T. denticola proteins . If protein toxicity is observed, consider using tightly regulated inducible expression systems or secretion-based approaches. For proper folding, co-expression with molecular chaperones may be beneficial. If disulfide bonds are critical (note the cysteine in the TDE_2151 sequence), consider expression in specialized E. coli strains like SHuffle or expression in the periplasm . Post-translational modifications present in native T. denticola might be absent in heterologous systems, potentially affecting protein function and requiring careful validation of recombinant protein activity.

How can I verify the structural integrity of purified recombinant TDE_2151?

Verification of structural integrity requires multiple analytical techniques. Circular dichroism spectroscopy can assess secondary structure content and proper folding of the purified protein . Thermal shift assays can determine protein stability and identify buffer conditions that maximize stability. Native PAGE or size-exclusion chromatography can assess oligomerization state and homogeneity. Intrinsic tryptophan fluorescence spectroscopy can provide information on tertiary structure and conformational changes upon ligand binding. For higher resolution structural analysis, consider X-ray crystallography or cryo-electron microscopy, though these require significant amounts of highly pure protein. Functional assays comparing recombinant protein with native protein (if accessible) provide the ultimate validation of structural integrity. For long-term storage, test different buffer compositions, pH values, and additives through accelerated stability studies to ensure the protein remains properly folded during storage.

How might TDE_2151 contribute to host-pathogen interactions in periodontal disease?

Although the specific role of TDE_2151 in host-pathogen interactions remains unknown, its investigation should be contextualized within T. denticola's virulence mechanisms. T. denticola produces numerous factors that interact with host proteins, including those involved in complement evasion (like FhbB), extracellular matrix degradation, and immune modulation . If TDE_2151 is surface-exposed, it may participate in adhesion to host tissues or immune evasion similar to other T. denticola proteins . Comparative analysis with virulence factors from other oral pathogens or related treponemes could provide functional insights. Experimental approaches should include testing TDE_2151's interaction with host components including fibronectin, as fibronectin binding is an important mechanism in T. denticola pathogenesis . Investigation of TDE_2151's potential role in inflammatory responses should include cytokine profiling in response to purified protein challenge of immune cells.

How can structural biology approaches advance our understanding of TDE_2151?

Structural characterization of TDE_2151 could provide critical insights into its function. Begin with computational structure prediction using AlphaFold2, followed by experimental validation through X-ray crystallography or NMR spectroscopy . Structure-guided mutagenesis of conserved residues can identify functionally important regions. Structural comparison with characterized proteins may reveal functional similarities not evident from sequence analysis alone. Protein-ligand interaction studies using techniques like hydrogen-deuterium exchange mass spectrometry can identify binding sites for potential substrates or interaction partners. Molecular dynamics simulations can provide insights into protein flexibility and conformational changes relevant to function. If TDE_2151 forms complexes with other proteins, structural studies of these complexes using techniques like cryo-electron microscopy could reveal mechanistic details of their functional interactions.

What research directions could elucidate the role of TDE_2151 in T. denticola biology and pathogenesis?

Future research on TDE_2151 should integrate multiple approaches to comprehensively understand its role. Transcriptomic and proteomic profiling comparing wild-type and TDE_2151 mutant strains under various conditions can identify affected pathways . Metabolomic analysis may reveal altered metabolic processes in mutant strains. In vivo models of periodontal disease using wild-type and mutant strains can assess the contribution of TDE_2151 to pathogenesis in a physiologically relevant context . Comparative genomics across clinical isolates could identify variants of TDE_2151 and correlate them with virulence phenotypes. Integration of TDE_2151 into the broader protein interaction network of T. denticola through systematic interaction studies would contextualize its function. Investigation of potential horizontal gene transfer and evolution of the UPF0178 family across different bacterial species could provide insights into its ancestral function and specialization in T. denticola.