Recombinant Treponema denticola Aspartate--tRNA ligase (aspS), partial

What is Treponema denticola and why is it significant in oral microbiome research?

Treponema denticola is an oral spirochete consistently found at significantly elevated levels in periodontal lesions and is recognized as a keystone pathogen in periodontitis . The strain ATCC 35405 (also designated as CIP 103919 or DSM 14222) is a whole-genome sequenced type strain originally isolated from a human periodontal pocket in Montreal . This bacterium produces several important proteins including methyl-accepting chemotaxis protein, major surface protein (Msp), and prolyl aminopeptidase, making it an important model organism for studying Treponema physiology and host-microbe interactions . T. denticola's contribution to periodontal disease progression through various virulence factors, particularly surface-expressed proteins, makes it a critical subject for molecular and genetic research.

What expression systems are typically used for recombinant T. denticola proteins?

• Deletion of signal peptide sequences and replacement with vector-encoded sequences

• Expression as inclusion bodies followed by purification and refolding

• Use of specialized E. coli strains designed for toxic protein expression

For in vivo studies, E. coli-T. denticola shuttle plasmids have been developed, though their efficiency varies between T. denticola strains .

What are the basic structural characteristics of T. denticola proteins that affect recombinant expression?

T. denticola proteins often contain prokaryotic signal sequences with potential cleavage sites for signal peptidase 1, as observed in the Msp protein . For aspS and similar proteins, attention to these structural elements is essential for successful expression. Many T. denticola surface proteins, like Msp, form oligomeric complexes that are critical for their function . The native structure of these proteins typically requires both N-terminal and C-terminal domains for proper oligomerization and surface expression . When designing expression constructs for aspS or other T. denticola proteins, researchers should consider:

• Presence of signal peptides (typically 20-30 amino acids at the N-terminus)

• Potential for oligomerization

• Post-translational modifications including potential N-glycosylation

• Functional domains that may be sensitive to truncation or modification

What are the key considerations when designing expression constructs for T. denticola proteins?

When designing expression constructs for T. denticola proteins including aspS, researchers should consider several critical factors:

• Signal peptide handling: The putative signal peptide sequence may need to be deleted and replaced by a vector-encoded peptide sequence to achieve high expression levels .

• Codon optimization: T. denticola has different codon usage patterns than E. coli, which may necessitate codon optimization for efficient expression.

• Expression toxicity: Full-length expression may be toxic to E. coli, requiring tightly regulated expression systems . For aspS specifically, toxicity assessment should be performed during initial expression trials.

• Terminal domains integrity: Both N and C termini may be critical for proper protein folding and function. Removal of as few as three C-terminal amino acids from Msp abrogated detection on the T. denticola cell surface, suggesting the importance of intact termini for proper expression and localization .

• Fusion tags: Strategic placement of purification or solubility tags is crucial, as tag location can impact protein folding and function.

How can toxicity issues be mitigated when expressing T. denticola proteins in E. coli?

Expression of T. denticola proteins, including potentially aspS, can be toxic to E. coli, as demonstrated with the Msp protein . Several strategies can mitigate this toxicity:

• Tightly regulated expression systems: Using the T7 RNA polymerase vector system with stringent control of basal expression is effective for minimizing toxicity before induction .

• Deletion of signal peptides: Replacing the native signal peptide with vector-encoded sequences can reduce toxicity while allowing high-level expression as inclusion bodies .

• Lower expression temperatures: Reducing growth temperature to 16-25°C during induction can decrease protein toxicity.

• Lower inducer concentrations: Titrating inducer concentration to find the optimal balance between expression yield and toxicity.

• Specialized host strains: Using E. coli strains designed for toxic protein expression, such as C41(DE3) or C43(DE3).

• Fusion to solubility-enhancing partners: Tags like MBP, SUMO, or Thioredoxin can reduce toxicity while improving solubility.

What purification strategies are most effective for T. denticola recombinant proteins?

For purification of recombinant T. denticola proteins like aspS, researchers should consider:

• Inclusion body purification: When proteins are expressed as inclusion bodies, denaturation and refolding protocols may be necessary. This approach was successful for Msp and could be applicable to aspS .

• Affinity chromatography: Tagging strategies (His, GST, MBP) facilitate purification to homogeneity, though tag placement must be carefully considered to maintain protein function.

• Size exclusion chromatography: Particularly important for oligomeric proteins to separate different assembly states.

• Ion exchange chromatography: Useful as an additional purification step based on the protein's isoelectric point.

• Native purification: For functional studies, maintaining native conformation throughout purification may be critical, especially for enzymatic proteins like aspS.

The purification strategy should align with the intended downstream applications, whether structural studies, enzymatic assays, or interaction analyses.

How do host restriction-modification systems affect transformation efficiency when working with T. denticola?

The restriction-modification (R-M) systems in T. denticola significantly impact transformation efficiency when introducing recombinant DNA. Recent improvements in genetic transformation methodology for T. denticola included:

• Modification of shuttle plasmids to remove recognition sites for Type II restriction enzyme TdeIII .

• Attention to appropriate methylation status of transforming plasmid DNA .

• Development of SyngenicDNA shuttle plasmids resistant to T. denticola R-M systems, which demonstrated greatly enhanced transformation efficiency .

These considerations would be crucial when attempting to express recombinant aspS in T. denticola. Research indicates that native plasmids are quite rare in T. denticola, suggesting that successful transformation requires careful consideration of R-M barriers .

What functional assays can verify activity of recombinant T. denticola proteins?

For functional validation of recombinant T. denticola proteins, including potential approaches for aspS, researchers can employ several strategies:

• Binding assays: For proteins like Msp, adherence to immobilized substrates (laminin, fibronectin) can confirm functionality . For aspS, aminoacylation assays would be appropriate to verify enzymatic activity.

• Competitive inhibition: Attachment of recombinant proteins may be decreased in the presence of soluble substrate, providing evidence of specific binding .

• Enhancement of bacterial attachment: Pretreatment of substrates with recombinant protein may increase bacterial attachment, as demonstrated with Msp . For aspS, complementation of aspS-deficient strains could demonstrate functionality.

• Enzymatic activity assays: For enzymes like aspS, specific assays measuring the rate of aminoacylation of tRNA^Asp would be essential.

• In vivo complementation: Introduction of recombinant protein or its encoding gene into mutant strains to rescue phenotypes provides strong evidence of functionality.

How can structural analysis enhance understanding of T. denticola protein function?

Advanced structural analysis of T. denticola proteins, including theoretical approaches for aspS, can provide critical insights:

• Domain mapping through mutagenesis: Fine-scale mutagenesis has revealed that both N and C termini, as well as centrally located epitopes, are required for native Msp oligomer expression . Similar approaches could identify critical domains in aspS.

• Structural modeling: Computational approaches can predict protein structure, as demonstrated with the proposal of Msp as a β-barrel protein similar to Gram-negative bacterial porins .

• Surface epitope identification: Identifying surface-exposed regions through protease treatment of intact cells can reveal functional domains, as seen with the 25 kDa polypeptide released from Msp after proteinase K treatment .

• Post-translational modification analysis: Evidence of N-glycosylation in Msp suggests the importance of assessing modifications in other T. denticola proteins, including potentially aspS .

• Oligomerization studies: Substitution experiments, such as replacing specific residues with FLAG tags, can determine regions essential for oligomer formation .

How do T. denticola proteins interact with host extracellular matrix components?

T. denticola proteins, particularly surface-exposed ones, frequently interact with host extracellular matrix (ECM) components. While specific information about aspS interactions is not available, patterns observed with other T. denticola proteins provide insight:

• Selective binding to ECM proteins: Recombinant Msp purified to homogeneity demonstrated selective adherence to immobilized laminin and fibronectin but not to bovine serum albumin .

• Competitive inhibition by soluble substrate: Attachment of recombinant protein may be decreased in the presence of soluble substrate, indicating specific binding mechanisms .

• Enhancement of bacterial attachment: Pretreatment of immobilized ECM components with recombinant protein increased T. denticola attachment, suggesting a bridging function .

• Proteolytic activity affecting ECM interactions: Dentilisin, a surface-expressed protease complex, has been shown to degrade endogenous ECM substrates and facilitate tissue penetration .

Understanding these interaction patterns helps in designing functional studies for newly characterized proteins like aspS, even if their primary function may not directly involve ECM binding.

What signaling pathways are activated by T. denticola proteins in host cells?

T. denticola proteins can trigger specific host signaling pathways, with important implications for periodontal disease progression:

• TLR2/MyD88 activation: T. denticola dentilisin, a surface-expressed protease complex comprised of three lipoproteins, activates TLR2-dependent mechanisms in host cells . This activation leads to upregulation of tissue-destructive genes in periodontal tissue .

• MMP expression regulation: Challenge with wild-type T. denticola (but not dentilisin-deficient strains) significantly upregulates matrix metalloproteinases (MMPs) 2, 11, 14, 17, and 28 in human periodontal ligament cells .

• Sp1 protein expression: T. denticola increases total Sp1 protein expression in periodontal ligament cells in a dentilisin-dependent manner .

• MyD88-dependent signaling: shRNA knockdown of MyD88 inhibits T. denticola-stimulated upregulation of MMPs, indicating the importance of this adaptor protein in signal propagation .

These findings highlight the complex interactions between T. denticola proteins and host cell signaling pathways, which should be considered when investigating the potential immunomodulatory effects of any T. denticola protein, including aspS.

What are the methodological challenges in studying T. denticola protein glycosylation?

Studying post-translational modifications like glycosylation in T. denticola proteins presents several methodological challenges:

• Detection of glycosylation: Evidence for N-glycosylation has been reported for Msp, suggesting this modification may be important for other T. denticola proteins as well .

• Characterization methods: Proteinase K treatment of intact cells has been used to release glycosylated fragments, such as the 25 kDa polypeptide containing the Msp surface epitope .

• Impact on recombinant expression: When expressing potentially glycosylated proteins like aspS in E. coli, researchers must consider that bacterial expression systems generally lack eukaryotic-like glycosylation machinery.

• Functional significance: Determining whether glycosylation affects protein function, stability, or immunogenicity requires careful comparative studies between glycosylated and non-glycosylated forms.

• Glycosylation site mapping: Mass spectrometry approaches combined with site-directed mutagenesis can help identify specific glycosylation sites and their importance for protein function.

What plasmid systems are most effective for T. denticola genetic studies?

Several plasmid systems have been developed for genetic studies in T. denticola, with varying efficiencies across strains:

For aspS studies, selecting the appropriate plasmid system based on the T. denticola strain is critical. Recent improvements in shuttle plasmids have facilitated routine transformation of the widely studied ATCC 35405 strain, which was previously challenging to transform .

How can codon optimization improve expression of T. denticola proteins?

Codon optimization can significantly improve heterologous expression of T. denticola proteins, including aspS:

• Codon bias analysis: T. denticola, like other spirochetes, may have codon usage preferences different from those of E. coli. Analyzing the codon adaptation index (CAI) of target genes can guide optimization.

• Rare codon replacement: Replacing rare codons with more abundant synonymous codons in the expression host can increase translation efficiency.

• GC content adjustment: Modifying the GC content while maintaining the amino acid sequence can improve mRNA stability and reduce secondary structure formation.

• Removal of regulatory elements: Eliminating potential internal Shine-Dalgarno sequences, RNA destabilizing elements, or cryptic splice sites can enhance expression.

• Expression validation: Comparing expression levels between native and codon-optimized sequences can verify the effectiveness of optimization strategies.

For aspS specifically, codon optimization could help overcome potential expression limitations in heterologous systems.

What are the considerations for signal peptide modification in T. denticola protein expression?

Signal peptide modification is a critical consideration for expression of T. denticola proteins:

• Identification of signal sequences: T. denticola proteins often contain prokaryotic signal sequences with potential cleavage sites for signal peptidase 1, as observed in Msp .

• Impact on expression: Full-length expression including the signal peptide can be toxic to E. coli, while deletion of the signal peptide and replacement with vector-encoded sequences can lead to high-level expression as inclusion bodies .

• Effect on localization: Signal peptides determine protein localization; their modification affects targeting to cellular compartments.

• Functional implications: For proteins like aspS, where enzymatic activity may be the primary function, signal peptide modification might have less impact on activity compared to surface-exposed proteins like Msp.

• Oligomerization effects: Signal peptide regions may contribute to protein-protein interactions. In Msp, substitution of a FLAG tag for residues 6-13 of mature Msp resulted in expression of full-length protein but absence of oligomers .