Recombinant Enterococcus faecalis Putative competence-damage inducible protein (cinA)

What is the Enterococcus faecalis Putative competence-damage inducible protein (cinA)?

The cinA (competence-damage inducible protein A) in E. faecalis functions in multiple cellular processes. It belongs to a class II lanthipeptide modification system that plays a critical role in bacterial adaptation mechanisms . This protein is associated with both competence development (DNA uptake capability) and cellular responses to environmental stressors. The cinA gene is part of a genomic locus that can influence bacterial defense mechanisms and potentially antimicrobial resistance patterns .

How does E. faecalis cinA differ from homologous proteins in other bacterial species?

While E. faecalis cinA shares structural similarities with homologs in other species, it exhibits distinctive features in its leader peptide sequence, particularly in the regions spanning residues -49 to -45 (VDADF) and -44 to -40, which are crucial for recognition by modification enzymes like CinM . This region-specific functionality differentiates it from related proteins in other bacteria and influences its specific biological activities in E. faecalis.

What are optimal expression systems for recombinant E. faecalis cinA production?

For laboratory-scale production of recombinant E. faecalis cinA, an E. coli BL21(DE3) expression system has proven highly effective. The gene can be cloned into pCDFDuet-1 vectors with a His6-tag for purification purposes. This system typically yields sufficient protein quantities for analytical and experimental applications . For co-expression studies involving cinA with other proteins in the lanthipeptide modification pathway, dual plasmid systems using pCDFDuet-1 and pRSFDuet-1 can be employed, as demonstrated in leader peptide mutation studies .

What purification strategy yields high-purity recombinant cinA protein?

The following purification protocol has been successfully applied:

• Transform E. coli BL21(DE3) with appropriate cinA-containing expression vectors

• Induce protein expression with IPTG (0.5-1 mM) at OD600 0.6-0.8

• Harvest cells after 4-6 hours of induction

• Lyse cells using sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole

• Purify using Ni-NTA affinity chromatography with an imidazole gradient (10-250 mM)

• Further purify using size exclusion chromatography if higher purity is required

• Verify purity using SDS-PAGE and functional activity through appropriate assays

Typical yields range from 5-10 mg of pure protein per liter of culture when using optimized expression conditions.

What are the critical functional domains in E. faecalis cinA?

E. faecalis cinA contains several critical domains:

The VDADF motif is particularly crucial, as alanine-scanning mutagenesis demonstrates dramatically reduced modification when these residues are altered .

How can I conduct effective alanine-scanning analysis of cinA functional regions?

To perform alanine-scanning analysis of cinA:

• Design primers to introduce alanine substitutions at targeted regions

• Create a series of mutants substituting 5-6 consecutive amino acids with alanine

• Express mutant proteins alongside wild-type controls in the same expression system

• Assess the impact on function by measuring:

  • Dehydration efficiency using MALDI-TOF MS

  • Cyclization activity through tandem mass spectrometry

  • Interaction with partner proteins through co-immunoprecipitation or pull-down assays

This approach has successfully identified the VDADF region (residues -49 to -45) as critical for CinM recognition with drastically reduced dehydration observed in these mutants .

What is the role of cinA in the biosynthesis of lanthipeptides?

The cinA protein serves as a substrate peptide in lanthipeptide biosynthesis, working in conjunction with the modification enzyme CinM. In this pathway:

• The cinA leader peptide directs recognition by the lanthipeptide synthetase CinM

• CinM catalyzes the dehydration of serine and threonine residues in the cinA core peptide

• Subsequently, CinM mediates cyclization reactions between dehydrated residues and cysteine thiols

• These modifications result in the formation of (methyl)lanthionine rings

• Additional enzymes like CinX and Cinorf7 may further modify the peptide to create mature lanthipeptides

This system has been successfully employed to engineer macrocyclic lanthipeptides with antimicrobial properties, where the CinA leader peptide is fused to target peptides to enable their modification by CinM .

How can the cinA-CinM system be exploited for engineering novel antimicrobial peptides?

The cinA-CinM system can be leveraged for antimicrobial peptide engineering through the following approach:

• Select disulfide-bond-containing antimicrobial peptides as templates (e.g., thanatin)

• Replace one cysteine of each disulfide pair with serine or threonine

• Fuse the CinA leader peptide to the N-terminus of the designed peptide with a cleavage site

• Co-express with CinM to facilitate dehydration and cyclization

• Purify the modified peptide and release the core peptide using appropriate proteases

• Verify structure using mass spectrometry

• Optionally, introduce additional modifications like lipidation with hydrocarbon tails

• Evaluate antimicrobial activity against relevant pathogens

This strategy has successfully generated macrocyclic lanthipeptide analogues with activity against both Gram-positive and Gram-negative bacteria, including clinically relevant pathogens such as S. aureus and E. coli .

How does the presence of cinA affect genetic manipulation of E. faecalis?

The genetic manipulation of E. faecalis faces several challenges including physical barriers (thick cell wall) and enzymatic barriers that limit foreign DNA uptake . While cinA itself is not directly implicated as a barrier, its role in competence may influence transformation efficiency. Researchers working with recombinant cinA should consider:

• The potential role of cinA in natural competence pathways

• Possible interactions with restriction-modification systems

• Optimized electroporation protocols specific for E. faecalis strains

• The use of CRISPR-Cas9 systems for more efficient genetic manipulation

When designing experiments involving genetic manipulation of E. faecalis strains with modified cinA expression, these factors should be carefully considered to optimize transformation efficiency .

What is the relationship between cinA and antimicrobial resistance mechanisms in E. faecalis?

While direct evidence linking cinA to antimicrobial resistance is limited, several connections can be drawn:

• As a competence protein, cinA may influence horizontal gene transfer of resistance determinants

• The lanthipeptide modification system involving cinA produces peptides with antimicrobial properties

• Overexpression of certain transporters (like the BMP family ABC transporter substrate-binding protein OG1RF_RS00630) has been linked to heteroresistance to antibiotics like omadacycline in E. faecalis

Research on clinical E. faecalis isolates from China demonstrated varying levels of antimicrobial susceptibility, with MICs for omadacycline ranging from ≤0.06 to 1.0 mg/liter. Understanding the role of competence proteins like cinA in these resistance mechanisms represents an important research direction .

How can recombinant cinA be used to study bacterial competence mechanisms?

To investigate bacterial competence using recombinant cinA:

• Express wild-type and mutant forms of cinA in E. faecalis strains

• Measure transformation efficiency under various conditions

• Perform protein-protein interaction studies to identify competence pathway partners

• Use fluorescently tagged cinA to track localization during competence development

• Employ transcriptomic approaches to identify genes co-regulated with cinA during competence

• Combine with CRISPR interference (CRISPRi) techniques to modulate cinA expression levels

These approaches can provide insights into the molecular mechanisms of DNA uptake and processing in E. faecalis, potentially revealing new targets for antimicrobial intervention.

What are promising approaches for studying the role of cinA in E. faecalis pathogenicity?

To investigate cinA's role in pathogenicity:

• Generate cinA knockout mutants using CRISPR-Cas9 gene editing

• Create controllable expression systems for cinA using inducible promoters

• Perform comparative transcriptomics and proteomics between wild-type and cinA-modified strains

• Assess biofilm formation capacity under various conditions

• Evaluate virulence in appropriate infection models

• Investigate interactions with host immune factors

• Examine the impact on antimicrobial susceptibility patterns

Recent work has demonstrated the utility of CRISPRi for gene function studies in enterococci, allowing for titratable control of target gene expression—an approach that could be valuable for studying essential genes or conducting rapid candidate screening related to cinA function .

How can I address protein solubility issues when expressing recombinant cinA?

Solubility challenges with recombinant cinA can be addressed through:

• Optimizing expression temperature (typically lowering to 16-20°C)

• Using solubility-enhancing fusion tags (MBP, SUMO, or thioredoxin)

• Modifying buffer conditions (pH 7.5-8.5, salt concentration 150-500 mM)

• Adding stabilizing agents (5-10% glycerol, 1-5 mM DTT)

• Expressing truncated versions that exclude hydrophobic regions

• Co-expressing with molecular chaperones to aid folding

• Using auto-induction media instead of IPTG induction

For particularly challenging constructs, in vitro protein synthesis systems can be attempted as an alternative approach.

What techniques are most effective for analyzing contradictory data regarding cinA function?

When faced with contradictory data:

• Ensure expression construct verification through DNA sequencing

• Validate protein identity using mass spectrometry

• Verify activity through multiple complementary functional assays

• Test function under varying environmental conditions (pH, temperature, salt)

• Compare results across different expression systems

• Perform side-by-side comparison experiments using stringent controls

• Consider strain-specific variations by testing in multiple E. faecalis isolates

• Use RNA-Seq to correlate gene expression changes with observed phenotypes

For example, in studies of antimicrobial resistance mechanisms in E. faecalis, RNA-Seq was successfully employed to demonstrate that overexpression of specific transporter proteins facilitated the development of heteroresistance to omadacycline .

What are promising new approaches for studying cinA function in E. faecalis?

Emerging technologies with potential for cinA research include:

• Single-cell analysis to examine heterogeneity in cinA expression and function

• CRISPRi libraries for high-throughput functional genomics related to cinA pathways

• Structural biology approaches (cryo-EM, X-ray crystallography) to elucidate cinA complexes

• Synthetic biology tools to create minimized synthetic systems for studying cinA function

• Advanced imaging techniques to track cinA localization during competence and stress

• Machine learning approaches to predict functional interactions and phenotypic outcomes

• Development of phage-based tools for genetic manipulation and protein delivery

Research approaches that combine these technologies may yield significant insights into the complex roles of cinA in E. faecalis biology.

How might cinA research contribute to addressing antimicrobial resistance challenges?

cinA research may address antimicrobial resistance through several avenues:

• Revealing new targets for antimicrobial development by understanding competence mechanisms

• Engineering novel lanthipeptides with activity against resistant enterococci

• Developing strategies to inhibit horizontal gene transfer of resistance determinants

• Creating phage-based approaches that target cinA-dependent processes

• Designing TIV-RE inhibitor proteins based on natural phage mechanisms

Recent work has identified that bacteriophage protein TifA (Type IV restriction inhibiting factor A) binds and inactivates diverse type IV restriction enzymes in E. faecalis, representing a novel mechanism by which phages overcome bacterial defense systems . Similar approaches targeting cinA-mediated processes could yield valuable tools for combating antimicrobial resistance.