Recombinant Enterococcus faecalis Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha (accA)

What is Enterococcus faecalis and why is it clinically significant?

Enterococcus faecalis is a species of gram-positive bacteria naturally found in the human gastrointestinal tract, mouth, and vagina. While it normally exists as a commensal organism in the intestines without causing harm, it can become pathogenic when it spreads to other parts of the body . E. faecalis is clinically significant because of its remarkable resilience—it can survive in hot, salty, or acidic environments—and its increasing antibiotic resistance .

The bacteria can cause serious infections if it enters the bloodstream, urinary tract, or surgical wounds. These infections can progress to life-threatening conditions including sepsis, endocarditis, and meningitis . E. faecalis infections are particularly problematic in healthcare settings, where they can spread between patients through contaminated equipment or poor hand hygiene practices. Individuals with compromised immune systems, such as those undergoing organ transplantation, kidney dialysis, or cancer treatment, are at increased risk of developing E. faecalis infections . The emergence of multi-drug resistant strains has further complicated treatment options, making research into novel targets like accA increasingly important.

What is the function of Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha (accA) in bacterial metabolism?

Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha (accA) encodes for a critical subunit of the acetyl-CoA carboxylase enzyme complex, which catalyzes the first committed step in fatty acid synthesis in bacteria . In E. faecalis and other bacteria, this enzyme is essential for the production of fatty acids and phospholipids that form the bacterial inner membrane structure .

The accA gene produces the carboxyl transferase subunit alpha of the acetyl-CoA carboxylase (ACCase) enzyme complex. This enzyme complex catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, which is the rate-limiting step in fatty acid synthesis pathway . Fatty acids produced through this pathway are essential components of the bacterial cell membrane, particularly the phospholipid bilayer of the inner membrane in gram-negative bacteria. The proper functioning of this membrane is crucial for cellular integrity, selective permeability, and ultimately, bacterial survival. Disruption of accA expression can lead to compromised membrane structure, altered cell permeability, and increased susceptibility to antibiotics .

How does recombinant expression of accA differ between E. faecalis and other bacterial species?

Recombinant expression of accA shows notable differences between E. faecalis and other bacterial species due to variations in gene structure, regulation mechanisms, and the bacterial acetyl-CoA carboxylase (ACCase) complex organization. When expressing recombinant accA, researchers must account for the unique characteristics of E. faecalis compared to model organisms like E. coli.

The bacterial ACCase enzyme complex contains distinct subunits that differ significantly from mammalian ACCase isoforms, making it an attractive target for antibiotic development . Specifically, bacterial ACCase complexes like those in E. faecalis have structural differences compared to human ACC isoforms (ACC1 and ACC2). While human isoforms have approximately 550 residues between the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, bacterial BC units typically have around 450 residues . These structural differences ensure that antimicrobial therapies targeting bacterial accA won't interfere with human ACCase enzymes, providing a safety advantage for therapeutic development.

Expression systems must be optimized specifically for E. faecalis accA, considering factors such as codon usage, protein folding, and potential toxicity to the host organism. When designing experiments to express recombinant E. faecalis accA, researchers should consider using inducible expression systems to control protein production levels and minimize potential toxic effects on the host cells.

How does antisense inhibition of accA affect bacterial antibiotic resistance profiles?

Antisense inhibition of accA significantly reduces bacterial antibiotic resistance by disrupting fatty acid synthesis and compromising membrane integrity, thereby facilitating antibiotic penetration into bacterial cells. Research demonstrates that E. coli expressing antisense RNA against accA shows markedly increased susceptibility to multiple antibiotic classes compared to control groups .

In experimental studies, E. coli cells expressing antisense RNA targeting accA displayed significantly larger zones of inhibition when exposed to antibiotics including tetracycline (10 μg/mL), carbenicillin (100 μg/mL), and chloramphenicol (25 μg/mL) . Statistical analysis confirmed this increased susceptibility with a paired t-test showing significant differences between treatment and control groups (P=0.000554, t-score 3.6, df=31, 95% CI, ) . The average zone of inhibition for bacteria with accA suppression was 20 mm, compared to just 6 mm in control samples without antisense RNA expression .

What is the relationship between accA inhibition and luxS expression in enteric bacteria?

The inhibition of accA through antisense RNA has been shown to suppress the expression of luxS, a gene encoding the S-ribosylhomocysteine lyase enzyme that plays crucial roles in bacterial quorum sensing and virulence . This relationship represents a significant molecular mechanism linking fatty acid synthesis to bacterial communication and pathogenicity.

LuxS is responsible for producing the autoinducer-2 (AI-2) signaling molecule, which mediates interspecies quorum sensing communication in many bacterial species. The suppression of luxS following accA inhibition indicates a regulatory connection between fatty acid metabolism and quorum sensing pathways . When researchers inhibited accA expression using antisense RNA, they observed a corresponding decrease in luxS expression, measured through quantitative PCR analysis of luxS-cDNA synthesized from E. coli cells expressing antisense RNA against accA under various glucose concentrations (25 μM, 5 μM, and control) .

This relationship has significant implications for bacterial pathogenicity since luxS regulates several virulence factors. The gene is essential for cell growth, intercellular quorum-sensing signal transfer, and biofilm formation . The discovery that accA inhibition suppresses luxS expression suggests that targeting accA could simultaneously disrupt bacterial membrane integrity and interfere with virulence mechanisms dependent on quorum sensing, potentially offering a dual-action approach to antimicrobial therapy.

What methodological approaches can be used to study the effects of recombinant accA expression on bacterial membrane composition?

Several sophisticated methodological approaches can be employed to investigate how recombinant accA expression affects bacterial membrane composition. These techniques provide complementary data on structural and functional changes in the membrane following manipulation of accA levels.

Lipidomic Analysis: Mass spectrometry-based lipidomics can characterize changes in phospholipid profiles and fatty acid composition resulting from altered accA expression. This approach can reveal specific lipid species affected by accA manipulation, providing insights into how fatty acid synthesis disruption impacts membrane structure.

Membrane Fluidity Assays: Fluorescence anisotropy measurements using probes like 1,6-diphenyl-1,3,5-hexatriene (DPH) can assess changes in membrane fluidity following accA modulation. Researchers can correlate these measurements with antibiotic susceptibility data to understand how membrane physical properties influence drug penetration.

Electron Microscopy: Transmission electron microscopy can visualize ultrastructural changes in bacterial membranes resulting from accA inhibition. This technique allows direct observation of membrane integrity, thickness, and potential disruptions.

Antibiotic Uptake Assays: Researchers can quantify the intracellular accumulation of fluorescently labeled antibiotics in bacteria with modulated accA expression. As demonstrated in previous studies, E. coli with suppressed accA shows increased antibiotic susceptibility due to compromised membrane integrity, facilitating greater antibiotic influx . This approach directly measures how accA affects drug penetration.

Gene Expression Analysis: Quantitative PCR and RNA-Seq can be used to analyze changes in expression of membrane-related genes following accA manipulation. Similar to the approach used to study luxS expression in response to accA inhibition, researchers can extract RNA, synthesize cDNA, and perform qPCR using primers specific for genes involved in membrane formation and maintenance .

What are optimal expression systems for producing recombinant E. faecalis accA?

When designing expression systems for recombinant E. faecalis accA, researchers must carefully consider host compatibility, protein solubility, and functional activity. Several expression systems have proven effective, each with distinct advantages depending on the research objectives.

E. coli Expression Systems: The pET expression system in E. coli BL21(DE3) strains provides high protein yields and inducible expression control through IPTG. For accA expression, the T7 promoter-based systems offer tight regulation, preventing potential toxicity from constitutive expression of this metabolically important enzyme. Researchers should consider using fusion tags like 6xHis or GST to facilitate purification and potentially enhance protein solubility.

Cell-Free Expression Systems: For rapid protein production and avoiding potential toxicity issues, cell-free protein synthesis systems based on E. coli extracts can express E. faecalis accA without cellular constraints. This approach is particularly useful for initial characterization studies and when testing antisense RNA inhibition strategies similar to those used in previous studies .

Conditional Expression in Native Host: For studying accA function in its native context, researchers can develop conditional expression systems in E. faecalis using tetracycline-responsive promoters or similar inducible systems. This approach allows manipulation of accA levels within the original organism, providing insights into physiological effects.

When optimizing expression conditions, researchers should carefully control temperature (typically 16-25°C for improved solubility), induction timing (mid-log phase), and inducer concentration. Adding specific chaperones or co-expressing with other ACCase subunits may improve proper folding and functional activity of the recombinant enzyme.

How can researchers effectively design antisense RNA strategies to inhibit accA expression?

Designing effective antisense RNA (asRNA) strategies to inhibit accA expression requires careful consideration of target sequence selection, delivery methods, and validation approaches. Based on successful strategies demonstrated in previous research, the following methodological framework is recommended:

Target Sequence Selection:

• Identify regions of the accA mRNA with high accessibility and minimal secondary structure, particularly around the translation initiation site and ribosome binding site.

• Design antisense sequences of 18-25 nucleotides complementary to these regions.

• Avoid sequences with significant complementarity to non-target genes to minimize off-target effects.

• Utilize computational tools to predict RNA secondary structures and accessibility of potential target regions.

Vector Construction:

• Clone designed asRNA sequences into plasmid vectors with appropriate antibiotic selection markers, such as the recombinant pHN1257 plasmid used in previous studies .

• Use inducible promoters to control asRNA expression levels.

• Consider incorporating ribozyme sequences to enhance asRNA stability.

Delivery and Selection:

• Transform constructs into target bacteria through electroporation or chemical transformation.

• Select transformants using appropriate antibiotics, such as kanamycin used in previous research .

• Culture selected colonies in liquid media and confirm asRNA expression.

Validation of Inhibition:

• Extract total RNA from bacterial samples expressing asRNA targeting accA.

• Synthesize cDNA using reverse transcriptase kits, following protocols similar to those used with the OneScript Reverse Transcriptase cDNA Synthesis kit .

• Perform quantitative PCR to measure accA mRNA levels, comparing cells with and without asRNA expression.

• Analyze ACCase enzyme activity through biochemical assays to confirm functional inhibition.

Phenotypic Analysis:

• Assess antibiotic susceptibility using disk diffusion assays with antibiotics such as tetracycline, carbenicillin, and chloramphenicol .

• Measure zones of inhibition to quantify antibiotic sensitivity changes.

• Analyze membrane integrity and fatty acid composition to confirm the mechanistic basis of observed phenotypes.

What techniques can be used to quantify changes in virulence factor expression following accA modulation?

Multiple complementary techniques can effectively quantify changes in virulence factor expression, particularly luxS, following accA modulation. Each method provides specific insights into the regulatory relationship between accA and virulence factors.

Quantitative PCR (qPCR):
qPCR remains the gold standard for precise quantification of gene expression changes. Following accA inhibition through antisense RNA, researchers can extract total RNA from bacterial samples, synthesize cDNA using reverse transcriptase, and perform qPCR with primers specific for virulence factors like luxS . This approach allows for direct measurement of transcript levels and can detect subtle changes in gene expression. The methodology should include appropriate reference genes for normalization and multiple biological replicates to ensure statistical validity.

RNA-Seq Analysis:
RNA-Seq provides a comprehensive view of transcriptome-wide changes following accA modulation. This approach can identify not only changes in known virulence factors but also reveal previously unrecognized genes affected by accA inhibition. Differential expression analysis between accA-inhibited and control samples can identify coordinated expression changes in virulence pathways.

Autoinducer-2 (AI-2) Assays:
Since luxS produces the AI-2 quorum sensing molecule, researchers can quantify AI-2 activity using reporter strains like Vibrio harveyi BB170. Cell-free supernatants from bacteria with modulated accA expression can be tested for AI-2 activity, providing a functional readout of luxS activity changes.

Biofilm Formation Assays:
Given luxS's role in biofilm formation, crystal violet staining assays can quantify changes in biofilm production following accA inhibition. This provides a phenotypic measurement of how accA modulation affects a luxS-dependent virulence trait.

Proteomics Analysis:
Mass spectrometry-based proteomics can detect changes in virulence factor protein levels following accA inhibition. This approach complements transcriptional analyses by confirming that mRNA changes translate to altered protein expression.

What statistical approaches are most appropriate for analyzing antibiotic susceptibility changes following accA inhibition?

When analyzing antibiotic susceptibility changes following accA inhibition, researchers should employ robust statistical methods that account for the nature of the data and experimental design. Based on previous research methodologies, the following statistical approaches are recommended:

Paired t-tests: This approach is particularly suitable when comparing antibiotic susceptibility between matched samples (with and without accA inhibition) as demonstrated in previous research (P=0.000554, paired t-test, t-score 3.6, df=31, 95% CI, ) . Paired tests are powerful when analyzing zone of inhibition measurements from the same bacterial strains under different treatment conditions.

Analysis of Variance (ANOVA): When comparing susceptibility across multiple antibiotics or different levels of accA inhibition, ANOVA followed by appropriate post-hoc tests (Tukey's HSD or Bonferroni) provides a comprehensive analysis while controlling for multiple comparisons.

Non-parametric alternatives: If data doesn't meet assumptions of normality, researchers should consider non-parametric alternatives such as the Wilcoxon signed-rank test (paired data) or Mann-Whitney U test (unpaired data).

Categorical analysis: For interpreting susceptibility categories (susceptible, intermediate, resistant), chi-square tests or Fisher's exact test can analyze the distribution shift following accA inhibition. Previous research demonstrated that 59% of accA-inhibited samples showed sensitivity to antibiotics compared to only 25% of control samples .

Effect size calculations: Beyond statistical significance, researchers should report effect sizes (Cohen's d or percentage differences) to quantify the magnitude of accA inhibition effects. Previous studies showed an 82% difference in antibiotic sensitivity between treatment and control groups .

Regression analysis: To understand the relationship between accA expression levels and antibiotic susceptibility, regression models can correlate quantitative measurements of accA inhibition (from qPCR data) with zone of inhibition measurements.

All statistical analyses should include appropriate sample sizes (minimum n=30 for parametric tests), clearly stated significance thresholds (typically p<0.05), and confidence intervals for important measurements.

How can researchers distinguish between direct effects of accA inhibition and secondary metabolic consequences?

Distinguishing between direct effects of accA inhibition and secondary metabolic consequences requires methodical experimental design and comprehensive analytical approaches. Researchers can implement the following strategies to differentiate primary from secondary effects:

Time-course experiments: By measuring changes at multiple time points following accA inhibition, researchers can establish the temporal sequence of effects. Direct consequences typically manifest earlier than secondary metabolic adaptations. For instance, monitoring changes in membrane phospholipid composition, followed by tracking luxS expression at regular intervals post-inhibition, can reveal which effects occur first .

Dose-response relationships: Systematically varying the degree of accA inhibition (using different concentrations of inducers for antisense RNA expression) allows researchers to determine whether effects on other pathways show proportional responses. Direct effects typically show clearer dose-dependency than downstream consequences. Previous research examining luxS expression following different levels of accA inhibition (using varying glucose concentrations) utilized this approach .

Metabolic rescue experiments: Supplementing the growth medium with products of the inhibited pathway can distinguish direct from indirect effects. For example, adding specific fatty acids to bypass the need for endogenous fatty acid synthesis in accA-inhibited bacteria would mitigate direct effects while leaving secondary consequences unchanged.

Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data provides a systems-level view of changes following accA inhibition. Pathway analysis of these integrated datasets can reveal direct targets versus secondary adaptations through network analysis.

Genetic complementation: Expressing accA from a plasmid in cells where the chromosomal gene is inhibited can confirm which phenotypes are directly related to accA function. Effects reversed by complementation are likely direct consequences of accA inhibition.

Mathematical modeling: Developing computational models of bacterial metabolism incorporating accA function can predict expected direct effects versus secondary adaptations, generating hypotheses that can be experimentally tested.

What are promising approaches for developing antimicrobial therapies targeting E. faecalis accA?

Research on E. faecalis accA presents several promising avenues for developing novel antimicrobial therapies that could overcome current resistance challenges. Based on established findings and emerging technologies, the following approaches show particular promise:

Antisense RNA therapeutics: Building on proven efficacy in laboratory settings, antisense RNA targeting accA could be developed into therapeutic agents . Delivery systems such as modified liposomes, cell-penetrating peptides, or bacteriophage vectors could be engineered to introduce antisense molecules into E. faecalis cells in clinical settings. This approach offers high specificity and potentially lower toxicity compared to traditional antibiotics.

Small molecule inhibitors: Structure-based drug design targeting the unique features of bacterial ACCase enzymes could yield selective inhibitors of accA function. The documented structural differences between bacterial and human ACCase enzymes (450 versus 550 residues between domains) provide an excellent basis for developing compounds that specifically target bacterial enzymes without affecting human counterparts .

CRISPR-Cas antimicrobials: CRISPR-Cas systems could be engineered to specifically target and cleave the accA gene in E. faecalis. Delivered via modified phages or nanoparticles, these systems could provide highly specific antimicrobial activity against E. faecalis while sparing beneficial microbiota.

Combination therapies: Given the finding that accA inhibition increases susceptibility to existing antibiotics, combination approaches could revitalize the efficacy of currently available drugs . Pairing subinhibitory concentrations of traditional antibiotics with accA-targeting agents could overcome resistance mechanisms while reducing required antibiotic doses, potentially minimizing side effects.

Immunotherapeutic approaches: Developing antibodies or vaccines targeting ACCase enzyme components exposed on the bacterial surface could provide an alternative strategy for neutralizing E. faecalis infections, particularly in high-risk hospitalized patients.

Biofilm disruption strategies: Since accA inhibition has been linked to suppression of luxS, which is essential for biofilm formation, targeting accA could provide a novel approach to disrupting E. faecalis biofilms in chronic infections . This could be particularly valuable for treating device-associated infections where biofilms present a major challenge.

How might E. faecalis develop resistance to accA-targeting therapies, and what counterstrategies can researchers employ?

Understanding potential resistance mechanisms to accA-targeting therapies is crucial for developing effective counterstrategies. Based on bacterial adaptation principles and existing knowledge of antibiotic resistance, researchers should consider the following resistance scenarios and corresponding mitigation approaches:

Potential Resistance Mechanisms:

• Target modification: E. faecalis could acquire mutations in the accA gene that maintain enzyme function while reducing binding affinity for inhibitors or antisense molecules.

• Compensatory metabolic pathways: Bacteria might upregulate alternative fatty acid acquisition mechanisms, such as increased uptake of exogenous fatty acids from the environment.

• Efflux pump overexpression: Enhanced expression of efflux systems could reduce intracellular concentration of small-molecule accA inhibitors.

• Degradation of antisense RNA: Increased expression of RNases that specifically degrade therapeutic antisense molecules could emerge.

• Biofilm-mediated resistance: Enhanced biofilm formation through alternative pathways could protect bacterial communities from accA-targeting therapies.

Counterstrategies:

• Multi-target approaches: Simultaneously targeting multiple components of the ACCase complex (not just accA) could raise the genetic barrier to resistance, requiring multiple simultaneous mutations for bacteria to develop resistance.

• Cycling or combination therapies: Rotating between different accA-targeting mechanisms or combining them with traditional antibiotics can prevent resistance development. The demonstrated synergy between accA inhibition and antibiotics like tetracycline, carbenicillin, and chloramphenicol provides a foundation for this approach .

• Adjuvant therapies: Inhibitors of fatty acid transport or alternative metabolic pathways could block potential compensatory mechanisms.

• Delivery system optimization: Developing delivery systems that rapidly achieve high local concentrations of inhibitors could overcome efflux-based resistance mechanisms.

• Resistance surveillance: Implementing systematic monitoring of clinical E. faecalis isolates for emerging resistance to accA-targeting therapies would allow early detection and intervention.

• Ecological approaches: Considering the role of the microbiome in preventing pathogen colonization, combining accA inhibitors with probiotics or prebiotics could provide ecological resistance to E. faecalis infections.