Recombinant Enterococcus faecalis Foldase protein prsA (prsA)

What is PrsA in Enterococcus faecalis?

PrsA in E. faecalis is an extracellular parvulin-like peptidyl-prolyl isomerase (PPIase) that functions as a membrane-anchored protein foldase . This protein plays a critical role in ensuring proper folding of secreted proteins as they exit the cell. Unlike in some other Gram-positive bacteria where multiple PrsA proteins may be produced, E. faecalis encodes only one PrsA family protein, which is not essential for viability . The protein is important for survival in high salt concentrations and virulence in model organisms such as Galleria mellonella .

How does PrsA function in protein folding?

PrsA functions as a peptidyl-prolyl isomerase that catalyzes the cis-trans isomerization of peptide bonds preceding proline residues in proteins, which is often a rate-limiting step in protein folding . As a membrane-anchored protein foldase, PrsA acts on secreted proteins as they emerge from the cell, ensuring they adopt their correct three-dimensional conformations. For example, PrsA is required for the proper folding and activity of the secreted metalloprotease gelatinase (GelE), though not for its expression . This role in protein folding is similar to that of PrsA homologs in other Gram-positive bacteria, where they ensure proper folding of secreted virulence factors and other proteins .

What is the relationship between PrsA and gelatinase (GelE) in E. faecalis?

PrsA is required for GelE activity but not for its gene expression or the expression of the Fsr quorum sensing system that regulates GelE . Research has demonstrated that disruption of prsA results in a GelE-negative phenotype on gelatin plates, and GelE activity in prsA::Tn supernatants is reduced approximately 70% compared to wild-type strains . While the Fsr quorum sensing system regulates the expression of gelE, PrsA operates post-translationally to ensure proper folding of the secreted GelE protein . PrsA likely acts on GelE as it is secreted from the cell, ensuring it adopts the correct conformation for enzymatic activity .

How can recombinant PrsA be expressed and purified for biochemical studies?

For expression and purification of recombinant E. faecalis PrsA:

• Clone the prsA gene (excluding the signal sequence and membrane anchor) into an expression vector such as pET or pGEX systems with appropriate affinity tags (His-tag or GST-tag).

• Transform the construct into a suitable E. coli expression strain like BL21(DE3) or derivatives.

• Induce protein expression with IPTG at optimal temperature (typically 16-25°C to enhance solubility).

• Lyse cells and purify using affinity chromatography corresponding to the attached tag.

• Further purify using size exclusion chromatography to obtain homogeneous protein.

• Confirm protein identity and purity using SDS-PAGE, Western blotting, and mass spectrometry.

• Assess folding quality using circular dichroism spectroscopy .

For functional studies, a PPIase activity assay using peptide substrates containing proline residues can be employed to confirm enzymatic activity of the purified protein.

What methods are available for creating prsA mutants in E. faecalis?

Several genetic approaches can be used to create prsA mutants in E. faecalis:

• Transposon mutagenesis: Previous studies have utilized transposon insertions in prsA (prsA::Tn) to study its function . This approach is useful for initial screening but lacks precision for targeted modifications.

• Allelic exchange: Using suicide vectors like pIMAY-Z, which has been successfully used in E. faecium and could be adapted for E. faecalis . This method allows for precise deletion or modification of the prsA gene.

• CRISPR-Cas9 systems: Recently developed for enterococci, CRISPR-Cas9 and Cas12a machinery provide efficient methods for genetic manipulation in E. faecalis . This approach allows for marker-less mutations and precise genomic editing.

• CRISPRi: For essential genes or rapid candidate screening, CRISPRi using catalytically inactive Cas9 (dCas9) can be employed to reduce gene expression without complete inactivation .

When working with clinical isolates, it's important to consider potential restriction-modification barriers that may impede DNA transfer, potentially requiring methylation of DNA or the use of conjugation from an appropriate donor strain .

How can the functional activity of PrsA be assessed in E. faecalis?

Several assays can be used to assess PrsA functional activity in E. faecalis:

• Gelatinase activity assays: Since PrsA is required for GelE activity, gelatinase activity can serve as an indirect measure of PrsA function:

  • Gelatin plate assays: Clear zones around colonies indicate active gelatinase

  • Quantitative dye release assays with supernatants from planktonic cells

• Protein secretion profiling: Analyze supernatant proteins using SDS-PAGE to identify changes in the exoprotein profile . For example, in the prsA::Tn mutant, there are observable differences in protein banding patterns, including the absence of the GelE band and changes in levels of other secreted proteins like SalB .

• Antibiotic susceptibility testing: Test sensitivity to cell wall-active antibiotics like vancomycin and oxacillin on agar plates, as prsA mutants show increased sensitivity compared to wild-type strains .

• Conjugative plasmid transfer efficiency: Measure transfer rates of conjugative plasmids, as deletion of prsA increases the efficiency of plasmid transfer .

• PPIase activity: Directly measure the peptidyl-prolyl isomerase activity using specific substrates in cell lysates or with purified protein .

What is the contribution of PrsA to E. faecalis virulence?

PrsA contributes to E. faecalis virulence through several mechanisms:

• Regulation of GelE activity: PrsA ensures proper folding of the secreted metalloprotease gelatinase (GelE), which is an important virulence factor contributing to autolysis, biofilm formation, and biofilm-associated antibiotic resistance .

• Impact on other secreted proteins: Disruption of prsA alters the exoprotein profile of E. faecalis, affecting levels of other secreted proteins including SalB, which contributes to cell envelope integrity and cephalosporin resistance .

• Biofilm formation: PrsA influences biofilm formation, which is critical for E. faecalis persistence and infection . A mutant with a transposon insertion in prsA was identified in a screen for defects in biofilm formation .

• Antibiotic resistance in biofilms: PrsA mediates biofilm-associated antibiotic resistance independently of GelE. Disruption of prsA leads to increased sensitivity to glycopeptides and oxacillin on agar plates, which could mimic biofilm growth conditions .

• Survival in challenging environments: PrsA is important for survival in high salt concentrations, which may enhance adaptability to environmental stresses encountered during infection .

In infection models, PrsA has been shown to be important for virulence in Galleria mellonella, supporting its role as a virulence factor .

How does PrsA influence antibiotic resistance in E. faecalis?

PrsA influences antibiotic resistance in E. faecalis through multiple mechanisms:

• Biofilm-specific resistance: PrsA mediates biofilm-associated antibiotic resistance independently of GelE. The prsA::Tn mutant shows increased sensitivity to glycopeptides (vancomycin) and β-lactams (oxacillin) on agar plates, even though no growth defect is observed in liquid culture at the same antibiotic concentrations . This suggests a biofilm-specific role in antibiotic resistance.

• Growth state-dependent effects: Interestingly, the influence of PrsA on antibiotic sensitivity varies between growth conditions. The prsA::Tn mutant shows strong growth inhibition on plates containing 2 μg/mL oxacillin or vancomycin, while no growth defect is observed in liquid culture at the same concentration . This suggests that growth on agar plates may more closely resemble biofilm growth than planktonic growth.

• Protein folding and cell wall integrity: As a peptidyl-prolyl isomerase, PrsA likely ensures proper folding of proteins involved in cell wall biosynthesis or antibiotic resistance mechanisms. In other Gram-positive bacteria like S. aureus, PrsA has been shown to be required for the stability of penicillin-binding proteins .

• Potential targets beyond GelE: The biofilm-specific antibiotic sensitivity of the prsA::Tn mutant suggests that other proteins that sense or respond to antibiotics during biofilm growth could be misfolded in the absence of PrsA .

These findings highlight the importance of identifying additional targets of PrsA foldase activity beyond GelE to better understand biofilm-specific antibiotic resistance in E. faecalis .

How can researchers differentiate between the direct and indirect effects of PrsA on cellular processes?

To differentiate between direct and indirect effects of PrsA on cellular processes:

• Complementation studies: Express wild-type PrsA in a prsA mutant strain and assess restoration of phenotypes. For example, expression of prsA from a pheromone-inducible plasmid has been shown to restore GelE activity on plates in a prsA::Tn mutant .

• Catalytically inactive mutants: Generate point mutations in the PPIase domain of PrsA that abolish catalytic activity but maintain protein structure. Compare phenotypes of strains expressing catalytically inactive PrsA with those of the prsA deletion mutant to distinguish between scaffold and enzymatic functions.

• Substrate identification approaches:

  • Perform co-immunoprecipitation studies with tagged PrsA to identify interacting proteins

  • Use comparative proteomics to identify proteins with altered abundance or post-translational modifications in wild-type versus prsA mutant strains

  • Employ crosslinking approaches to capture transient PrsA-substrate interactions

• Domain-specific studies: Create chimeric proteins with domains from PrsA homologs from other bacteria to identify which domains are responsible for specific functions.

• Context-dependent phenotyping: Assess phenotypes under various growth conditions (planktonic vs. biofilm, different media, stress conditions) to identify condition-specific effects that may point to different substrate proteins .

• Time-course experiments: Monitor changes in gene expression and protein abundance over time following disruption of prsA to distinguish immediate (likely direct) from delayed (likely indirect) effects.

What is known about the structural features of E. faecalis PrsA and how do they compare to homologs in other bacteria?

While detailed structural information specific to E. faecalis PrsA is limited in the provided search results, we can infer several features based on homology to PrsA proteins in other Gram-positive bacteria:

• Domain architecture: E. faecalis PrsA likely contains:

  • An N-terminal signal sequence directing secretion

  • A lipoprotein anchor at the N-terminus for membrane attachment

  • A parvulin-like PPIase domain that catalyzes prolyl isomerization

  • Chaperone domains that assist in protein folding

• Membrane localization: PrsA in E. faecalis is described as membrane-anchored, similar to its homologs in other Gram-positive bacteria . This localization positions it to interact with proteins as they are secreted through the membrane.

• Comparison with homologs: Unlike B. subtilis, where prsA is essential and cannot be deleted without compensatory mutations or increased Mg²⁺ in growth medium, E. faecalis produces only one PrsA family protein that is not essential for viability . This contrasts with bacteria like B. anthracis, L. monocytogenes, and group A Streptococcus, which produce multiple PrsA proteins .

• Substrate specificity: While in group A Streptococcus, PrsA is required for proper folding of the secreted exotoxin SpeB and is encoded adjacent to speB, in E. faecalis, prsA is in a distal location relative to gelE . This suggests potentially different evolutionary pressures on substrate specificity.

• Functional differences: Unlike in group A Streptococcus, where both PrsA and trigger factor (Tig) are required for refolding of SpeB, Tig is not required for GelE activity in E. faecalis OG1RF . This indicates potential differences in the folding mechanisms between these organisms.

Further structural studies including X-ray crystallography or cryo-EM of E. faecalis PrsA would be valuable to better understand its specific structural features and substrate interactions.

What research gaps exist in our understanding of PrsA function in E. faecalis?

Several important research gaps exist in our understanding of PrsA function in E. faecalis:

• Comprehensive substrate identification: Beyond GelE, the full range of proteins that require PrsA for proper folding remains largely unknown . Identifying these substrates would provide insight into the broader impact of PrsA on cellular physiology and virulence.

• Mechanism of biofilm-specific antibiotic resistance: While PrsA mediates biofilm-associated antibiotic resistance, the specific proteins and mechanisms involved are not fully understood . Identifying the proteins that sense or respond to antibiotics during biofilm growth that could be misfolded in the absence of PrsA is an important area for future research.

• Structural studies: Detailed structural information specific to E. faecalis PrsA is lacking, including potential unique features that distinguish it from homologs in other bacteria.

• Regulation of PrsA expression: Little is known about how prsA expression is regulated in response to environmental conditions or during different phases of infection. During rabbit subdermal abscess infection, expression of prsA increased after 2 hours but the long-term dynamics are less clear .

• Potential for therapeutic targeting: Given its role in virulence and antibiotic resistance, PrsA could be a target for novel therapeutic approaches, but research in this direction is limited.

• In vivo relevance: While PrsA has been shown to be important for virulence in the Galleria mellonella model, its significance in mammalian infection models and human infections requires further investigation.

• Discrepancy between genotype and phenotype: The relationship between PrsA and GelE may help explain observations of clinical isolates that are GelE-negative despite possessing the gelE gene, but this requires further investigation .

• Interaction with host factors: How PrsA-dependent secreted proteins interact with host factors during infection remains poorly understood.

Addressing these gaps would significantly advance our understanding of the role of PrsA in E. faecalis physiology, pathogenesis, and antibiotic resistance.

What controls should be included when evaluating the impact of PrsA on protein secretion and folding?

When evaluating the impact of PrsA on protein secretion and folding, several controls should be included:

• Strain controls:

  • Wild-type parent strain (positive control)

  • prsA mutant strain (experimental)

  • prsA mutant complemented with wild-type prsA (restoration control)

  • prsA mutant complemented with catalytically inactive prsA (distinguishes between structural and enzymatic functions)

  • Mutants of specific secreted proteins (e.g., ΔgelE) to distinguish PrsA effects from effects of its substrates

• Expression controls:

  • qRT-PCR to verify that gene expression of putative substrates (e.g., gelE) is unaffected in the prsA mutant, confirming that observed effects are post-translational

  • Western blotting to assess protein levels in cellular and secreted fractions

• Growth condition controls:

  • Multiple growth media (e.g., TSB-D, MM9-YEG) to identify media-dependent effects

  • Different growth phases (log, stationary) to capture temporal dynamics

  • Planktonic versus biofilm growth to identify context-specific effects

  • Stress conditions (e.g., antibiotics, salt) to assess condition-dependent responses

• Fraction controls:

  • Analysis of multiple cellular fractions (supernatant, cell wall, protoplast) to differentiate secretion defects from folding defects

  • Purification of specific proteins from wild-type and mutant backgrounds to directly assess folding state

• Activity assays:

  • Multiple substrate assays for functional activity (e.g., dye release assays for GelE)

  • Biological activity assays (e.g., conjugative plasmid transfer efficiency)

  • Antibiotic susceptibility testing in both liquid and solid media

• Structural analyses:

  • Circular dichroism to assess secondary structure of purified proteins

  • Limited proteolysis to evaluate folding differences

  • Thermal stability assays to measure protein stability

These comprehensive controls help distinguish direct effects of PrsA on protein folding from indirect effects on gene expression, protein stability, or cellular physiology.

How should researchers address the differences in PrsA-dependent phenotypes observed between liquid culture and solid media?

To address the differences in PrsA-dependent phenotypes observed between liquid culture and solid media, researchers should consider the following approaches:

• Experimental design considerations:

  • Always test phenotypes in both conditions (liquid and solid) to capture environment-specific effects

  • Include time-course sampling to differentiate between transient and stable phenotypes

  • Use multiple methods to assess the same phenotype (e.g., disk diffusion, broth microdilution, and time-kill assays for antibiotic susceptibility)

  • Vary cell densities in liquid culture to determine if the differences are related to cell density rather than growth mode

• Growth environment characterization:

  • Monitor oxygen gradients in both settings using oxygen-sensitive probes

  • Measure pH changes in microenvironments

  • Assess nutrient availability and diffusion in both conditions

  • Examine cell-to-cell contact and aggregation patterns

• Biofilm characterization:

  • Determine if growth on agar plates resembles biofilm growth by analyzing matrix production

  • Compare gene expression profiles between planktonic, plate, and biofilm growth

  • Develop intermediate growth models (e.g., static liquid culture biofilms, flow cells) to bridge the gap between fully planktonic and solid media growth

• Physiological state analysis:

  • Assess cell wall thickness and composition across growth conditions

  • Examine protein localization using fluorescent reporters

  • Measure the activity of secreted enzymes under different growth conditions

  • Use electron microscopy to visualize ultrastructural differences

• Gene expression and proteomic approaches:

  • Compare transcriptomes and proteomes across growth conditions to identify differentially expressed genes and proteins

  • Use ribosome profiling to assess translation differences

  • Employ RNA-seq and proteomics to identify condition-specific regulatory networks

• Data interpretation:

  • Consider that bacterial physiology differs fundamentally between liquid and solid growth

  • Interpret antibiotic susceptibility in the context of growth mode-specific effects

  • Recognize that clinical relevance may depend on growth mode (e.g., biofilm formation during infection)

• Translation to in vivo models:

  • Compare phenotypes in liquid, solid, and animal infection models

  • Determine which in vitro condition better predicts in vivo behavior

  • Develop ex vivo models that more accurately reflect host niches

By systematically addressing these aspects, researchers can better understand the mechanistic basis for the observed differences and determine which experimental conditions most accurately reflect the physiological state of E. faecalis during infection.

What approaches can be used to identify the full complement of proteins that require PrsA for proper folding?

To identify the full complement of proteins that require PrsA for proper folding in E. faecalis, researchers can employ several complementary approaches:

• Comparative secretome analysis:

  • Use mass spectrometry-based proteomics to compare supernatant proteins from wild-type, prsA mutant, and complemented strains

  • Apply quantitative techniques such as SILAC, iTRAQ, or TMT labeling for accurate quantification of protein abundance changes

  • Look for proteins with altered mobility on SDS-PAGE that might indicate misfolding or incomplete processing

  • Analyze not only presence/absence but also post-translational modifications that might be affected by improper folding

• Protein stability and solubility profiling:

  • Compare soluble versus insoluble protein fractions in wild-type versus prsA mutant strains

  • Employ thermal proteome profiling (TPP) to identify proteins with altered thermal stability in the absence of PrsA

  • Use pulse-chase experiments with radioactive labeling to assess protein turnover rates

• Structural biology approaches:

  • Apply hydrogen/deuterium exchange mass spectrometry to identify proteins with altered folding dynamics

  • Use limited proteolysis coupled with mass spectrometry to detect conformational differences

  • Employ circular dichroism to assess secondary structure changes in candidate substrates

• Direct interaction studies:

  • Perform co-immunoprecipitation with tagged PrsA to identify interacting partners

  • Use crosslinking approaches to capture transient interactions between PrsA and substrates

  • Apply bacterial two-hybrid systems to screen for potential interactions

  • Employ surface plasmon resonance or microscale thermophoresis to measure binding affinities

• Functional genomics screens:

  • Conduct synthetic lethal screens to identify genes that become essential in a prsA mutant background

  • Perform suppressor screens to identify mutations that restore function to a prsA mutant

  • Use transposon sequencing (Tn-seq) to identify genes with altered fitness contributions in the absence of PrsA

• Computational prediction:

  • Identify proteins with high proline content, particularly in regions predicted to be structurally important

  • Look for proteins with signal sequences indicating secretion

  • Analyze conservation of potential substrates with known PrsA substrates in other Gram-positive bacteria

• Phenotypic assays:

  • Systematically test activities of known secreted enzymes in wild-type versus prsA mutant backgrounds

  • Assess changes in antibiotic resistance profiles that might indicate misfolding of specific resistance determinants

  • Examine biofilm formation and cell surface properties that depend on properly folded surface proteins

Integration of these multiple approaches would provide a comprehensive view of the PrsA-dependent folding network in E. faecalis and identify direct versus indirect effects of PrsA disruption.

Could PrsA serve as a target for novel antimicrobial development against E. faecalis?

PrsA has several characteristics that make it a potentially attractive target for novel antimicrobial development against E. faecalis:

• Virulence attenuation: Disruption of prsA reduces virulence in model organisms like Galleria mellonella, suggesting that inhibiting PrsA could attenuate E. faecalis pathogenicity .

• Impact on antibiotic resistance: PrsA mediates biofilm-associated antibiotic resistance, and its disruption increases sensitivity to glycopeptides and β-lactams in biofilm-like growth conditions . PrsA inhibitors could potentially serve as antibiotic adjuvants to enhance the efficacy of existing antibiotics against biofilm-associated infections.

• Role in stress adaptation: PrsA contributes to survival in high salt concentrations and other stress conditions, suggesting that its inhibition could reduce bacterial fitness in host environments .

• Conservation and specificity: PrsA is conserved across Gram-positive bacteria but shows structural and functional differences from human PPIases, potentially allowing for selective targeting .

• Surface accessibility: As a membrane-anchored protein involved in folding secreted proteins, PrsA may be more accessible to inhibitors than intracellular targets, potentially eliminating the need for compounds to cross the cell membrane .

Challenges and considerations for targeting PrsA include:

• Non-essentiality: PrsA is not essential for E. faecalis viability under standard laboratory conditions, suggesting that inhibitors would be anti-virulence agents rather than bactericidal compounds . This might necessitate combination therapy with traditional antibiotics.

• Potential for resistance: Mechanisms of resistance to PrsA inhibitors would need to be explored, particularly given the adaptability of enterococci.

• Cross-reactivity: Ensuring specificity against bacterial versus human PPIases would be essential to avoid off-target effects.

• Delivery to biofilms: Since PrsA's role appears most significant in biofilm growth, effective delivery of inhibitors to biofilms would be crucial for efficacy.

Development strategies could include high-throughput screening of small molecule libraries against recombinant PrsA, structure-based drug design based on the PPIase domain, and phenotypic screening for compounds that mimic prsA mutation phenotypes in biofilm formation and antibiotic sensitivity .

How might the interaction between PrsA and antibiotic resistance mechanisms influence clinical treatment strategies?

The interaction between PrsA and antibiotic resistance mechanisms could influence clinical treatment strategies in several important ways:

• Biofilm-specific targeting: The prsA mutant shows increased sensitivity to glycopeptides (vancomycin) and β-lactams (oxacillin) specifically during biofilm-like growth on solid media, but not in liquid culture . This suggests that targeting PrsA could be particularly effective against biofilm-associated infections, which are notoriously difficult to treat with conventional antibiotics.

• Antibiotic selection considerations:

  • The increased sensitivity of prsA mutants to specific antibiotics suggests that certain drug classes (glycopeptides, β-lactams) might be more effective when combined with PrsA inhibitors

  • Clinical isolates with naturally occurring prsA mutations might show differential susceptibility to antibiotics, potentially informing personalized treatment approaches

• Combination therapy potential:

  • PrsA inhibitors could serve as antibiotic adjuvants that sensitize E. faecalis biofilms to existing antibiotics

  • Sequential therapy approaches might be effective (e.g., PrsA inhibitor to disrupt biofilm integrity followed by conventional antibiotics)

  • Targeting multiple folding pathways simultaneously (e.g., PrsA and other chaperones) might provide synergistic effects

• Diagnostic implications:

  • Assessing prsA status in clinical isolates could potentially predict biofilm formation capacity and antibiotic susceptibility profiles

  • Developing rapid tests for PrsA activity could help guide treatment decisions

• Resistance development considerations:

  • Understanding how PrsA contributes to adaptation to antibiotic exposure could help predict and prevent resistance development

  • Monitoring changes in prsA expression or mutation during antibiotic treatment could provide early warning of developing resistance

• Therapeutic development directions:

  • Rather than directly inhibiting PrsA enzyme activity, disrupting its interaction with specific substrates involved in antibiotic resistance might provide more targeted approaches

  • Identifying and targeting the specific proteins that require PrsA for proper folding and that contribute to antibiotic resistance could provide alternative therapeutic strategies

• Special patient populations:

  • Given PrsA's role in salt tolerance, its targeting might be particularly effective in certain infection sites with specific ionic environments

  • Patients with biofilm-associated infections (e.g., catheter-associated UTIs, endocarditis) might particularly benefit from therapies addressing PrsA function

These considerations highlight the potential for targeting PrsA as part of sophisticated anti-infective strategies against E. faecalis, particularly for biofilm-associated infections that are resistant to conventional antibiotic treatments .