Recombinant Enterococcus faecalis Protein translocase subunit SecA (secA), partial

What is the quaternary structure of E. faecalis SecA and how does it differ from SecA in other bacterial species?

E. faecalis SecA exists predominantly as a monomer in solution, with a molecular weight of approximately 110 kDa as determined by analytical ultracentrifugation and size-exclusion chromatography. Electron microscopy of negatively stained EFSecA shows particles with a diameter of 5-6 nm, consistent with a monomeric form. This represents a significant difference from SecA proteins of Escherichia coli and Bacillus subtilis, which typically exist as dimers under similar conditions .

What are the key structural features of crystallized E. faecalis SecA?

Crystallization studies of E. faecalis SecA have yielded crystals that diffract to 2.4 Å resolution. These crystals belong to the monoclinic space group C2, with the following unit-cell parameters:

• a = 203.4 Å

• b = 49.8 Å

• c = 100.8 Å

• α = γ = 90.0°

• β = 119.1°

For successful crystallization, researchers engineered a K6N mutation in E. faecalis SecA to reduce proteolytic sensitivity. The wild-type protein was prone to N-terminal proteolysis, losing five to six amino acids due to the presence of a lysine-lysine motif at positions 6 and 7. By mimicking the B. subtilis SecA sequence and replacing lysine 6 with asparagine, researchers were able to produce the intact protein for structural studies .

How does SecA mediate protein translocation across bacterial membranes?

SecA serves as the essential ATPase component of the bacterial Sec pathway, driving protein translocation through the SecYEG channel. The working mechanism has been debated between two primary models:

• Brownian Ratchet Model: SecA acts as a regulatory protein whose Two-Helix Finger (THF) senses the sizes of residues in polypeptide substrates. When large side chains are encountered, SecA converts to an ATP-bound state, opening the SecY channel to allow passage. After ATP hydrolysis, the channel closes .

• Push-Slide Model: SecA functions as an active motor rather than a passive regulator. The THF directly interacts with and moves polypeptide substrates, similar to pore loops in AAA ATPases. Upon ATP binding, the THF contacts the polypeptide substrate and pushes it toward the channel. As ATP hydrolyzes, the THF retracts while the clamp holds the polypeptide tightly, preventing backward movement .

Recent structural evidence supports a mechanism with striking similarity to how helicases move DNA/RNA substrates. The structures of active SecA-SecY with a moving protein substrate in both ADP and ATP states reveal common structural features shared with other protein translocation systems targeted by antibiotics .

How is SecA spatially distributed within E. faecalis cells during different stages of the cell cycle?

SecA in E. faecalis demonstrates distinct localization patterns that change as cells progress through division. Immunofluorescence microscopy studies after lysozyme-mediated cell-wall degradation and detergent-mediated membrane permeabilization reveal that:

• Early Division Stage: SecA predominantly localizes to the equatorial mid-cell, appearing as single foci at the septum .

• Late Division Stage: SecA appears in a single- or multi-focal pattern at nascent sites of cell division .

This localization pattern is consistent with observations made by immunoelectron microscopy (IEM), confirming that SecA colocalizes with both sortase A (SrtA) and sortase C (SrtC) at discrete foci, often near the septum .

The spatial restriction of SecA to specific regions in E. faecalis is significant because it coordinates the secretion and assembly of virulence factors. This focal concentration may enhance the efficiency of protein translocation and subsequent processing by sortase enzymes .

What is the relationship between SecA and sortase enzymes in E. faecalis virulence?

SecA and Sortase A (SrtA) in E. faecalis exhibit coordinated localization to discrete domains near the septum or nascent septal sites throughout the cell cycle. This colocalization is functionally significant for virulence factor secretion and assembly .

The process follows a distinct pathway:

• SecA mediates the translocation of proteins with signal peptides across the cytoplasmic membrane via the Sec pathway.

• For proteins destined to be cell wall-anchored virulence factors, sortase enzymes (particularly SrtA) recognize specific sorting signals and catalyze the covalent attachment of these proteins to the peptidoglycan cell wall .

Disruption of these localized secretion and sorting sites, for example by cationic antimicrobial peptides like human β-defensins, can impair virulence factor assembly. β-defensins interact with E. faecalis at discrete septal foci, disrupting the sites of localized secretion and sorting. This suggests that focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly .

How do cationic antimicrobial peptides affect SecA localization and function?

Human β-defensins interact with E. faecalis at discrete septal foci, coinciding with the localization sites of SecA and sortase enzymes. This interaction leads to disruption of the localized secretion and sorting machinery, with several important consequences:

• Mislocalization of sortase enzymes and associated secretion machinery, including SecA

• Impaired attachment of virulence factors to the cell wall

• Reduced pathogen virulence

The bacteria attempt to counteract this targeting through modification of anionic lipids by the multiple peptide resistance factor (MprF) protein. MprF adds cationic residues to anionic lipids as a general cationic peptide resistance strategy, which:

• Limits focal defensin targeting in E. faecalis

• Renders the bacterium more resistant to killing by defensins

• Reduces susceptibility to focal targeting by cationic antimicrobial peptides

This evidence suggests a paradigm in which focal targeting by antimicrobial peptides is directly linked to their killing efficiency and to disruption of virulence factor assembly through SecA and sortase disruption.

Why is SecA considered a promising target for antimicrobial development against E. faecalis?

SecA presents several advantages as an antimicrobial target against E. faecalis and other bacteria:

• Essential Function: SecA plays an indispensable role in protein secretion and is essential for bacterial survival across a broad spectrum of bacteria. The SecA gene in E. faecalis is critical for viability .

• No Human Homologue: Unlike SecYEG, there are no SecA counterparts in mammalian cells, reducing the risk of off-target effects in human hosts .

• Membrane Accessibility: As a membrane protein in its functional form, SecA inhibitors can directly access their target without needing to enter the cytoplasmic space. This advantage reduces concerns about drug permeation and intracellular concentration, which are common challenges in antimicrobial development .

• Impact on Efflux Pumps: Most efflux pumps, particularly in Gram-negative bacteria, consist of membrane proteins with signal peptides. Inhibition of SecA could affect the assembly of functional efflux systems, potentially addressing the challenge of multidrug resistance (MDR) .

• Virulence Factor Secretion: SecA is essential for the secretion of bacterial toxins and virulence factors, making it a target that could reduce pathogenicity even at sub-lethal inhibitory concentrations .

What experimental approaches are recommended for screening potential SecA inhibitors?

When developing an experimental pipeline for screening SecA inhibitors, researchers should consider:

• In vitro ATPase Assays: Measuring the ATPase activity of purified recombinant E. faecalis SecA in the presence of potential inhibitors. Consider using a truncated enzyme assay system, as truncated SecA may be more relevant to the functional form in cellular membranes (the intact SecA has an inhibitory C-terminal domain) .

• Protein Translocation Assays: Evaluating the ability of compounds to inhibit SecA-dependent translocation of model substrates across membrane vesicles.

• Bacterial Growth Inhibition: Testing compounds for their ability to inhibit E. faecalis growth, with confirmation that growth inhibition correlates with SecA inhibition.

• Periplasmic Accessibility Assays: Assessing whether SecA inhibitors can access the protein from the periplasmic (in Gram-negative) or extracellular (in Gram-positive) side of the membrane, which has implications for bypassing efflux pumps .

• Virulence Factor Secretion Analysis: Measuring the impact of potential inhibitors on the secretion of specific virulence factors to demonstrate target engagement in living cells.

• Resistance Development Monitoring: Evaluating the frequency and mechanisms of resistance development against SecA inhibitors.

The key issues to address in these experiments include determining whether SecA inhibition is sufficient to achieve antimicrobial effects, understanding the consequences of attenuated virulence factor secretion, and confirming the accessibility of the functional form of SecA to inhibitors .

What are the optimal conditions for cloning and expressing recombinant E. faecalis SecA?

Based on successful approaches described in the literature, the following protocol is recommended for cloning and expressing recombinant E. faecalis SecA:

• Gene Engineering:

  • Clone the SecA gene from E. faecalis genomic DNA

  • Consider introducing a K6N mutation to reduce proteolytic sensitivity at the N-terminus (the lysine-lysine motif at positions 6-7 makes the wild-type protein highly susceptible to N-terminal proteolysis)

  • Add a C-terminal hexahistidine tag to facilitate purification

• Expression System:

  • Use E. coli as the host organism

  • Place the engineered gene under the control of a T5 promoter and lac operator

  • The construct should direct the synthesis of a protein of approximately 97 kDa

• Purification Strategy:

  • Implement immobilized metal affinity chromatography (IMAC) using the C-terminal His-tag

  • Include 10 mM DTT in buffers to prevent formation of dimers via intermolecular disulfide bridges

  • Verify protein integrity by mass spectrometry and/or Edman degradation to confirm intact N-terminus

• Quality Control:

  • Analyze quaternary structure by analytical ultracentrifugation and size-exclusion chromatography

  • Verify monomeric state (expected ~110 kDa) and morphology by electron microscopy (particles should be 5-6 nm in diameter)

What techniques can be used to study SecA localization in E. faecalis cells?

Several complementary techniques have proven effective for studying SecA localization in E. faecalis:

• Immunoelectron Microscopy (IEM):

  • Allows visualization of SecA in thin sections of E. faecalis

  • Bypasses barriers to antibody penetration presented by the cell wall and membrane

  • Provides high-resolution localization data at the nanometer scale

  • Enables colocalization studies with other proteins like sortase enzymes

• Immunofluorescence Microscopy (IFM):

  • Requires lysozyme-mediated cell-wall degradation and detergent-mediated membrane permeabilization

  • Use antibodies raised against E. faecalis SecA (α-SecA) or epitope-tagged versions

  • Classify cells into size stages to track localization throughout the cell cycle

  • Enables visualization of SecA distribution patterns in whole cells at different division stages

• Epitope Tagging:

  • Express SecA with human influenza HA tag

  • Verify that the tagged protein is fully functional

  • Allows tracking using commercially available antibodies

• Fluorescent Protein Fusions:

  • Generate SecA fusions with fluorescent proteins like GFP

  • Enable live-cell imaging of SecA dynamics

  • Consider potential functional impacts of the fusion on SecA activity and localization

These techniques can be combined with treatments, such as exposure to antimicrobial peptides, to study how different conditions affect SecA localization and function in E. faecalis.

What genetic approaches can be used to study SecA function in E. faecalis?

Several genetic approaches have been employed to study SecA function:

• Transposon Mutagenesis:

  • TnS insertions can be used to inactivate the secA gene

  • This approach allows for mapping of essential regions within the gene

  • Can help identify domains critical for function

• Specialized Transducing Phages:

  • Lambda phages carrying the secA gene or portions of it can be constructed

  • These phages can be used to complement secA mutations in conditional lethal strains

  • Enable genetic mapping and analysis of the secA locus

• Recombinant DNA Techniques:

  • Cloning of secA gene fragments into vectors

  • Analysis by restriction enzyme digestion to determine minimum gene requirements

  • For example, the E. coli secA gene was found to be encoded by portions of two EcoRI fragments of 2.8 and 0.8 kb in size

• Conditional Lethal Mutations:

  • Temperature-sensitive (Ts) mutations in secA

  • Allow for functional analysis under permissive and non-permissive conditions

  • The secA51(Ts) allele has been used as a standard secA mutant for complementation studies

• Dominance Testing:

  • Determination whether mutations are recessive or dominant to wild-type alleles

  • Provides insights into the nature of the mutation and its effect on protein function

  • For example, the secA5(Ts) mutation has been shown to be recessive to the wild-type allele

These genetic tools have been instrumental in identifying and characterizing the secA gene product as a 92-kilodalton polypeptide and understanding its essential role in protein secretion.

How can researchers differentiate between the effects of SecA inhibition on bacterial viability versus virulence?

To distinguish between impacts on viability and virulence, researchers should implement a multi-faceted experimental approach:

• Dose-Dependent Analysis:

  • Test inhibitors at a range of concentrations to identify:

    • Minimum inhibitory concentration (MIC) for growth inhibition

    • Sub-MIC concentrations that may affect virulence but not growth

  • Measure growth curves to detect subtle effects on growth rate versus complete inhibition

• Virulence Factor Secretion Assays:

  • Quantify secreted virulence factors in culture supernatants using:

    • ELISA for specific proteins

    • Proteomics analysis to identify global secretion changes

    • Activity assays for enzymatic virulence factors

  • Compare secretion profile changes at sub-MIC concentrations

• Functional Assays:

  • Evaluate bacterial adhesion to host cells or extracellular matrix components

  • Measure biofilm formation capacity

  • Assess invasion of host cells where applicable

  • Test resistance to host defense mechanisms

• Animal Models:

  • Compare infection outcomes with:

    • Untreated E. faecalis

    • E. faecalis treated with SecA inhibitors at sub-MIC concentrations

    • E. faecalis treated with SecA inhibitors at MIC or higher

  • Monitor bacterial burden, host response, and disease progression

• Genetic Controls:

  • Use conditional SecA mutants (if available) to correlate phenotypes

  • Compare SecA inhibitor effects with known antibiotics targeting different cellular processes

This comprehensive approach can help determine whether SecA inhibitors primarily kill bacteria or reduce their virulence, which has important implications for antimicrobial strategy development and potential resistance emergence.

What are the structural and mechanistic differences between E. faecalis SecA and Sec systems in other bacterial species?

E. faecalis SecA exhibits several notable differences compared to Sec systems in other bacteria:

• Quaternary Structure:

  • E. faecalis SecA exists predominantly as a monomer in solution

  • E. coli and B. subtilis SecA typically exist as dimers under similar conditions

  • The monomeric state of E. faecalis SecA is maintained even in the absence of detergents or high salt concentrations that might disrupt protein-protein interactions

• N-terminal Sensitivity:

  • E. faecalis SecA contains a lysine-lysine motif at positions 6-7 that renders it highly sensitive to N-terminal proteolysis

  • Researchers have found that replacing lysine 6 with asparagine (K6N mutation) improves stability, mimicking the sequence found in B. subtilis SecA

• SecA Homologues:

  • While most bacteria have only one SecA homologue, some Gram-positive pathogens have two SecA homologues (SecA1 and SecA2)

  • SecA1 is the conventional SecA, critical for secretion of many proteins with Sec-dependent signal peptides and essential for viability

  • SecA2 is less conserved, involved in secretion of specific virulence-related proteins, and generally not essential

  • The literature does not specifically mention whether E. faecalis has one or two SecA homologues

• Interaction with Antimicrobial Peptides:

  • E. faecalis SecA localization sites are targets for cationic antimicrobial peptides like human β-defensins

  • The multiple peptide resistance factor (MprF) protein in E. faecalis adds cationic residues to anionic lipids to protect against this targeting

Understanding these differences is crucial for developing species-specific SecA inhibitors and for predicting the broader applicability of such inhibitors across bacterial species.

How does SecA function differ between single-spanning and multi-spanning membrane proteins in Enterococcus?

Research has revealed interesting distinctions in how SecA mediates the assembly of different types of membrane proteins:

• Loop Size Dependency:

  • For multiple-spanning membrane proteins (like YidC), SecA dependency correlates with periplasmic loop size

  • When large periplasmic loops connecting transmembrane domains are reduced to less than 30 amino acids, multiple-spanning membrane proteins can become SecA-independent

• Single-Spanning Membrane Proteins:

  • Surprisingly, single-spanning membrane proteins maintain SecA-dependency regardless of periplasmic loop size

  • Even when periplasmic loops are reduced to as few as 13 amino acids, single-spanning membrane proteins still require SecA for complete assembly

• Coordination Mechanisms:

  • For membrane proteins with large periplasmic loops, the SecY translocon must coordinate:

    • Signal Recognition Particle (SRP)-dependent targeting and integration of transmembrane domains

    • SecA-dependent translocation of periplasmic loops

These findings challenge the conventional model that suggested ATP hydrolysis by SecA is required only for translocation of periplasmic loops larger than 30 amino acids. The maintained SecA-dependency of single-spanning membrane proteins with small periplasmic loops suggests that SecA may play additional roles in their assembly beyond simply providing energy for translocation of large periplasmic domains .

What approaches can be used to quantify SecA activity in experimental settings?

Researchers studying SecA can employ several quantitative approaches to measure its activity:

• ATPase Activity Assays:

  • Measure ATP hydrolysis rates using:

    • Colorimetric assays for phosphate release

    • Coupled-enzyme assays (e.g., pyruvate kinase/lactate dehydrogenase)

    • Radiometric assays with [γ-32P]ATP

  • Compare basal ATPase activity versus translocation-stimulated activity in the presence of:

    • Model preproteins

    • SecYEG proteoliposomes

    • Lipid membranes

• Real-Time Translocation Assays:

  • Monitor movement of fluorescently labeled preproteins across membranes

  • Measure protection of translocated domains from external proteases

  • Track accessibility of reporter domains during translocation

• Binding Affinity Measurements:

  • Determine SecA interactions with:

    • Nucleotides (ATP, ADP) using isothermal titration calorimetry

    • Preproteins using fluorescence anisotropy

    • SecYEG using surface plasmon resonance

  • Calculate dissociation constants (Kd) for each interaction

• Conformational Change Analysis:

  • Monitor SecA structural changes during the ATPase cycle using:

    • Intrinsic tryptophan fluorescence

    • FRET with strategically placed fluorophores

    • Limited proteolysis to detect exposed domains

  • Correlate conformational changes with different stages of the translocation cycle

• qPCR-Based Detection Methods:

  • While not directly measuring SecA activity, quantitative PCR techniques similar to those used for Enterococcus detection might be adapted to:

    • Measure expression levels of SecA

    • Quantify translocation of SecA-dependent substrates

These quantitative approaches provide complementary information about different aspects of SecA function and can be combined to gain comprehensive insights into the mechanism of SecA-mediated protein translocation.

What are the critical parameters for successful crystallization of E. faecalis SecA?

Based on published crystallization studies, the following parameters are critical for successful crystallization of E. faecalis SecA:

• Protein Engineering:

  • Introduce the K6N mutation to prevent N-terminal proteolysis

  • Add a C-terminal hexahistidine tag for purification

  • Verify protein integrity by mass spectrometry and/or Edman degradation

• Purification Quality:

  • Ensure high purity (>95% homogeneity) as assessed by SDS-PAGE

  • Verify monomeric state by analytical ultracentrifugation and size-exclusion chromatography

  • Include 10 mM DTT in buffers to prevent formation of intermolecular disulfide bridges that could lead to unwanted dimerization

• Crystallization Conditions:

  • Successful crystals were obtained using the vapor-diffusion technique

  • Crystal formation yielded monoclinic crystals belonging to space group C2

  • Diffraction quality extends to 2.4 Å resolution

• Derivative Preparation:

  • For phase determination, selenomethionine derivatives may be prepared

  • This involves expressing the protein in minimal media with selenomethionine substituting for methionine

• Crystal Parameters:

  • Unit-cell parameters:

    • a = 203.4 Å

    • b = 49.8 Å

    • c = 100.8 Å

    • α = γ = 90.0°

    • β = 119.1°

These parameters provide important guidelines for researchers attempting to reproduce or extend crystallographic studies of E. faecalis SecA, which is essential for structure-based drug design efforts targeting this protein.

How does the ATP hydrolysis cycle of SecA correlate with different stages of protein translocation?

The ATP hydrolysis cycle of SecA is intricately coordinated with the protein translocation process, following specific stages:

• Initiation Phase:

  • SecA binds to the SecYEG channel and the signal sequence of the preprotein

  • ATP binding to SecA induces conformational changes that:

    • Open the SecY channel

    • Allow insertion of the signal sequence and early mature regions of the preprotein

  • This initiates the translocation process

• Translocation Cycle:

  • In the ATP-bound state:

    • The Two-Helix Finger (THF) of SecA makes direct contacts with the polypeptide substrate

    • SecA pushes the polypeptide toward and through the channel

    • The SecY channel is in an open conformation

  • After ATP hydrolysis:

    • The THF retracts

    • The clamp domain of SecA holds the polypeptide chain tightly

    • This prevents backward movement of the substrate

    • The SecY channel may adopt a more closed conformation

  • Upon ADP release and ATP rebinding:

    • SecA resets for the next cycle

    • The process repeats, progressively moving the polypeptide through the channel

• Mechanistic Models:

  • The "Push-Slide Model" proposes that SecA actively moves the polypeptide in discrete steps coupled to ATP hydrolysis

  • Recent structural evidence indicates SecA moves protein substrates using a mechanism strikingly similar to how helicases move DNA/RNA substrates

• Termination:

  • Once translocation is complete, SecA dissociates from the SecYEG complex

  • The channel returns to its resting state

Understanding this correlation between ATP hydrolysis and translocation stages is crucial for designing inhibitors that target specific states of the SecA cycle, potentially leading to more effective antimicrobial agents.

How might structural knowledge of E. faecalis SecA inform drug design strategies?

Structural insights into E. faecalis SecA provide several promising avenues for rational drug design:

• Exploiting Monomeric Nature:

  • Unlike E. coli and B. subtilis SecA that function as dimers, E. faecalis SecA exists predominantly as a monomer

  • This unique quaternary structure could be exploited to design inhibitors specific to E. faecalis SecA

  • Targeting interfaces that prevent dimerization or stabilize the monomeric form could provide selectivity

• ATP Binding and Hydrolysis Domains:

  • The ATPase activity of SecA is essential for its function

  • Structural knowledge of the nucleotide-binding domains could guide design of:

    • Competitive ATP analogs

    • Allosteric inhibitors that prevent conformational changes required for ATP hydrolysis

    • Compounds that lock SecA in specific conformational states

• Interfacial Regions:

  • Focus on the interface between SecA and the SecYEG translocation channel

  • Target the regions of SecA that interact with preprotein substrates

  • Design inhibitors that disrupt these essential protein-protein interactions

• Accessible Periplasmic Domains:

  • Evidence suggests that some SecA bound at SecYEG is accessible from the periplasm

  • This accessibility could be exploited to design inhibitors that target SecA from the extracellular space

  • Such an approach might bypass the need for compounds to penetrate the cytoplasmic membrane

• Species-Specific Features:

  • The K6N mutation introduced to stabilize E. faecalis SecA highlights a region of proteolytic sensitivity

  • This region and other species-specific features could be targeted for selective inhibition

  • Compare with SecA structures from other pathogens to identify unique binding pockets

High-resolution crystal structures of E. faecalis SecA in different conformational states (e.g., with ATP, ADP, or transitional analogs) would further enhance structure-based drug design efforts against this promising antimicrobial target.

What is known about resistance mechanisms against SecA inhibitors?

While SecA inhibitors are still in developmental stages rather than clinical use, several potential resistance mechanisms can be predicted based on current knowledge:

Understanding these potential resistance mechanisms is crucial for developing SecA inhibitor strategies that maintain long-term efficacy, such as combination therapies or inhibitors designed to maintain activity against likely resistant variants.

How might the localization of SecA in E. faecalis be exploited for targeted antimicrobial delivery?

The distinct localization pattern of SecA in E. faecalis provides innovative opportunities for targeted antimicrobial delivery:

• Septal-Targeting Delivery Systems:

  • SecA predominantly localizes to the equatorial mid-cell and septum during cell division

  • Nanoparticles or delivery vehicles that preferentially accumulate at bacterial division septa could concentrate SecA inhibitors at their site of action

  • This approach mimics the natural targeting of antimicrobial peptides like β-defensins to these same regions

• Charge-Based Targeting:

  • Human β-defensins interact with E. faecalis at discrete septal foci, coinciding with SecA localization

  • Cationic peptides or polymers could serve as delivery vehicles for SecA inhibitors

  • The bacterial defense mechanism of adding cationic residues to anionic lipids (via MprF) suggests that anionic targeting might bypass this resistance mechanism

• Dual-Action Conjugates:

  • Development of molecules combining:

    • A septal-targeting moiety (derived from antimicrobial peptides)

    • A SecA inhibitor payload

  • Such conjugates could enhance local inhibitor concentration while potentially disrupting both membrane integrity and protein secretion

• Sortase-SecA Co-targeting:

  • Since SecA colocalizes with sortase enzymes, dual inhibitors targeting both systems could provide synergistic effects

  • This approach would simultaneously disrupt protein secretion and cell wall attachment of virulence factors

• Cell Cycle-Dependent Delivery:

  • SecA localization changes throughout the cell cycle, appearing as single foci at early division stages and in multi-focal patterns at late division stages

  • Time-release formulations could optimize inhibitor exposure during periods of maximal SecA activity and accessibility