Recombinant ESX-1 secretion-associated protein EspE (espE)

What is the ESX-1 secretion system and what is its significance?

The ESX-1 (ESAT-6 system 1) is a specialized secretion system found in pathogenic mycobacteria including Mycobacterium tuberculosis and Mycobacterium marinum. It represents a critical virulence mechanism encoded by the extended region of difference 1 (extRD1) locus in these organisms. The ESX-1 secretion apparatus is required for mycobacterial virulence and the secretion of multiple effector proteins that collectively enable the bacteria to control and exploit host cells as replication-permissive niches . This secretion system facilitates the delivery of these effector proteins across the complex mycobacterial cell envelope, allowing them to interact with host components and modify the host environment to benefit bacterial survival and replication .

What is EspE and what role does it play in mycobacterial pathogenesis?

EspE (ESX-1 secretion-associated protein E) is a substrate protein of the ESX-1 secretion system in pathogenic mycobacteria. It belongs to "group III" of the ESX-1 substrates and works closely with another substrate, EspF. EspE and EspF are required for mycobacterial virulence and may be responsible for phagosomal lysis, a critical step in mycobacterial pathogenesis that allows bacteria to escape from the phagosome into the cytosol . Additionally, EspE and EspF function as negative regulators of ESX-1 substrate gene expression, forming part of a feedback mechanism that controls the secretion of other ESX-1 substrates . This dual function as both effector proteins and regulators makes them particularly significant in mycobacterial virulence strategies.

How is EspE secreted through the ESX-1 system?

EspE secretion through the ESX-1 system follows a hierarchical process and depends on specific protein-protein interactions. Critically, EspE requires EspF for secretion, as they form a protein complex . The secretion mechanism involves:

• EspE and EspF interaction through specific residues that form salt bridges and hydrophobic interactions between their α-helices, creating a helical bundle .

• EspF interaction with EsxA (another ESX-1 substrate), which may facilitate targeting or transport of the EspE/EspF complex .

• Dependence on "group I" substrates (including EsxA, EsxB, PPE68, and MMAR_2894) and "group II" substrates (EspB, EspJ, and EspK) for proper secretion .

The hierarchical nature of ESX-1 secretion means that EspE and EspF (group III substrates) are secreted after the group I and group II substrates, highlighting a sequential and coordinated process of protein export .

What structural features enable EspE to interact with other ESX-1 substrates?

EspE contains several structural features that facilitate its interaction with other ESX-1 substrates, particularly EspF:

• N-terminal domain: The N-terminal half of EspE contains α-helices that mediate protein-protein interaction with EspF, forming a helical bundle .

• Specific interaction sites: Critical residues within these α-helices form:

  • Salt bridges (e.g., involving D83 in EspE and R62 in EspF)

  • Hydrophobic core interactions that stabilize the EspE-EspF complex

• C-terminal domain: EspE has a largely disordered C-terminal half containing several predicted α-helical regions. Interestingly, this region may overlay the EspF-binding site, potentially regulating EspF interaction . Different structural prediction methods (AlphaFold2 and RoseTTAFold) provide varying models of how this C-terminal domain interacts with the N-terminal domain, with RoseTTAFold suggesting the C-terminal α-helices may overlay the EspF-binding site .

These structural features enable EspE to form specific interactions required for both its secretion and function in the ESX-1 system.

How do mutations in EspE affect its secretion and function?

Mutations in specific residues of EspE significantly impact its secretion and function in distinct ways:

These mutation studies provide valuable insights into structure-function relationships and could guide the development of strategies to interfere with EspE function for therapeutic purposes.

What experimental approaches can be used to study EspE-EspF interactions?

Several experimental approaches can be employed to study the interactions between EspE and EspF:

• Structural modeling and prediction:

  • Computational modeling using tools like AlphaFold2 and RoseTTAFold to predict protein structures and interactions .

  • These predictions can guide the identification of key residues involved in protein-protein interactions.

• Site-directed mutagenesis (SDM):

  • Generate point mutations in specific residues predicted to be involved in interactions .

  • For example, disrupting predicted salt bridges (EspED83R and EspFR62D) to test their importance.

• Complementation studies:

  • Create unmarked deletion strains (ΔespE or ΔespF) .

  • Complement these strains with wild-type or mutant versions of the genes on integrating plasmids .

  • This approach allows testing of how specific mutations affect function in vivo.

• Protein expression and detection:

  • Western blot analysis to detect protein production and secretion .

  • Use of appropriate controls:

    • RpoB (β subunit of RNA polymerase) as a loading control for cell-associated fractions and a lysis control for secreted fractions .

    • MPT-32 (a protein secreted independently of ESX-1) as a loading control for both cell-associated and secreted fractions .

• Transcript analysis:

  • Reverse transcriptase quantitative PCR (RT-qPCR) to verify transcription of genes in different strains .

  • This helps distinguish between effects on transcription versus post-transcriptional processes.

• Functional assays:

  • Hemolytic activity assays to assess the functional consequences of mutations .

  • This correlates with the ability to cause phagosomal lysis during infection.

These methodologies provide complementary approaches to understand both the structural basis and functional significance of EspE-EspF interactions.

How can recombinant EspE protein be produced and purified for in vitro studies?

While the search results do not provide specific protocols for EspE recombinant production, a methodological approach based on standard protein expression and purification techniques, combined with insights from the provided research, would include:

• Gene cloning and construct design:

  • Clone the espE gene into an appropriate expression vector.

  • Consider including a fusion tag (His-tag, GST-tag, or MBP-tag) to facilitate purification.

  • When designing constructs, consider the structural information available:

    • Full-length constructs may present challenges due to the disordered C-terminal region .

    • N-terminal domain constructs might be more stable for structural studies.

• Expression system selection:

  • E. coli-based expression systems are commonly used for mycobacterial proteins.

  • For proteins that form complexes, consider co-expression with partners (e.g., EspF).

  • Alternative systems like mycobacterial expression hosts might provide better folding for native conformation.

• Optimization of expression conditions:

  • Test different growth temperatures, induction conditions, and media formulations.

  • For mycobacterial proteins, lower temperatures (16-20°C) often improve solubility.

• Purification strategy:

  • Affinity chromatography based on the fusion tag.

  • Ion exchange chromatography, exploiting EspE's charge properties.

  • Size exclusion chromatography for final polishing and to assess oligomeric state.

• Protein complex formation:

  • For studying EspE-EspF interactions, consider:

    • Co-expression and co-purification approaches.

    • In vitro reconstitution of complexes from individually purified components.

    • Validation of complex formation using techniques like size exclusion chromatography, native PAGE, or analytical ultracentrifugation.

• Quality control:

  • Circular dichroism spectroscopy to assess secondary structure content.

  • Dynamic light scattering to evaluate sample homogeneity.

  • Limited proteolysis to identify stable domains.

• Functional validation:

  • Develop binding assays to verify interactions with partners like EspF.

  • Consider in vitro assays that might reflect EspE's role in pore formation or membrane interactions.

This methodological framework would need to be optimized based on specific research goals and the characteristics of EspE observed during expression trials.

How does EspE fit into the hierarchical secretion model of ESX-1?

EspE occupies a specific position within the hierarchical secretion model of ESX-1, with important implications for understanding mycobacterial virulence regulation:

• Hierarchical classification:

  • EspE and EspF are classified as "group III" substrates in the ESX-1 secretion system .

  • This classification reflects both the order of secretion and the dependencies between different substrate groups.

• Secretion dependencies:

  • Group I substrates (EsxA, EsxB, PPE68, and MMAR_2894) are secreted first and are required for the secretion of all other substrates .

  • Group II substrates (EspB, EspJ, and EspK) require group I substrates for secretion .

  • Group III substrates (EspE and EspF) require both group I and group II substrates for their secretion .

• Internal hierarchy within group II:

  • Within group II, there is a further hierarchy: EspJ is required for EspK secretion, and EspK is required for EspB secretion .

  • This suggests a sequential assembly or transport process.

• Regulatory feedback loop:

  • A critical aspect of EspE and EspF's position in the hierarchy is their regulatory function:

  • EspE and EspF negatively regulate ESX-1 substrate gene expression .

  • When espE or espF genes are deleted, there is increased secretion of group I and group II substrates .

  • This creates a feedback mechanism where the last substrates to be secreted regulate the expression of earlier substrates.

• Comparative regulatory systems:

  • This regulatory arrangement has parallels in other bacterial secretion systems, such as type III secretion systems, where similar increases in substrate secretion following the loss of regulatory substrates have been reported .

This hierarchical model helps explain how mycobacteria coordinate the complex process of ESX-1 secretion, ensuring that substrates are secreted in the correct order and proportions for optimal virulence.

What is known about the interaction between EspE, EspF, and the EsxA/EsxB heterodimer?

The interaction between EspE, EspF, and the EsxA/EsxB heterodimer represents a complex network of protein-protein interactions that facilitate ESX-1 secretion:

• EspF-EsxA interaction:

  • The interaction between EspF and EsxA is a key molecular bridge connecting the EspE/EspF pair to the EsxA/EsxB heterodimer .

  • This interaction is mediated primarily through:

    • The flexible N-termini of EspF and EsxA, which are predicted to form short, six-residue β-strands, resulting in a two-stranded, parallel, β-sheet between the two proteins .

    • Additional side chain interactions between the C-terminal α-helices of EspF and EsxA .

• Functional significance of these interactions:

  • The EspF-EsxA interaction appears to facilitate either targeting or transport of the EspE/EspF substrate pair to the extracellular environment .

  • This interaction provides a direct connection between the EspE/EspF complex and the core secretion machinery that interacts with EsxA/EsxB.

• Role of EsxB in the interaction network:

  • While EspF interacts with EsxA, the flexible C-terminus of EsxB remains free to interact with the EccCb1 ATPase .

  • This arrangement allows the EsxA/EsxB heterodimer to both interact with the secretion machinery (via EsxB) and with the EspE/EspF complex (via EsxA).

• Model for sequential interactions:

  • A proposed model suggests that EspE and EspF first interact with each other, which is required for their secretion .

  • The EspE/EspF complex then interacts with the EsxA/EsxB heterodimer via EspF-EsxA interaction .

  • This facilitates the targeting or transport of EspE/EspF through the ESX-1 machinery .

• Additional interaction possibilities:

  • It is likely that EspF or EspE also interact with other substrates, including the group II substrates (EspB, EspJ, or EspK) .

  • These additional interactions may further stabilize the secretion complex or regulate the order of substrate secretion.

This intricate network of interactions ensures the coordinated secretion of multiple ESX-1 substrates, which collectively contribute to mycobacterial virulence.

How do EspE and EspF regulate ESX-1 gene expression and substrate secretion?

EspE and EspF exert important regulatory functions on ESX-1 gene expression and secretion, operating through mechanisms that are distinct from their role in secretion:

• Negative regulation of ESX-1 substrate expression:

  • EspE and EspF negatively regulate the expression of genes encoding ESX-1 substrates .

  • When espE or espF genes are deleted, there is increased secretion of group I and group II substrates compared to wild-type strains .

• Separation of regulatory and secretory functions:

  • A key finding is that the protein-protein interactions required for EspE and EspF secretion (such as the salt bridge between EspE and EspF) are dispensable for their regulatory functions .

  • This suggests that different domains or mechanisms are involved in these two distinct functions.

• Regulatory mechanism:

  • While the exact mechanism of regulation is not fully detailed in the search results, it appears to involve feedback control:

    • As group III substrates that are secreted after groups I and II, EspE and EspF can modulate the expression of earlier substrates.

    • This creates a regulatory circuit that could help balance the relative amounts of different substrates.

• Parallel regulatory systems:

  • This regulatory arrangement has parallels in other bacterial secretion systems, such as type III secretion systems, where similar regulatory mechanisms have been documented .

• Experimental evidence:

  • Deletion mutants of espE or espF show increased secretion of other ESX-1 substrates .

  • Even when EspE and EspF mutations prevent their secretion, they retain their regulatory functions .

  • This suggests that the regulatory activity occurs inside the bacterial cell and doesn't require secretion of these proteins.

This regulatory function adds another layer of complexity to the ESX-1 system, highlighting how mycobacteria finely tune their virulence mechanisms through sophisticated feedback controls.

How might targeting EspE function lead to novel anti-tuberculosis therapeutic strategies?

Targeting EspE function presents several promising avenues for novel anti-tuberculosis therapeutic development:

• Disruption of ESX-1 secretion:

  • Inhibiting EspE function could disrupt the ESX-1 secretion system, which is essential for M. tuberculosis virulence .

  • Potential approaches include:

    • Small molecule inhibitors targeting the EspE-EspF interaction interface, particularly the salt bridge and hydrophobic core regions identified in structural studies .

    • Peptide mimetics that compete with natural binding partners.

• Interference with regulatory functions:

  • EspE and EspF negatively regulate ESX-1 substrate gene expression .

  • Therapeutics that enhance this negative regulation could potentially decrease virulence factor secretion.

  • Alternatively, compounds that target the specific regulatory domains or mechanisms of EspE/EspF could disrupt the balanced expression of virulence factors.

• Inhibition of phagosomal escape:

  • EspE and EspF are implicated in phagosomal lysis , a critical step in M. tuberculosis pathogenesis.

  • Preventing this function could trap bacteria within phagosomes, making them more vulnerable to host defense mechanisms.

  • This approach might be particularly effective in combination with drugs that enhance phagosomal maturation or acidification.

• Vaccine development:

  • Understanding EspE structure and function could inform the development of attenuated strains for vaccine candidates.

  • Recombinant EspE protein or engineered variants might serve as subunit vaccine components that stimulate protective immunity.

• Diagnostic applications:

  • Detection of EspE or antibodies against it might serve as biomarkers for active TB infection.

  • This could potentially help distinguish between latent and active infection states.

Future research should focus on resolving the complete structure of EspE, including its C-terminal domain, developing high-throughput screens for inhibitors of EspE-EspF interaction, and validating these approaches in animal models of tuberculosis infection.

What are the key unresolved questions about EspE function and regulation?

Several critical questions about EspE function and regulation remain unresolved and represent important areas for future research:

• Complete structural characterization:

  • The full structure of EspE, particularly the largely disordered C-terminal half, remains poorly understood .

  • Different structural prediction methods give conflicting results for how the C-terminal domain interacts with the N-terminal domain .

  • Resolving this structure would provide insights into both secretory and regulatory functions.

• Precise regulatory mechanism:

  • While it's established that EspE and EspF negatively regulate ESX-1 substrate gene expression , the exact molecular mechanisms remain unclear.

  • How do these proteins influence gene expression? Do they interact with transcription factors, affect mRNA stability, or operate through other mechanisms?

  • What domains or specific residues are responsible for the regulatory function?

• Transport mechanism through the cell envelope:

  • The exact path and mechanism by which EspE traverses the complex mycobacterial cell envelope remains incompletely understood .

  • Does EspE interact with other components of the cell wall during secretion?

  • What conformational changes might occur during the secretion process?

• Role in phagosomal lysis:

  • While EspE and EspF are implicated in phagosomal lysis , their exact mechanism of action remains unclear.

  • Do they form pores, disrupt membrane integrity through enzymatic activity, or recruit other factors?

  • How do they cooperate with other ESX-1 substrates in this process?

• Integration with other virulence mechanisms:

  • How does EspE function coordinate with other mycobacterial virulence factors?

  • Is there cross-talk between the ESX-1 system and other secretion systems or virulence mechanisms?

• Potential as biomarkers:

  • Could EspE or antibodies against it serve as biomarkers for TB infection or disease progression?

  • How does EspE expression or secretion correlate with different phases of infection?

• Host recognition and response:

  • How do host cells recognize and respond to EspE?

  • Does EspE interact with specific host receptors or cellular components?

  • What immune responses are elicited by EspE, and how might these be harnessed for vaccine development?

Addressing these questions will require integrated approaches combining structural biology, genetics, biochemistry, cell biology, and immunology to fully understand the complex roles of EspE in mycobacterial pathogenesis.