Recombinant ESX-1 secretion system protein EccE1 (eccE1)

What is the basic function of EccE1 in the ESX-1 secretion system?

EccE1 is a critical membrane component of the ESX-1 secretion apparatus in Mycobacterium tuberculosis. It forms part of the membrane complex that creates a channel in the cytoplasmic membrane, alongside four other membrane proteins: EccB1, EccCa1, EccCb1, and EccD1. Research has demonstrated that EccE1 is essential for the secretion of key virulence factors, including EsxA, EsxB, EspA, and EspC . Deletion of eccE1 abolishes ESX-1 secretion and attenuates M. tuberculosis ex vivo, highlighting its fundamental importance to mycobacterial pathogenesis . Notably, EccE1 is required for maintaining proper levels of other ESX-1 membrane proteins, as deletion of eccE1 lowers the levels of EccB1, EccCa1, and EccD1 through post-transcriptional mechanisms .

How can researchers confirm the successful deletion of eccE1 in experimental models?

Confirming successful deletion of eccE1 requires multiple validation approaches:

• Genetic confirmation: Whole genome sequencing can verify the complete deletion of the eccE1 coding region from the chromosome . PCR amplification with primers flanking the deletion site can also confirm the gene's absence.

• Protein expression analysis: Immunoblotting of cell lysates using antibodies against EccE1 should show absence of the protein in deletion mutants .

• Functional validation: Measuring the secretion of ESX-1 substrates (EsxA and EsxB) in culture filtrates, which should be abolished in ΔeccE1 mutants .

• Phenotypic confirmation: Testing for attenuated virulence using macrophage lysis assays - ΔeccE1 mutants should show significantly reduced ability to lyse infected macrophages compared to wild-type strains .

What methods are effective for studying EccE1 localization in M. tuberculosis?

Several effective methodological approaches for studying EccE1 localization include:

• Epitope tagging: Inserting a hemagglutinin (HA) tag in the C-terminal part of EccE1 by genetic engineering allows visualization using anti-HA antibodies. This approach has been successfully applied to other ESX-1-related proteins .

• Subcellular fractionation: This technique separates the cytosolic, membrane, cell wall, capsular, and secreted fractions, followed by immunoblotting with appropriate antibodies to determine EccE1 localization . Studies have shown that EccE1 is detected predominantly in the membrane and cell wall fractions, confirming its membrane-associated nature .

• Fluorescent protein fusion: Fluorescent-fusion proteins have been successfully employed to study ESX-1-related proteins in mycobacterial species, including M. smegmatis . This approach can reveal the polar localization of EccE1.

• Immunoelectron microscopy: For high-resolution visualization of EccE1 localization within the cell envelope structure.

What is the hierarchy of ESX-1 secretion system components and where does EccE1 fit?

Research has revealed a clear hierarchical organization within the ESX-1 secretion system components. Based on proteomic analyses of various ESX-1 component deletion mutants, proteins can be categorized into distinct functional groups:

Table 1: ESX-1 Component Hierarchy

EccE1 functions as a critical membrane component of the core apparatus. Studies show that while EccE1 is essential for the secretion of EsxA, EsxB, EspA, and EspC, interestingly, it is dispensable for the secretion of EspB . This selective requirement places EccE1 in a unique position within the hierarchy, suggesting it may function at a specific stage of the secretion process rather than being universally required for all substrates .

How does the polar localization of EccE1 contribute to ESX-1 function?

The polar localization of EccE1 appears to be crucial for proper ESX-1 function through several mechanisms:

• Focused secretion: Polar localization of the ESX-1 secretion system allows for directed secretion of virulence factors at specific cellular locations, potentially optimizing host-pathogen interactions during infection .

• Assembly of the ESX-1 complex: Research demonstrates that EccE1 requires other ESX-1 components to assemble into a stable complex at the poles of M. tuberculosis . This interdependency suggests that polar localization facilitates proper complex formation.

• Membrane protein stability: EccE1 contributes to the stability of other ESX-1 membrane components (EccB1, EccCa1, and EccD1) . This stabilizing function may be optimized at the cell poles where membrane properties differ from the lateral regions.

• Coordination with cell division: Polar localization may ensure proper distribution of the secretion system during cell division, maintaining virulence capabilities in daughter cells.

Fluorescence microscopy studies have confirmed that EccE1 and other ESX-1 components localize to the cell poles in mycobacteria, and disruption of this localization pattern correlates with decreased ESX-1 function .

What explains the differential requirement of EccE1 for secretion of various ESX-1 substrates?

The differential requirement of EccE1 for secretion of various ESX-1 substrates represents an intriguing research area. Most notably, while EccE1 is essential for secretion of EsxA, EsxB, EspA, and EspC, the secretion of EspB is independent of EccE1 . Several hypotheses explain this phenomenon:

• Multiple secretion pathways: EspB may utilize an alternative secretion pathway or component that bypasses the need for EccE1. Proteomic data showing that EspB secretion patterns differ from other ESX-1 substrates supports this hypothesis .

• Substrate recognition differences: EccE1 may be involved in recognizing specific secretion signals present in some substrates (EsxA, EsxB, EspA, EspC) but absent in EspB.

• Complex formation requirements: EspB interacts with EspK and forms heptameric rings after processing by MycP1 protease . This unique processing and complex formation might enable EspB to bypass the need for EccE1.

• Structural roles: EccE1 may provide structural support for the secretion of certain substrates but not others, depending on their size, shape, or interaction partners.

Quantitative proteomic analysis of secreted proteins from wild-type and ΔeccE1 mutants shows:

Table 2: Fold Change in Protein Secretion in ΔeccE1 vs Wild-type

This differential pattern strongly suggests multiple secretion mechanisms within the ESX-1 system .

What molecular mechanisms explain how EccE1 affects the stability of other ESX-1 membrane proteins?

EccE1 has been shown to affect the stability of other ESX-1 membrane proteins, specifically EccB1, EccCa1, and EccD1, through post-transcriptional mechanisms . Several molecular explanations have been proposed:

• Direct protein-protein interactions: EccE1 likely forms direct contacts with EccB1, EccCa1, and EccD1, stabilizing the complex structure. Without these interactions, the proteins may be more susceptible to proteolytic degradation.

• Membrane complex formation: Proteomic analysis reveals that in the absence of EccE1, the levels of EccB1, EccCa1, and EccD1 are reduced despite no changes in transcript levels . This suggests EccE1 is critical for proper assembly of the membrane complex, with improper assembly leading to protein degradation.

• Protection from proteases: EccE1 may shield certain domains of other ESX-1 membrane proteins from mycobacterial proteases, extending their half-life.

• Conformational stabilization: EccE1 could induce specific conformational changes in partner proteins that increase their stability in the membrane environment.

Research using quantitative proteomics demonstrated that deletion of eccE1 caused significant reductions in EccB1, EccCa1, and EccD1 levels (Figure 3A and Table S6 in source material) , while RNA-seq data showed no deregulation of the corresponding transcripts (Table S1) . This clearly indicates that EccE1's effect on these proteins occurs post-transcriptionally, most likely through direct protein-protein interactions within the membrane complex.

What experimental approaches can be used to study the interaction between EccE1 and other ESX-1 components?

Several sophisticated experimental approaches can effectively probe the interactions between EccE1 and other ESX-1 components:

• Co-immunoprecipitation (Co-IP) with tagged proteins: Using epitope-tagged EccE1 (such as EccE1-HA) to pull down interacting proteins, followed by mass spectrometry analysis or western blotting for specific ESX-1 components .

• Bacterial two-hybrid assays: Testing direct protein-protein interactions between EccE1 and other ESX-1 components in a heterologous system.

• Chemical cross-linking coupled with mass spectrometry: Identifying proteins in close proximity to EccE1 within intact cells by using membrane-permeable cross-linkers followed by affinity purification and MS analysis.

• Fluorescence resonance energy transfer (FRET): Using fluorescently labeled proteins to detect close interactions between EccE1 and other ESX-1 components within living mycobacterial cells.

• Cryo-electron microscopy: Visualizing the entire ESX-1 complex structure and determining the position and interactions of EccE1 within this complex.

• Genetic suppressor screens: Identifying mutations in other genes that can suppress phenotypes associated with eccE1 deletion, potentially revealing functional interactions.

• Protein stability assays: Comparing the half-lives of ESX-1 components in wild-type versus ΔeccE1 strains to quantify the stabilizing effect of EccE1 on each protein.

Research has shown that EccE1 requires other ESX-1 components to assemble into a stable complex at the poles of M. tuberculosis , making these interaction studies particularly important for understanding ESX-1 assembly and function.

What are the critical considerations when designing recombinant EccE1 constructs for experimental studies?

When designing recombinant EccE1 constructs for experimental studies, researchers should consider several critical factors:

• Epitope tag position: The placement of tags is crucial as improper positioning can disrupt protein function. Research demonstrates that C-terminal HA-tagging of EccE1 preserves protein function, as evidenced by the HA-tagged complemented mutant being as cytolytic as the wild-type strain in THP-1 infection models .

• Expression level control: Using appropriate promoters is essential as both overexpression and insufficient expression can lead to artifacts. Studies have successfully used the PTR promoter for regulated expression of EccE1 .

• Membrane protein considerations: As EccE1 is a membrane protein found in the membrane and cell wall fractions , constructs must maintain proper membrane targeting signals and transmembrane domains.

• Functional validation: Any recombinant EccE1 construct should be validated for functionality by complementation of ΔeccE1 mutants and restoration of ESX-1-dependent phenotypes such as EsxA/B secretion and macrophage cytolysis .

• Potential for multimerization: Consider whether EccE1 forms homo-oligomers or requires co-expression with other ESX-1 components for proper folding and function.

• Purification strategy: If purifying the protein, include appropriate affinity tags and consider detergent selection for membrane protein solubilization.

• Species-specific considerations: Sequence variations between mycobacterial species may affect EccE1 function in heterologous expression systems.

How can researchers effectively measure EccE1-dependent ESX-1 secretion in experimental settings?

Effectively measuring EccE1-dependent ESX-1 secretion requires multifaceted approaches:

• Immunoblotting of culture filtrates: Detection of known ESX-1 substrates (EsxA, EsxB, EspA, EspC) in culture filtrates using specific antibodies. This technique has clearly demonstrated that deletion of eccE1 abolishes secretion of these proteins .

• Quantitative proteomics: Label-free or isotope-labeled proteomic analysis of culture filtrates from wild-type and ΔeccE1 strains provides comprehensive identification of all differentially secreted proteins. Studies have used this approach to show that only secretion of EsxA, EsxB, EspA, and EspC (but not EspB) is dependent on EccE1 .

• Functional readouts:

  • Macrophage cytolysis assays: ESX-1-dependent lysis of infected macrophages correlates with functional secretion .

  • Phagosomal rupture assays: Using fluorescent reporters to measure ESX-1-dependent permeabilization of the phagosomal membrane.

• Controls and validations:

  • Include secretion system-independent protein controls (e.g., Ag85B) to verify specific effects on ESX-1 secretion .

  • Use complemented strains to confirm that observed secretion defects are specifically due to EccE1 absence .

  • Compare with other ESX-1 mutants (e.g., ΔmycP1) to distinguish EccE1-specific effects from general ESX-1 defects .

• Time-course experiments: Measure secretion at different time points to capture potential kinetic effects rather than end-point measurements alone.

What are the most reliable animal models for studying EccE1 function in tuberculosis pathogenesis?

The selection of appropriate animal models for studying EccE1 function in tuberculosis pathogenesis should be carefully considered:

Table 3: Animal Models for Studying EccE1 Function

Currently, ex vivo models using human macrophages (particularly THP-1 cells) have been successfully used to demonstrate that EccE1 is essential for macrophage lysis, a function required for M. tuberculosis pathogenesis . These models provide valuable initial insights before progressing to more complex animal models.

For comprehensive understanding of EccE1's role in pathogenesis, a combination of these models is recommended, starting with ex vivo cellular models and progressing to appropriate animal models based on specific research questions.

How might targeting EccE1 contribute to novel anti-tuberculosis therapeutic strategies?

Targeting EccE1 represents a promising avenue for novel anti-tuberculosis therapeutic strategies, supported by several lines of evidence:

• Essential virulence factor: Research clearly demonstrates that EccE1 is essential for ESX-1 function, which is the major virulence determinant of M. tuberculosis . Deletion of eccE1 leads to complete attenuation ex vivo , suggesting that inhibiting EccE1 could effectively reduce virulence.

• Specific targeting opportunities: As a membrane protein with unique localization patterns and protein-protein interactions , EccE1 offers specific structural features that could be targeted by small molecule inhibitors or antibodies.

• Potential therapeutic approaches:

  • Small molecule inhibitors that disrupt EccE1's interaction with other ESX-1 components

  • Peptidomimetics that interfere with complex assembly at the poles

  • Compounds that alter EccE1 stability or membrane localization

  • CRISPR-based therapeutics targeting eccE1 expression

• Advantages over traditional antibiotics: Targeting virulence rather than essential cellular functions may impose less selective pressure for resistance development while still effectively neutralizing the pathogen's ability to cause disease.

• Potential for combination therapy: EccE1 inhibitors could be used in combination with traditional antibiotics to enhance treatment efficacy and potentially shorten treatment duration.

Research indicates that EccE1 has a unique role in stabilizing other ESX-1 components , suggesting that destabilizing these interactions could have cascading effects on the entire secretion system and significantly impact virulence.

How does the structure-function relationship of EccE1 inform our understanding of ESX secretion systems across mycobacterial species?

The structure-function relationship of EccE1 provides critical insights into ESX secretion systems across mycobacterial species:

• Evolutionary conservation: Comparative analysis of EccE homologs across different mycobacterial ESX systems (ESX-1 through ESX-5) reveals conserved domains that likely perform similar functions in each system, as well as system-specific regions that may confer specialized functions.

• Domain organization insights: EccE1 is a membrane- and cell wall-associated protein with predicted transmembrane domains. Understanding which domains interact with other ESX-1 components can illuminate the general architecture of ESX systems.

• Functional conservation: Studies in different mycobacterial species (including M. tuberculosis and M. marinum) show similar requirements for EccE in ESX-1 function , suggesting fundamental mechanistic conservation despite some species-specific adaptations.

• Substrate specificity mechanisms: The finding that EccE1 is required for secretion of some substrates (EsxA, EsxB, EspA, EspC) but not others (EspB) provides clues about how substrate recognition and processing might differ across various ESX systems.

• Assembly and localization: The polar localization of EccE1 and its requirement for other ESX-1 components to form a stable complex may represent a common feature of multiple ESX systems, informing our understanding of how these complexes assemble in different mycobacterial species.

Research comparing ESX-1 with other ESX systems reveals hierarchical organization patterns that may be consistent across different secretion systems, with EccE homologs potentially playing similar structural or stabilizing roles in each system.

What are the most effective protocols for purifying recombinant EccE1 while maintaining its native conformation?

Purifying recombinant EccE1 while preserving its native conformation presents significant challenges due to its membrane-associated nature . The following methodological approach has proven effective:

• Expression system selection:

  • Mycobacterial expression systems (e.g., M. smegmatis) provide a native-like membrane environment

  • E. coli-based systems with specialized membrane protein expression strains (C41/C43) can be used with careful optimization

• Construct design:

  • C-terminal tagging (His6 or HA) has been validated for EccE1

  • Consider using fusion partners that enhance solubility while maintaining native structure

  • Include TEV protease cleavage sites to remove tags if necessary for functional studies

• Cell lysis and membrane preparation:

  • Gentle cell disruption methods (French press or sonication)

  • Differential centrifugation to isolate membrane fractions

  • DNase/RNase treatment to reduce viscosity

• Membrane protein solubilization:

  • Screen multiple detergents at various concentrations:

    • DDM (n-Dodecyl β-D-maltoside): 0.5-1%

    • LMNG (Lauryl maltose neopentyl glycol): 0.01-0.1%

    • Digitonin: 0.5-1%

  • Consider nanodisc or amphipol reconstitution for enhanced stability

• Purification strategy:

  • Affinity chromatography (Ni-NTA for His-tagged constructs)

  • Size exclusion chromatography to separate monomeric from aggregated protein

  • Ion exchange chromatography as a polishing step

• Quality control:

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to evaluate stability

  • Functional reconstitution assays if applicable

• Co-expression considerations:

  • Co-expression with interacting partners (EccB1, EccCa1, EccD1) may enhance stability

  • Research has shown that EccE1 requires other ESX-1 components to form a stable complex

For native complex isolation, alternative approaches include pulling down the entire ESX-1 complex using appropriately tagged components, which may better preserve the native structural context of EccE1.

What standardized assays can quantitatively measure the impact of EccE1 mutations on ESX-1 function?

Several standardized assays can quantitatively assess the impact of EccE1 mutations on ESX-1 function:

• Secretion quantification assays:

  • ELISA-based quantification of key ESX-1 substrates (EsxA, EsxB) in culture filtrates

  • Western blotting with densitometry for semi-quantitative analysis

  • Quantitative mass spectrometry with isotope labeling for comprehensive secretome analysis

• Functional cellular assays:

  • Macrophage cytotoxicity assays using lactate dehydrogenase (LDH) release as a quantitative readout

  • Phagosomal rupture assays using FRET-based reporters or β-lactamase-based systems

  • Intracellular bacterial survival assays in macrophages

• Protein interaction analysis:

  • Quantitative co-immunoprecipitation to measure interaction strength with other ESX-1 components

  • Surface plasmon resonance (SPR) with purified components

  • Microscale thermophoresis for interaction studies in complex environments

• Localization assays:

  • Quantitative fluorescence microscopy to measure the degree of polar localization

  • Subcellular fractionation followed by quantitative western blotting to determine membrane vs. cytosolic distribution

• Protein stability measurements:

  • Pulse-chase experiments to determine half-life of EccE1 variants

  • Thermal shift assays for purified proteins

  • Proteolytic susceptibility assays

Statistical considerations for mutation analysis:

When analyzing multiple EccE1 mutations, researchers should:

• Include appropriate wild-type and negative controls (ΔeccE1)

• Perform complementation with wild-type EccE1 to confirm phenotype rescue

• Use multiple biological replicates (minimum n=3)

• Apply appropriate statistical tests (ANOVA with post-hoc tests for multiple comparisons)

• Consider the magnitude of effect relative to complete deletion (partial vs. complete loss of function)