Recombinant Uncharacterized PE-PGRS family protein PE_PGRS35 (PE_PGRS35)

What is the domain architecture of PE_PGRS35 and how does it influence functional analyses?

PE_PGRS35 (Rv1983) is a 558 amino acid protein with three distinct structural domains that must be considered in experimental design:

The domain-specific functions necessitate different experimental approaches. When studying PE_PGRS35, researchers should consider domain-specific constructs to isolate functions attributable to each region. The PE domain typically maintains structural integrity, while the PGRS domain mediates host interactions. Notably, the C-terminal domain with its aspartic proteinase motif is critical for enzymatic activity .

How do structural variations in PE_PGRS35 across Mycobacterium tuberculosis strains impact experimental design?

Natural genetic variations in PE_PGRS35 must be addressed when designing experiments:

• Some clinical isolates contain a 1-bp indel that disrupts the entire C-terminal domain while maintaining the PE and PGRS domains .

• Frameshift mutations affect protease function specifically, with truncated recombinant PE_PGRS35 lacking the C-terminal domain losing enzymatic activity .

• Strain selection is therefore critical - researchers should sequence the gene from their working strain before conducting functional studies to avoid misinterpretation of results.

When comparing strains, it's advisable to include positive and negative controls that account for these natural variations. Complementation experiments with functional domain deletion mutants can help isolate the contribution of each domain to observed phenotypes .

What are the optimal expression systems for producing functional recombinant PE_PGRS35?

The expression system significantly impacts PE_PGRS35 functionality:

For studying protease activity, expression in mycobacterial hosts or insect cells is recommended to preserve the aspartic proteinase functionality of the C-terminal domain. E. coli systems may be suitable for structural studies of individual domains but may not preserve the native conformation required for enzymatic activity .

What methodological approaches resolve the challenges of PE_PGRS35 purification?

Purification of full-length PE_PGRS35 presents unique challenges due to its glycine-rich PGRS domain:

Researchers should validate purified protein by both SDS-PAGE and Western blotting, as the unusual amino acid composition of the PGRS domain can cause aberrant migration patterns .

How can researchers effectively assay the protease activity of PE_PGRS35?

To characterize PE_PGRS35's protease activity:

• Substrate selection: LipY is a validated substrate for PE_PGRS35 (its M. marinum homolog PecA). Design assays using recombinant LipY to monitor cleavage at the YxxxD/E secretion motif within the PE domain or in the linker domain .

• Activity detection methods:

  • SDS-PAGE analysis showing the appearance of cleavage products

  • Fluorescence resonance energy transfer (FRET) peptides spanning the cleavage sites

  • Mass spectrometry to identify exact cleavage positions

• Reaction conditions optimization: Assays should include:

  • pH range testing (optimal range typically 6.0-7.0)

  • Metal ion dependency evaluation (test with EDTA and various divalent cations)

  • Temperature range assessment (30-37°C range recommended)

• Control experiments:

  • Include catalytically inactive mutants (mutations in the aspartic proteinase motif)

  • Test truncated versions lacking the C-terminal domain as negative controls

  • Compare with other PE_PGRS proteins to assess specificity

The protease activity occurs at the mycobacterial cell surface, so cell-based assays using intact mycobacteria expressing PE_PGRS35 provide more physiologically relevant results than purified protein assays alone .

What experimental approaches effectively characterize the immunological properties of PE_PGRS35?

PE_PGRS35 likely has immunomodulatory functions similar to other PE_PGRS proteins, though its specific mechanisms differ from homologs like PE_PGRS33:

• T-cell epitope mapping:

  • Synthetic peptides representing predicted epitopes in the PE domain can be used to stimulate peripheral blood mononuclear cells (PBMCs) from tuberculosis patients

  • Quantify cytokine release (particularly IFN-γ) to assess immunogenicity

  • Focus on predicted epitopes in the PE domain, as the PGRS domain typically lacks conventional T-cell epitopes

• Host receptor interaction studies:

  • While PE_PGRS33 directly interacts with TLR2 to trigger TNF-α secretion, PE_PGRS35's specific receptor interactions remain undetermined

  • Use co-immunoprecipitation or surface plasmon resonance with potential host receptors

  • Verify interactions through cell-based reporter assays using receptor-deficient cell lines

• Immunological outcome measurements:

  • Cytokine profiling (TNF-α, IL-12, IFN-γ)

  • Cell death pathway analysis (apoptosis vs. necrosis)

  • Phagosome maturation assessment

When designing these experiments, consider domain-specific constructs, as the PE domain, PGRS domain, and C-terminal domain may have distinct immunological properties .

How can researchers reconcile conflicting data regarding PE_PGRS35's role in mycobacterial pathogenesis?

Several approaches help address contradictory findings:

• Strain-specific analysis: PE_PGRS35 function varies between strains due to genetic polymorphisms. Sequence the gene from your experimental strain and compare with reference data, focusing on:

  • Frameshift mutations affecting the C-terminal domain

  • Polymorphisms in the PGRS domain that might alter host interactions

  • Presence of intact protease motifs

• Methodological standardization:

  • Discrepancies often arise from different experimental systems (in vitro vs. in vivo)

  • Use comparable expression levels across studies

  • Standardize protein production methods

  • Create a comprehensive panel of functional domain deletion mutants

• Integrated experimental approach:

  • Combine biochemical assays (protease activity)

  • Cell biology (localization, trafficking)

  • Immunology (host response)

  • In vivo infection models (using both knockout and complemented strains)

• Collaborative cross-validation:

  • Establish research consortia using standardized materials

  • Perform parallel experiments in different laboratories

  • Create a repository of validated reagents and strains

These approaches help determine whether apparent contradictions represent true biological complexity or methodological differences .

What advanced techniques can elucidate the evolutionary selection pressures on PE_PGRS35?

Understanding evolutionary dynamics requires sophisticated analytical approaches:

• Comprehensive sequence analysis:

  • Calculate domain-specific nucleotide diversity (π) and dN/dS ratios

  • The PE domain shows conservation (low dN/dS)

  • The PGRS domain exhibits higher variability

  • The C-terminal domain with protease function shows evidence of selection (dN/dS = 1.7)

• Population genomics approach:

  • Sequence PE_PGRS35 across diverse clinical isolates representing different lineages

  • Identify lineage-specific polymorphisms and indels

  • Map mutations to functional domains

  • Correlate with geographic distribution and disease phenotypes

• Experimental evolution:

  • Subject M. tuberculosis to controlled selective pressures (host immunity, drug pressure)

  • Monitor changes in PE_PGRS35 sequence over multiple passages

  • Assess functional consequences of evolved variants

• Comparative genomics with mycobacterial species:

  • Analysis of PE_PGRS35 homologs across pathogenic and non-pathogenic mycobacteria

  • Reconstruction of ancestral sequences

  • Documentation of key acquisition/loss of functional elements

These approaches reveal how PE_PGRS35 balances conservation of essential functions with adaptation to host pressures .

How does PE_PGRS35 functionally differ from other PE_PGRS proteins, and what methodologies best demonstrate these distinctions?

Comparative functional analysis requires:

• Parallel functional assays:

  • Compare protease activity with other PE_PGRS proteins containing similar C-terminal domains

  • PE_PGRS35 exhibits aspartic proteinase activity while PE_PGRS33 primarily functions in immune modulation

  • Test substrate specificity using recombinant PE proteins and lipases

• Domain swapping experiments:

  • Create chimeric proteins exchanging domains between PE_PGRS35 and other family members

  • Assess how domain swapping affects:

    • Protease activity

    • Cellular localization

    • Immunogenicity

    • Host cell interactions

• Differential expression analysis:

  • Compare expression patterns during infection phases

  • Unlike PE_PGRS30, which is upregulated during chronic infection, PE_PGRS35's expression pattern remains less characterized

• Structural biology approach:

  • Compare structural predictions across PE_PGRS family members

  • Model how structural differences impact function

  • Validate through mutagenesis of key residues

The evidence suggests that despite sequence homology, PE_PGRS proteins have evolved distinct functions, with PE_PGRS35 specializing in protease activity while others like PE_PGRS33 primarily mediate immune interactions .

What considerations are essential when designing knockout and complementation studies for PE_PGRS35?

Genetic manipulation studies require careful design:

• Knockout strategy optimization:

  • Use homologous recombination or CRISPR-Cas9 approaches

  • Verify complete gene deletion through PCR and sequencing

  • Confirm absence of protein expression via Western blotting

  • Check for compensatory upregulation of other PE_PGRS genes

• Complementation considerations:

  • Use integrative vectors for stable expression

  • Test both native promoter and inducible systems

  • Create domain deletion series:

    • Full-length PE_PGRS35

    • PE domain only

    • PE+PGRS domains without C-terminal domain

    • C-terminal domain with secretion signal

• Phenotypic analysis breadth:

  • Assess growth in various media conditions

  • Measure survival in macrophages

  • Examine phagosome maturation

  • Test virulence in animal models

  • Evaluate immunological responses

• Potential pitfalls:

  • Polar effects on neighboring genes

  • Irregular complementation levels

  • Strain-specific variations in phenotype

  • Redundancy among PE_PGRS family members

These approaches help determine the specific contribution of PE_PGRS35 to mycobacterial physiology and pathogenesis, distinguishing its role from the broader PE_PGRS family functions .

What emerging technologies could advance our understanding of PE_PGRS35 function?

Several cutting-edge approaches may resolve current knowledge gaps:

• Cryo-electron microscopy:

  • Determine the full 3D structure of PE_PGRS35

  • Visualize substrate binding and processing

  • Identify conformational changes during protease activation

• Single-cell analysis in infection models:

  • Monitor PE_PGRS35 expression in individual bacteria during infection

  • Correlate expression with bacterial physiological state

  • Track consequences of PE_PGRS35 activity on host cell responses

• Proximity labeling proteomics:

  • Identify interaction partners of PE_PGRS35 in living bacteria

  • Map the complete substrate repertoire beyond LipY

  • Discover associated regulatory proteins

• Humanized mouse models:

  • Test PE_PGRS35 function in the context of human immune components

  • Evaluate contribution to granuloma formation and maintenance

  • Assess impact on bacterial persistence and reactivation

These approaches will help complete our understanding of PE_PGRS35's roles in mycobacterial physiology and pathogenesis .

How might knowledge of PE_PGRS35 inform novel therapeutic strategies for tuberculosis?

Translational applications of PE_PGRS35 research include:

• Drug development approaches:

  • Design specific inhibitors targeting the aspartic proteinase domain

  • Create peptidomimetics that block key substrate interactions

  • Develop compounds that interfere with surface localization

• Vaccine strategy considerations:

  • Evaluate PE_PGRS35 as a potential vaccine antigen

  • Design constructs focusing on the PE domain's immunogenic epitopes

  • Avoid the highly variable PGRS domain in vaccine formulations

• Biomarker development:

  • Investigate PE_PGRS35 protease products as diagnostic markers

  • Monitor anti-PE_PGRS35 antibodies during disease progression

  • Correlate PE_PGRS35 genetic variants with treatment outcomes

• Host-directed therapy potential:

  • Target host pathways affected by PE_PGRS35 activity

  • Modulate responses to PE_PGRS35-mediated lipid processing

  • Develop adjunct therapies that complement conventional antibiotics

Understanding PE_PGRS35's role in mycobacterial physiology and host interactions may reveal vulnerabilities that can be exploited for therapeutic intervention .