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Updated: Jun 13, 2025
Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria
Published on: December 8, 2023
PE/PPE Proteome and ESX-5 Substrate Spectrum in Mycobacterium marinum
Lili Yan1, Hiu Ying Lai1, Thomas Chun Ning Leung1
1School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China.
Abstract:
PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.
Insights
This study enhanced proteomic methods to identify more PE/PPE proteins in Mycobacterium marinum, revealing novel insights into their expression and interaction within the ESX-5 secretion system.
Area of Science:
- Microbiology
- Proteomics
- Mycobacterial Pathogenesis
Background:
- PE/PPE proteins are crucial for pathogenic mycobacteria, involved in survival and host interaction via the ESX-5 secretion system.
- Limited knowledge exists regarding the expression and secretion of PE/PPE proteins due to challenges in their identification.
- Classical proteomic methods using trypsin are insufficient for identifying PE/PPE proteins due to low arginine/lysine content and sequence homology.
Purpose of the Study:
- To develop and apply an improved proteomic strategy for comprehensive identification and characterization of PE/PPE proteins in *Mycobacterium marinum*.
- To investigate the interactions within the ESX-5 secretion system and identify PE-PPE protein partners.
- To elucidate the expression patterns and localization of PE/PPE proteins.
Main Methods:
- Utilized a modified proteomic approach combining endoproteinase AspN and trypsin for *Mycobacterium marinum* proteome characterization.
- Employed EspG5 pulldown assays to identify proteins associated with the ESX-5 secretion system.
- Performed correlation analysis between protein abundance and phylogenetic relationships to predict PE/PPE protein interactions.
Main Results:
- Identified 27 unique PE/PPE proteins, particularly PE_PGRS proteins, using AspN digests, significantly increasing coverage of the PE/PPE protein pool.
- EspG5 pulldown assays successfully retrieved 44 ESX-5-associated PPE proteins, representing 85% of the identified PPE proteome.
- Discovered evidence of PE-PPE protein pairs and mapped critical residues for EspG5-PPE complex formation, validating interactions with PPE31 and PPE32.
Conclusions:
- The enhanced proteomic platform substantially increases the identification of PE/PPE proteins, overcoming limitations of traditional methods.
- The study provides a deeper understanding of PE/PPE protein expression, secretion, and interactions within the ESX-5 system.
- Findings contribute to elucidating the functional roles of PE/PPE proteins in mycobacterial pathogenicity and host-pathogen dynamics.
