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Selective enrichment of mycobacterial proteins from infected host macrophages
Ajit G Chande1, Zaved Siddiqui1, Mukul Kumar Midha2
1Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi-India.
Abstract:
Upon infection, Mycobacterium tuberculosis (Mtb) deploys specialized secretion machinery to deliver virulent proteins with the capacity to modulate a variety of host-cellular pathways. Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host. Here, we introduce an Mtb-selective protein labeling method based on genetic incorporation of azidonorleucine (Anl) through the expression of a mutant methionyl-tRNA synthetase. Exclusive incorporation of Anl, into native Mtb proteins, provided a click handle to pull out low abundant secretory proteins from the lysates of infected cells. Further, temporal secretome profiling, upon infection with strains of varying degree of virulence, revealed the proficiency of virulent Mtb to secrete chaperones. This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence. The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.
Insights
Mycobacterium tuberculosis (Mtb) uses a novel labeling method to identify virulence factors within host cells. This technique reveals how Mtb secretes chaperones to suppress host cell stress, aiding virulence.
Area of Science:
- Microbiology
- Molecular Biology
- Immunology
Background:
- Mycobacterium tuberculosis (Mtb) infection involves the secretion of virulent proteins that modulate host cellular pathways.
- Identifying intracellular Mtb proteins is challenging due to the abundance of host proteins.
- Selective enrichment of Mtb virulence effectors is crucial for understanding pathogenicity.
Purpose of the Study:
- To develop a method for selectively labeling and enriching Mtb proteins within infected host cells.
- To investigate the Mtb secretome and its role in modulating host cellular responses, particularly during infection with virulent strains.
- To understand the mechanisms underlying Mtb virulence, including the suppression of host endoplasmic reticulum (ER) stress.
Main Methods:
- Genetic incorporation of azidonorleucine (Anl) into native Mtb proteins using a mutant methionyl-tRNA synthetase.
- Utilizing click chemistry to selectively pull out low-abundant Mtb secretory proteins from infected cell lysates.
- Temporal secretome profiling of Mtb strains with varying virulence levels.
Main Results:
- The Anl labeling method successfully enriched low-abundant Mtb secretory proteins.
- Virulent Mtb strains were found to secrete chaperones.
- Secreted chaperones contributed to Mtb-specific suppression of ER stress in host macrophages, indicating a role in virulence.
Conclusions:
- The Anl labeling approach provides a powerful tool for studying the Mtb secretome and host-pathogen interactions.
- Mtb virulence involves the secretion of chaperones that suppress host ER stress.
- This method facilitates future imaging and proteomic studies to elucidate Mtb pathogenicity determinants.
