Selective enrichment of mycobacterial proteins from infected host macrophages.

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.

• Authors: Ajit G Chande, Zaved Siddiqui, Mukul Kumar Midha, Varsha Sirohi, Srikanth Ravichandran, Kanury V S Rao
• Journal: Scientific reports (Sci Rep) Vol. 5 Pg. 13430 (Aug 25 2015) ISSN: 2045-2322 [Electronic] England
• PMID: 26303024 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Virulence Factors

Topics

• Bacterial Proteins (biosynthesis, genetics, isolation & purification)
• Cell Line
• Gene Expression Profiling (methods)
• Humans
• Macrophages (chemistry, microbiology)
• Mycobacterium tuberculosis (physiology)
• Staining and Labeling (methods)
• Virulence Factors (genetics, isolation & purification, metabolism)