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.

Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity

“…Identification of the BfrB Binding Protein in Human Macrophages-One of the effectors identified here, BfrB, was previously found to be required for Mtb survival in macrophages (41,51) and is enriched from infected macrophages (43), suggesting that BfrB may play an important role in the interaction with the host. To identify the macrophage binding partners of BfrB, we purified GST-tagged BfrB and incubated it with PMA-treated THP-1 lysate before performing GST pull-down and silver staining ( Fig.…”

“…Previous work has suggested that bacterial proteins smaller than 70 KDa can cross the host phagosomal membrane (61). Although the exact mechanism by which BfrB secreted across the Mtb membrane to macrophage remains unclear (43,44), the disassembly of Mtb BfrB triggered by pupylation may be important in this regard.…”

“…We focused on BfrB (Rv3841), a ferritin-like protein that was known to play a key role in the maintenance of Mtb iron homeostasis (41). Interestingly, BfrB was recently identified as a secreted protein that is enriched from infected macrophages and is also present in the exosomes of patients, suggesting that it may play additional roles in the interactions with the host (42)(43)(44). However, the mechanism by which BfrB functions in the modulation of the host immune response is poorly known.…”

S2 from Equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3

“…HEK293T cells were pulsed with 50µM myristic acid-azide (Life Technologies) for 24 hours and the incorporated azide moiety was conjugated to the alkyne immobilized on a matrix using the Click-iT protein enrichment kit (Life Technologies), as reported earlier (35). HA-tagged proteins in the enriched fraction were detected by immunoblotting.…”

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

“…Recently however, enrichment of bacterial secretomes using in vitro infection models was reported [92,93]. Both studies used biorthogonal labeling of bacterial proteins with ANL to isolate the secretome ( Figure 3B).…”