Screening Mycobacterium tuberculosis Secreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms. IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

SopF, a phosphoinositide binding effector, promotes the stability of the nascent Salmonella-containing vacuole

“…yEGFP-SopF was produced at equivalent levels in all yeast strains upon galactose induction (S3C Fig). Notably, the steady-state subcellular localization of SopF in yeast (internal membrane sites; Fig 3A, S3B Fig) and mammalian cells (plasma membrane; Figs 2C and 3B) differed, similar to what has previously been reported for some secreted bacterial proteins [58,65]. This may be a consequence of imaging in live (yeast) versus formaldehyde-fixed (HeLa) cells.…”

Effectors Targeting the Unfolded Protein Response during Intracellular Bacterial Infection

“…This suggests that M. tuberculosis inhibits Perk signaling to delay apoptosis of the host cell thereby promoting bacterial replication. The Mpt64 protein ( Rv1980c ) has also been shown to localize to the ER to inhibit CHOP expression [ 39 ], although whether it inhibits upstream phosphorylation of Perk or eIF2α was not tested.…”

Proteomic Profiling Reveals the Architecture of Granulomatous Lesions Caused by Tuberculosis and Mycobacterium avium Complex Lung Disease

“…Mycobacteria have several protein secretion systems including type VII secretion system for their housekeeping growth and modulation of infected host immune system (Ligon et al, 2012). By comparison with secreted proteins of M. tuberculosis reported previously (Penn et al, 2018;Stamm et al, 2019), we found that twenty-two M. tuberculosis proteins among 614 detected ones were indicated to be presumably secreted proteins (Supplementary Figure S8 and Supplementary Table S4). Six of these proteins are Mce family proteins which are secreted or on the cell surface to facilitate host cell entry (Chitale et al, 2001;Saini et al, 2008;Zhang et al, 2018).…”

“…FIGURE S8 | Venn diagram illustrating the number of proteins in common among the present (This_study) and previously reported studies (Penn et al, 2018;Stamm et al, 2019). The gene lists for Stamm_2019 and Penn_2018 were reported by Penn et al (2018) and Stamm et al (2019). TABLE S1 | Excel file of the raw and imputed data of all proteomic analyses for identification of human, M. tuberculosis, and MAH proteins in mycobacterial granulomatous lesions.…”