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Screening Mycobacterium tuberculosis secreted proteins identifies Mpt64 as eukaryotic membrane-binding virulence factor

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 secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb exported proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 exported 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 exported 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 was necessary for Mtb survival in primary human macrophages. These data highlight the importance of exported proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms. Importance Advances have been made to identify exported 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 exported proteins for eukaryotic membrane binding, host subcellular localization and interactions with host vesicular trafficking. In addition, we observed the localization of one exported protein, Mpt64, 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 contributes to the virulence of M. tuberculosis during infection of primary human macrophages.