Alto, N. M. | Search bioRxiv

bioRxiv2018

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.\n\nImportanceAdvances 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.

microbiology↗

bioRxiv2017

Actin assembly-inducing protein ActA promotes FcγRIa-mediated Listeria internalization

Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of human listeriosis. While the ability of L. monocytogenes to enter and survive in professional phagocytes is critical to establish a successful infection, mechanisms of invasion are poorly understood. Our previous investigation into the role of type I interferon-stimulated genes in bacterial infection revealed that the human immunoglobulin receptor Fc{gamma}RIa served as a L. monocytogenes invasion factor. Fc{gamma}RIa-mediated L. monocytogenes entry occurred independently of immunoglobulin interaction or bacterial internalins. However, the bacterial determinants that mediate Fc{gamma}RIa interaction remain unclear. Using a comparative genomics approach, we identify actin assembly-inducing protein ActA as a pathogen specific ligand of Fc{gamma}RIa. Fc{gamma}RIa enhanced entry of pathogenic L. monocytogenes and L. ivanovii strain but not non-pathogenic L. innocua. We found that the major virulence regulator PrfA is required for pathogen entry into Fc{gamma}RIa-expressing cells and identify its gene target actA as the critical Listeria ligand. ActA alone was sufficient to promote entry into Fc{gamma}RIa-expressing cells, and this function is independent of its actin nucleating activity. Together, these studies present an unexpected role of ActA beyond its canonical function in actin-based motility and expand our understanding of Listeria strategies for host cell invasion.\n\nImportanceListeria monocytogenes is a food-borne bacterial pathogen and a causative agent of listeriosis with up to 50% mortality rate in immunocompromised individuals. While the mechanisms of Listeria entry into non-phagocytic cells have been extensively characterized, the details of phagocytic cell invasion are still poorly understood. We have recently discovered that human immunoglobulin receptor Fc{gamma}RIa mediates Listeria uptake by monocytic cells. This process occurred independently of canonical immunoglobulin interactions as well as classic Listeria internalization factors. Importantly, molecular determinants of Listeria-Fc{gamma}RIa interaction leading to bacterial entry, remained unknown. In this study, we demonstrate that Listeria virulence factor actin-assembly inducing protein ActA is required for Fc{gamma}RIa-mediated entry. Further, ActA was found to be sufficient for the internalization, suggesting its role as a bacterial ligand of Fc{gamma}RIa. Together, these findings expand our knowledge of mechanisms that Listeria has evolved to exploit cellular signaling pathways and immune defense of the host.

microbiology↗