Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. Mtb has evolved to survive inside macrophages. However, the precise molecular mechanisms employed by Mtb to adapt to and manipulate the host environment remain unknown. To date, few Mtb secreted virulence factors have been discovered. Because many eukaryotic cellular processes are membrane-dependent, and many bacterial virulence factors target host membranes, we hypothesized that Mtb secretes virulence factors that target eukaryotic membranes.
We collated proteomic and genomic data for putative Mtb secreted proteins with possible membrane binding activity. We then screened 200 proteins in two independent assays. We first tested the ability of individual proteins to associate with eukaryotic membranes using a unique temperature sensitive yeast screen. We further characterized these membrane-associated Mtb effectors for their subcellular localization within mammalian host cells by using a fluorescence colocalization study with a panel of organelle markers. Hits were subsequently tested in a variety of biochemical, cellular and pathogenesis assays.
The initial yeast screen identified 48 out of 200 proteins that associated with eukaryotic membranes. The majority of these membrane-associated Mtb effectors co-localized with the endoplasmic reticulum. Four membrane-associated effectors co-localized with mitochondria. Since mitochondrial stress is an important determinant of the outcome of macrophage infection with Mtb, ongoing studies are focused on the role of these mitochondrial-localizing effectors in modulating mitochondrial function and Mtb pathogenesis.
Conclusion: We have identified multiple novel Mtb membrane binding effector proteins. Four effectors target mitochondria, an organelle whose function in innate immunity to intracellular pathogens is not well known. Identifying the molecular functions of Mtb secreted proteins will improve our understanding of how Mtb modifies its intracellular niche for long-term survival.
M. U. Shiloh, None