Methods: 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 next tested the ability of individual Mtb proteins to alter the host secretory pathway. Hits were subsequently tested in a variety of biochemical, cellular and pathogenesis assays, including the ability of Mtb mutants to survive in macrophages and in mice.
Results: The initial yeast screen identified 48 out of 200 proteins while the second secretion assay further identified 20 candidate Mtb proteins that either enhanced or inhibited the eukaryotic general secretory pathway. Five proteins were membrane associated and altered host secretion. All five are previously uncharacterized proteins and we are currently studying these proteins through a series of biochemical and cell biological assays. One protein, CES1, is secreted into macrophages and localizes to the endoplasmic reticulum. Through genetic truncation experiments, we determined that CES1 interacts with membranes through an N-terminal domain. Finally, a deletion mutant of CES1 in Mtb is attenuated for growth in macrophages in vitro and after aerosol infection in vivo.
Conclusion: We have identified multiple novel Mtb membrane binding effector proteins. One of these proteins, CES1, is vital for Mtb pathogenesis in macrophages and mice. Understanding the molecular functions of Mtb secreted proteins will improve our understanding of how Mtb modifies its intracellular niche for long-term survival.
C. E. Stamm,
N. M. Alto, None
M. U. Shiloh, None