902. Identification and Characterization of Novel Mycobacterium tuberculosis Secreted Virulence Proteins
Session: Oral Abstract Session: Opening the Hood and Looking at the Mechanisms
Friday, October 28, 2016: 9:15 AM
Room: 388-390
Background: 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.

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.

Chelsea E. Stamm, B.S.1, Bethany Weigele, PhD2, Neal M. Alto, PhD2 and Michael U. Shiloh, MD, PhD1, (1)Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, (2)Microbiology, University of Texas Southwestern Medical Center, Dallas, TX

Disclosures:

C. E. Stamm, None

B. Weigele, None

N. M. Alto, None

M. U. Shiloh, None

Findings in the abstracts are embargoed until 12:01 a.m. CDT, Wednesday Oct. 26th with the exception of research findings presented at the IDWeek press conferences.