Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.
Author Summary Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis, one of the world's most deadly infections. The host immune system can't eradicate Mtb because it grows within macrophages, cells that normally kill bacteria. One of the intracellular survival strategies of Mtb is to avoid delivery to lysosomes, a phenomenon described over 40 years ago, but for which the mechanism and molecular details remain incomplete. Mtb possess specialized secretion systems (Type VII secretion systems; TSSS) that transfer particular proteins out of the bacteria, but how these proteins promote infection is not well understood. In this study, we used a high stringency yeast two-hybrid system to identify interactions between secreted effectors from Mtb and human host factors. We identified ninety-nine such interactions and focused our attention on the interaction between EsxH, secreted by Esx-3, a TSSS of Mtb, and Hrs, a component of the host ESCRT machinery. We provide evidence that Mtb EsxH directly targets host Hrs to disrupt delivery of bacteria to lysosomes. Thus, this study demonstrates the role of a TSSS effector and the ESCRT machinery in what is one of the central features of tuberculosis pathogenesis, thereby providing molecular insight into why humans can't clear Mtb infection.