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.

Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe. We found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of lipopolysaccharide O antigen enables vesicles to bypass clathrin-mediated endocytosis, which enhances both their entry rate and efficiency into host cells. Collectively, our findings highlight the composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections.

Outer membrane vesicles (OMVs) are nanosized proteoliposomes derived from the outer membrane of Gram-negative bacteria. They are ubiquitously produced both in culture and during infection, and are now recognized to play crucial roles during host-microbe interactions. OMVs can transport a broad range of chemically diverse cargoes, including lipids and lipopolysaccharides, membrane embedded and associated proteins and small molecules, peptidoglycan and nucleic acids. Particularly virulence factors such as adhesins and toxins are often enriched in OMVs. Here, we discuss a variety of ways in which OMVs facilitate host-microbe interactions, including their contributions to biofilm formation, nutrient scavenging, and modulation of host cell function. We particularly examine recent findings regarding outer membrane vesicle – host cell interactions in the oral cavity and the gastrointestinal tract.

Outer membrane vesicles (OMVs) are nanosized proteoliposomes derived from the outer membrane of Gram-negative bacteria. They are ubiquitously produced both in culture and during infection, and are now recognized to play crucial roles during host-microbe interactions. OMVs can transport a broad range of chemically diverse cargoes, including lipids and lipopolysaccharides, membrane embedded and associated proteins and small molecules, peptidoglycan and nucleic acids. Particularly virulence factors such as adhesins and toxins are often enriched in OMVs. Here, we discuss a variety of ways in which OMVs facilitate hostmicrobe interactions, including their contributions to biofilm formation, nutrient scavenging, and modulation of host cell function. We particularly examine recent findings regarding outer membrane vesicle-host cell interactions in the oral cavity and the gastrointestinal tract.