Mycobacterium tuberculosis (Mtb) survives within macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. Here we show that by inducing miR-33 and its passenger strand miR-33*, Mtb inhibits integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promotes autophagy flux through derepression of key autophagy effectors such as ATG5, ATG12, LC3B and LAMP1 and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, enhancing lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit utilized by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

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.

Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH-and temperature-dependent pore-forming activity of LLO, which is unique among cholesteroldependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca 2 þ oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca 2 þ uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

' success as a pathogen comes from its ability to evade degradation by macrophages. Normally macrophages clear microorganisms that activate pathogen-recognition receptors (PRRs) through a lysosomal-trafficking pathway called "LC3-associated phagocytosis" (LAP). Although activates numerous PRRs, for reasons that are poorly understood LAP does not substantially contribute to control. LAP depends upon reactive oxygen species (ROS) generated by NADPH oxidase, but fails to generate a robust oxidative response. Here, we show that CpsA, a LytR-CpsA-Psr (LCP) domain-containing protein, is required for to evade killing by NADPH oxidase and LAP. Unlike phagosomes containing wild-type bacilli, phagosomes containing the Δ mutant recruited NADPH oxidase, produced ROS, associated with LC3, and matured into antibacterial lysosomes. Moreover, CpsA was sufficient to impair NADPH oxidase recruitment to fungal particles that are normally cleared by LAP. Intracellular survival of the Δ mutant was largely restored in macrophages missing LAP components (, ,, ,, or ) but not in macrophages defective in a related, canonical autophagy pathway (, , or). The Δ mutant was highly impaired in vivo, and its growth was partially restored in mice deficient in NADPH oxidase, , or, demonstrating that CpsA makes a significant contribution to the resistance of to NADPH oxidase and LC3 trafficking in vivo. Overall, our findings reveal an essential role of CpsA in innate immune evasion and suggest that LCP proteins have functions beyond their previously known role in cell-wall metabolism.