The vesicle-forming host ESCRT complex promotes multiplication of Rhodococcus equi in macrophages

ABSTRACT Rhodococcus equi is a facultative intracellular pathogen of foals and immunocompromised humans, causing predominantly lung disease. Key to its multiplication in macrophages is the neutralization of the pH in the Rhodococcus -containing vacuole (RCV) through the secreted membrane-permeabilizing protein virulence-associated protein A (VapA). Here, we show that the host endosomal sorting complex required for transport (ESCRT) promotes intracellular multiplication of R. equi through vesicle formation in the endocytic pathway and a hitherto undescribed pathway of ESCRT-dependent microautophagy directly on the RCV. Microautophagy generates intra-RCV vesicles containing the majority of the RCV transmembrane proteins lysosome-associated membrane protein 1 and the Niemann-Pick disease type C1 protein. This vesiculation pathway also leads to the accumulation of otherwise cytosolic ubiquitin in the RCV. Formation of microautophagic vesicles is independent of the ESCRT factor ALG2-interacting protein X (ALIX) but depends on the ESCRT-III-associated protein vacuole protein sorting 4 (VPS4). In summary, our data indicate that ESCRT provides a link between host vesicle formation in the endocytic pathway, microautophagy, and bacterial intracellular multiplication. IMPORTANCE Very little is known about which host constituents promote intracellular growth of the facultative intracellular pathogen Rhodococcus equi within its main target cell, the macrophage. Here, we present evidence that host proteins of the so-called endosomal sorting complex required for transport (ESCRT) complex are important for R. equi multiplication. This ESCRT complex generates host membrane vesicles, which invaginate into the pathogen-containing vacuole in a process reminiscent of microautophagy. This process can lead to a strong enrichment of lysosomal transmembrane proteins and ubiquitinated host proteins in the pathogen-containing vacuole and has not been described before for any microbe. Our observations are of pathological, pharmacological, and cell biological interest.