Invading pathogens provoke the autophagic machinery and, in a process termed xenophagy, the host cell survives because autophagy is employed as a safeguard for pathogens that escaped phagosomes. However, some pathogens can manipulate the autophagic pathway and replicate within the niche of generated autophagosome-like vesicles. By automated fluorescence-based high content analyses, we demonstrate that Staphylococcus aureus strains (USA300, HG001, SA113) stimulate autophagy and become entrapped in intracellular PtdIns(3)P-enriched vesicles that are decorated with human WIPI-1, an essential PtdIns(3)P effector of canonical autophagy and membrane protein of both phagophores and autophagosomes. Further, agr-positive S. aureus (USA300, HG001) strains were more efficiently entrapped in WIPI-1 positive autophagosome-like vesicles when compared to agr-negative cells (SA113). By confocal and electron microscopy we provide evidence that single- and multiple-Staphylococci entrapped undergo cell division. Moreover, the number of WIPI-1 positive autophagosome-like vesicles entrapping Staphylococci significantly increased upon (i) lysosomal inhibition by bafilomycin A1 and (ii) blocking PIKfyve-mediated PtdIns(3,5)P2 generation by YM201636. In summary, our results provide evidence that the PtdIns(3)P effector function of WIPI-1 is utilized during xenophagy of Staphylococcus aureus. We suggest that invading S. aureus cells become entrapped in autophagosome-like WIPI-1 positive vesicles targeted for lysosomal degradation in nonprofessional host cells. 1. Introduction Macroautophagy (hereafter autophagy) is a cytoprotective cellular degradation mechanism for long-lived proteins and organelles [1]. Autophagy is specific to eukaryotic cells and important for cellular survival by enabling a constitutive clearance and recycling of cytoplasmic material (basal autophagy). Crucial to the process of autophagy is the fact, that cytoplasmic material is stochastically degraded. Portions of the cytoplasm become randomly sequestered in unique, double-membrane vesicles, autophagosomes. Autophagosomes are generated by elongation and closure of a membrane precursor, the phagophore. Subsequently, autophagosomes fuse with lysosomes to acquire acidic hydrolases for cargo degradation [2]. This stochastic constitutive form of autophagy provides constant clearance of the cytoplasm. Upon stress, such as starvation, the autophagic activity is induced above basal level to compensate nutrient shortage by providing monomeric constituents, such as amino acids, and energy. Conversely, under
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