Use of RNA interference inDrosophilaS2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen

Abstract

Three genome-wide RNA interference screens were performed in Drosophila S2 cells to dissect the contribution of host processes to Listeria monocytogenes entry, vacuolar escape, and intracellular growth. Among the 116 genes identified, several host pathways previously unrecognized as playing a role in listerial pathogenesis were identified: knockdowns affecting vacuolar trafficking to and from the multivesicular body bypassed the requirement for the essential pore-forming toxin listeriolysin O in mediating escape from phagocytic vacuoles and knockdowns affecting either subunit of serine palmitoyltransferase, a key enzyme in ceramide and sphingolipid biosynthesis, enhanced the toxicity of listeriolysin O expressed in the host cell cytosol, leading to lack of appropriate toxin activity compartmentalization and host cell death. Genomewide RNA interference screens using Drosophila S2 cells proved to be a powerful approach to dissect host-pathogen interactions.Listeria monocytogenes ͉ listeriolysin O ͉ multivesicular bodies ͉ serine palmitoyltransferase I nfectious diseases caused by intracellular pathogens are responsible for an enormous amount of worldwide morbidity and mortality. These pathogens exploit the basic processes of host cells to establish their intracellular niche (1). Listeria monocytogenes, a facultative intracellular Gram-positive bacterial pathogen, thrives in the cytosol of host cells. The intracellular life cycle of L. monocytogenes has been well defined (2) and can be summarized as follows. Bacteria enter cells by either phagocytosis or bacteria-mediated internalization. Subsequent to internalization, the bacteria produce a cholesterol-dependent pore-forming cytolysin, termed listeriolysin O (LLO), and two phospholipases C (PLCs) that mediate rupture of the resulting phagosome, thereby allowing bacteria access to the rich milieu of the host cytosol. Once in the cytosol, bacteria grow rapidly and exploit a host system of actin-based motility to move intracellularly and spread from cell to cell. Mutants lacking LLO cannot escape from the phagosome, whereas those lacking PLCs are partially defective in escape. In some mammalian epithelial cells, however, a requirement for LLO can be bypassed, and in that case, PLCs are required for vacuolar escape (3, 4). Nevertheless, LLO is absolutely required for pathogenicity and is essential in the vast majority of cells analyzed. However, LLO is a doubleedged sword that can kill the host cell if expressed inappropriately. Mutations affecting its acidic pH optimum or in a PESTlike sequence result in inappropriate LLO expression in the cytosol, leading to plasma membrane damage, premature cell death, and severe attenuation in experimental listeriosis (5-7).Although much has been learned about the cellular microbiology of L. monocytogenes infection, the characterization of host processes contributing to pathogenesis has been hampered by the lack of tools for whole-genome genetic manipulations of the host. Many unanswered questions remain, such as how do bacteria e...

ESCRT factors restrict mycobacterial growth

“…While the ESCRT machinery restricts the growth of M. smegmatis, genome-wide screens in Drosophila found that CG8055, Vps28, and Vps4 were required for intracellular growth of Listeria monocytogenes (8,9), suggesting that diminished ESCRT activity does not make cells generally susceptible to bacterial infection. However, when these cells were infected with a mutant strain of L. monocytogenes that normally gets trapped in the vacuole because of a defect in the cytolysin, which is required for escape from the phagosome (LLO), the bacteria were better able to escape.…”

“…Arguing against this possibility, phagosomal escape of LLO-minus Listeria requires the bacterial phospholipases. Thus, loss of phagosome integrity in ESCRT-depleted cells still depends on bacterial factors (9). Furthermore, we examined by electron microscopy the intracellular location of M. smegmatis in S2 cells that had been depleted of Tsg101 (SI Fig.…”

“…We developed a model of infection using Mycobacterium fortuitum and Drosophila S2 cells, a macrophagelike cell line that is amenable to RNAi. Such an approach, using RNAi in combination with a Drosophila tissue culture model of infection, has proved a valuable strategy for dissecting the host contribution to infection for a number of pathogens (7)(8)(9)(10)(11)(12). We used M. fortuitum as a model mycobacterial pathogen because, like M. tuberculosis, it restricts phagosome fusion with lysosomes (13), suggesting common virulence properties with other mycobacteria.…”

Confronting physiology: how do infected flies die?

“…Listeria lacking LLO are also unable to escape the phagosome during infections of S2 cells in vitro (Cheng and Portnoy, 2003). The Portnoy group not only characterized known aspects of LLO function, but also then performed a large-scale RNAi screen to alter the course of a Listeria infection of cultured Drosophila cells (Cheng et al, 2005). By comparing the effects of different RNAis on wild-type bacteria and mutants in LLO, they identified several fly genes specifically involved in LLO pathogenesis.…”

“…Theoretical arguments aside, however, many non-physiological microbes cause unexpectedly interesting infections in the fly. Some particularly odd examples include: Listeria monocytogenes (which has elegant temperature regulation of virulence and might not be expected to be virulent at less than 30°C), Streptococcus pneumoniae (a common inhabitant of our airways that has to be grown under CO 2 and probably never encounters the fly), and Chlamydia trachomatis (a highly co-evolved obligate intracellular pathogen) (Cheng and Portnoy, 2003;Mansfield et al, 2003;Agaisse et al, 2005;Cheng et al, 2005;Elwell and Engel, 2005;Pham et al, 2007).…”