The Helicobacter pylori vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using ␤-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, ␤-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.

Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.Serratia marcescens is a gram-negative enteric bacterium that can function as an opportunistic pathogen within immunocompromised hosts (7,14,23,24,41). S. marcescens is a source of nosocomial infections, in part because the organism readily adheres to invasive hospital instrumentation, such as catheters, endoscopes, and intravenous tubing (1,17,27,32,34), and is relatively resistant to standard sterilization and disinfection protocols (3,8,52,53). Resistances to ␤-lactams, cephalosporins, and aminoglycosides have been reported, thereby complicating treatment of S. marcescens nosocomial infections (5,6,16,35,56).Upon introduction into the host, S. marcescens can infect numerous sites, including the urinary (42, 60) and respiratory (2) epithelia, muscle and subcutaneous tissues (17), the kidneys (25, 42), the lungs (13, 47), and also the heart and pericardium (58, 59). In addition, S. marcescens eye infections are common and are a frequent cause of keratitis (4, 29-31, 36, 65). In general, S. marcescens infections induce inflammation and fever, but fatal bacteremia can develop in patients weakened by previous infection, surgery, or immunosuppression (24,59,64,67). Despite numerous reported S. marcescens infections and the emergence of antibiotic-resistant strains (7,14,23,62), the virulence mechanisms of this organism are poorly understood.Carbonell and coworkers reported that S. marcescens culture filtrates exhibited pronounced in vitro cytotoxicity to cultured mammalian cells (11,12). Importantly, cytotoxicity was detected to various extents in all the strains that were tested, regardless of biotype or serotype (12). However, the cytotoxic factor or factors in S. marcescens culture filtrates were not identi...