The food-borne pathogen Listeria monocytogenes is known to colonize the lumen of the gallbladder in infected mice and to grow rapidly in this environment (J. Hardy et al., Science 303:851-853, 2004). However, relatively little is known about the mechanisms utilized by the pathogen to survive and grow in this location. We utilized gallbladder bile (GB bile) isolated directly from porcine gallbladders as an ex vivo model of gallbladder growth. We demonstrate that GB bile is generally nontoxic for bacteria and can readily support growth of a variety of bacterial species including L. monocytogenes, Lactococcus lactis, Salmonella enterica serovar Typhimurium, and Escherichia coli. Significantly, L. monocytogenes grew at the same rate as the nonpathogenic species Listeria innocua, indicating that the pathogen does not possess specialized mechanisms that enable growth in this environment. However, when we reduced the pH of GB bile to pH 5.5 in order to mimic the release of bile within the small intestine, the toxicity of GB bile increased significantly and specific resistance mechanisms (Sigma B, BSH, and BilE) were essential for survival of the pathogen under these conditions. In order to identify genetic loci that are necessary for growth of L. monocytogenes in the gallbladder, a mariner transposon bank was created and screened for mutants unable to replicate in GB bile. This led to the identification of mutants in six loci, including genes encoding enzymes involved in purine metabolism, amino acid biosynthesis, and biotin uptake. Although GB bile does not represent a significant impediment to bacterial growth, specific metabolic processes are required by L. monocytogenes in order to grow in this environment.

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.

Summary The bacterial pathogen Listeria monocytogenes uses actin-based motility to spread from infected human cells to surrounding healthy cells. Cell-cell spread involves the formation of thin extensions of the host plasma membrane (‘protrusions’) containing motile bacteria. In cultured enterocytes, the Listeria protein InlC promotes protrusion formation by binding and antagonizing the human scaffolding protein Tuba. Tuba is a known activator of the GTPase Cdc42. In this work, we demonstrate an important role for Cdc42 in controlling Listeria spread. Infection of the enterocyte cell line Caco-2 BBE1 induced a decrease in the level of Cdc42-GTP, indicating that Listeria downregulates this GTPase. Genetic data involving RNA interference indicated that bacterial impairment of Cdc42 may involve inhibition of Tuba. Experiments with dominant negative and constitutively activated alleles of Cdc42 demonstrated that the ability to inactivate Cdc42 is required for efficient protrusion formation by Listeria. Taken together, these findings indicate a novel mechanism of bacterial spread involving pathogen-induced downregulation of host Cdc42.

Many bacterial pathogens subvert mammalian type IA phosphoinositide 3-kinase (PI3K) in order to induce their internalization into host cells. How PI3K promotes internalization is not well understood. Also unclear is whether type IA PI3K affects different pathogens through similar or distinct mechanisms. Here, we performed an RNA interference (RNAi)-based screen to identify components of the type IA PI3K pathway involved in invasin-mediated entry of Yersinia enterocolitica, an enteropathogen that causes enteritis and lymphadenitis. The 69 genes targeted encode known upstream regulators or downstream effectors of PI3K. A similar RNAi screen was previously performed with the food-borne bacterium Listeria monocytogenes. The results of the screen with Y. enterocolitica indicate that at least nine members of the PI3K pathway are needed for invasin-mediated entry. Several of these proteins, including centaurin-␣1, Dock180, focal adhesion kinase (FAK), Grp1, LL5␣, LL5␤, and PLD2 (phospholipase D2), were recruited to sites of entry. In addition, centaurin-␣1, FAK, PLD2, and mTOR were required for remodeling of the actin cytoskeleton during entry. Six of the human proteins affecting invasin-dependent internalization also promote InlBmediated entry of L. monocytogenes. Our results identify several host proteins that mediate invasin-induced effects on the actin cytoskeleton and indicate that a subset of PI3K pathway components promote internalization of both Y. enterocolitica and L. monocytogenes. Many microbial pathogens exploit host signal transduction pathways in order to cause disease (1-4). One key pathway subverted by bacterial, viral, and protozoan pathogens involves a mammalian lipid kinase called type IA phosphoinositide 3-kinase (PI3K) (3). Type IA PI3K plays a critical role in internalization of several pathogens into host cells. These microbes include bacteria that cause anthrax (Bacillus anthracis) (5), respiratory infections (Pseudomonas aeruginosa and Chlamydia pneumoniae) (6, 7), and food-borne disease (Campylobacter jejuni and Listeria monocytogenes) (3,4,8). Type IA PI3K also promotes entry of Ebola virus (9), influenza A virus (10), and parasites causing Chagas' disease (Trypanosoma cruzi) (11) or toxoplasmosis (Toxoplasma gondii) (12). Overall, the mechanism by which this mammalian lipid kinase controls infection by these diverse pathogens is not well understood.Mammalian type IA PI3K is an heterodimeric enzyme composed of a 110-kDa catalytic subunit and a 85-kDa regulatory subunit (13). This PI3K is coupled to growth factor, cytokine, or cell adhesion receptors and controls a variety of processes, including cell growth, survival, and motility (13,14). Type IA PI3K promotes its biological effects through at least two mechanisms, the best understood of which involves lipid kinase activity. This PI3K produces phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], a lipid second messenger. PI(3,4,5)P 3 is converted by phosphatases to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ]. Together, PI(3,4,5...

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria promotes its internalization into some human cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. The interaction of InlB with the Met receptor stimulates host signaling pathways that promote cell surface changes driving bacterial uptake. One human signaling protein that plays a critical role in Listeria entry is type IA phosphoinositide 3-kinase (PI 3-kinase). The molecular mechanism by which PI 3-kinase promotes bacterial internalization is not understood. Here we perform an RNA interference (RNAi)-based screen to identify components of the type IA PI 3-kinase pathway that control the entry of Listeria into the human cell line HeLa. The 64 genes targeted encode known upstream regulators or downstream effectors of type IA PI 3-kinase. The results of this screen indicate that at least 9 members of the PI 3-kinase pathway play important roles in Listeria uptake. These 9 human proteins include a Rab5 GTPase, several regulators of Arf or Rac1 GTPases, and the serine/threonine kinases phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTor), and protein kinase C-. These findings represent a key first step toward understanding the mechanism by which type IA PI 3-kinase controls bacterial internalization.

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The bacterial pathogen Listeria monocytogenes causes foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. The signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and protein kinase C-␣ (PKC-␣) in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multiprotein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into cells of the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and hypoxia-inducible factor 1␣ (HIF-1␣) and the mTORC2 substrate PKC-␣ each control Listeria uptake. Genetic or pharmacological inhibition of PKC-␣ reduced the internalization of Listeria and the accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-␣ to be host factors exploited by Listeria to promote infection. PKC-␣ controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor.KEYWORDS InlB, Listeria monocytogenes, Met receptor, protein kinase C, actin, mTOR L isteria monocytogenes is a foodborne pathogen capable of causing serious infections resulting in meningitis or abortions (1). Critical for disease is the ability of Listeria to induce its internalization into nonphagocytic mammalian cells in the intestine, liver, or placenta (2). One of the major pathways of internalization of Listeria into human cells is mediated by interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met (3). InlB-dependent entry involves remodeling of the host plasma membrane through actin polymerization (4, 5). Substantial progress in characterizing InlB-mediated uptake has been made, resulting in the identification of several host signal transduction pathways that control actin polymerization and perhaps other physiological processes during entry (4). Despite this progress, a detailed understanding of the molecular mechanism of InlB-dependent internalization of Listeria remains incomplete.The human type IA phosphoinositide 3-kinase (PI3K) pathway is one of the major signaling networks controlling InlB-mediated entry (4,(6)(7)(8). Recent RNA interference (RNAi) screens identified 13 components of the type IA PI3K pathway involved in InlB-dependent internalization of Listeria (9, 10). One host protein demonstrated in this work to play an important role in Listeria entry is mTOR (mammalian ...

SUMMARY Listeria monocytogenes is a food-borne pathogen that uses actin–dependent motility to spread between human cells. Cell-to-cell spread involves the formation by motile bacteria of plasma membrane-derived structures termed ‘protrusions’. In cultured enterocytes, the secreted Listeria protein InlC promotes protrusion formation by binding and inhibiting the human scaffolding protein Tuba. Here we demonstrate that protrusions are controlled by human COPII components that direct trafficking from the endoplasmic reticulum. Co-precipitation experiments indicated that the COPII proteins Sec31A and Sec13 interact directly with a Src Homology 3 domain in Tuba. This interaction was antagonized by InlC. Depletion of Sec31A or Sec13 restored normal protrusion formation to a Listeria mutant lacking inlC, without affecting spread of wild-type bacteria. Genetic impairment of the COPII component Sar1 or treatment of cells with brefeldin A affected protrusions similarly to Sec31A or Sec13 depletion. These findings indicated that InlC relieves a host-mediated restriction of Listeria spread otherwise imposed by COPII. Inhibition of Sec31A, Sec13, or Sar1 or brefeldin A treatment also perturbed the structure of cell-cell junctions. Collectively, these findings demonstrate an important role for COPII in controlling Listeria spread. We propose that COPII may act by delivering host proteins that generate tension at cell junctions.