Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.     

Early intracellular events during internalization of Listeria monocytogenes by J774 cells.

The gram-positive bacillus Listeria monocytogenes gains entry into host cells through a phagosome membrane that forms around entering bacteria. During the early stages of internalization the invading bacteria appear to modify the protein composition of the forming phagosome membrane in J774 cells. MHC class II molecules on the cell surface and exposed surface molecules available for biotinylation are excluded from the bacteria-host cell membrane interface and from the forming phagosome. This exclusion of MHC class II molecules from the early phagosome may partially help to explain previous reports suggesting that L. monocytogenes is able to interfere with antigen presentation. Inside the host cell, MHC class II molecules are delivered to the phagosome membrane. This is followed by delivery of LAMP 1, a marker of late endocytic compartments, and fusion with low-pH compartments. The bacteria then escape into the cell cytoplasm, possibly assisted by rapid delivery of this low-pH environment.     

Structural Details of Human Tuba Recruitment by InlC of Listeria monocytogenes Elucidate Bacterial Cell-Cell Spreading

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InlA- but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins

To determine the contribution of the previously identified internalins, InlA, InlB, InlC, InlE, InlG, and InlH, to internalization of Listeria monocytogenes by non-professional phagocytic mammalian cells, we constructed mutants with various combinations of deletions in the respective inl genes. Internalization of these mutants into the epithelial-like Caco-2 and the microvascular endothelial HBMEC cell lines were studied. Deletion of the inlGHE gene cluster, or of the single genes, led to a two to fourfold increased internalization by HBMEC and other non-phagocytic mammalian cells. Invasion into HBMEC was totally blocked in the absence of InlB, and InlB-dependent internalization did not require the presence of any of the other internalins. Internalization by Caco-2 cells was reduced to a level of about 1% in the absence of InlA and InlB, and was most efficient in the presence of InlA, InlB and InlC and in the absence of InlG, InlH and InlE. InlB and InlA, in each case in the absence of the other internalins, led (compared with the wild-type strain) to reduced internalization of about 20% and less than 10% respectively. InlA-dependent internalization (in the absence of InlB) required the additional function of InlC and InlGHE. The deletion of inlGHE enhanced the expression of InlA and InlB. The increased amount of InlA led to an increase in early association of L. monocytogenes with Caco-2 cells without enhancing its uptake in the absence of the other internalins, whereas the larger amount of InlB did not enhance early association of L. monocytogenes with HBMEC but led to an increase in internalization of L. monocytogenes. The results suggest that InlB is able to induce phagocytosis in HBMEC and (at a lower efficiency) in Caco-2 cells by itself, but InlA needs the supportive functions of the other internalins to trigger phagocytosis. None of these internalins seems to be required for cell-to-cell spread by L. monocytogenes, as shown by microinjection of Caco-2 cells with appropriate inl mutants.     

Dithiothreitol enhances Listeria monocytogenes mediated cell cytotoxicity

The hybridoma Ped-2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6 hr. The cytotoxicity effect on Ped-2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0-2 mM) that is known to activate LLO was investigated to make the Ped-2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0-50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5 mM gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5-2 hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria-mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA+ hlyA+) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2-3 hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15 min. This indicated that LLO activity, which is responsible for Ped-2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2 hr, which were comparable to the 5-hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria-mediated Ped-2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user-friendly rapid assay to screen cytopathogenic properties of Listeria species.     

Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.     

Mammalian peptidoglycan recognition protein TagL inhibits Listeria monocytogenes invasion into epithelial cells

Peptidoglycan recognition proteins are a family of evolutionary conserved proteins that play a basic role in the innate immunity of insects, but their role in the immunity of mammals remains unclear. To elucidate its functions, a mouse member of the peptidoglycan recognition proteins family, TagL, was stably expressed in colon adenocarcinoma HT29 cells, and its effect on the invasion and intracellular growth of the enteroinvasive pathogenic bacterium Listeria monocytogenes was assessed. The expression of TagL substantially impaired bacterial invasion and early intracellular growth. The observed effects were partly caused by a loss of viability by intraphagosomal bacteria. Efficient phagosome escaping but not efficient invasion helped bacteria to overplay TagL.     

A role for ActA in epithelial cell invasion by Listeria monocytogenes

We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes . An in frame Δ actA mutant was constructed in a hyperinvasive strain of prfA * genotype, in which all genes of the PrfA-dependent virulence regulon, including actA , are highly expressed in vitro . Loss of ActA production in prfA * bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the Δ actA prfA * mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA * bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro ) or prfA *Δ actA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA * strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.     

Listeria monocytogenes listeriolysin O and phosphatidylinositol-specific phospholipase C affect adherence to epithelial cells

Listeria monocytogenes, a foodborn intracellular animal and human pathogen, produces several exotoxins contributing to virulence. Among these are listeriolysin O (LLO), a pore-forming cholesterol-dependent hemolysin, and a phosphatidylinositol-specific phospholipase C (PI-PLC). LLO is known to play an important role in the escape of bacteria from the primary phagocytic vacuole of macrophages, and PI-PLC supports this process. Evidence is accumulating that LLO and PI-PLC are multifunctional virulence factors with many important roles in the host-parasite interaction other than phagosomal membrane disruption. LLO and PI-PLC may induce a number of host cell responses by modulating signal transduction of infected cells via intracellular Ca2+ levels and the metabolism of phospholipids. This would result in the activation of host phospholipase C and protein kinase C. In the present study, using Bacillus sub tilis strains expressing LLO, PI-PLC, and simultaneously LLO and PI-PLC, we show that LLO and PI-PLC enhance bacterial binding to epithelial cells Int407, with LLO being necessary and PI-PLC playing an accessory role. The results of this work suggest that these two listerial proteins act on epithelial cells prior to internalization.     

Mitogen-activated protein kinases are required for effective infection of human choroid plexus epithelial cells by Listeria monocytogenes

Listeria monocytogenes, a Gram-positive bacterium, can cause meningitis after invading the human central nervous system. The blood-cerebrospinal fluid barrier (BCSFB), located at the epithelium of the choroid plexus, is a possible entry site for L. monocytogenes into the brain, and in vitro L. monocytogenes invades human choroid plexus epithelial papilloma (HIBCPP) cells. Although host cell signal transduction subsequent to infection by L. monocytogenes has been investigated, the role of mitogen-activated protein kinases (MAPK) is not clarified yet. We show that infection with L. monocytogenes causes activation of the MAPKs Erk1/2 and p38 preferentially when bacteria are added to the physiologically more relevant basolateral side of HIBCPP cells. Deletion of the listerial virulence factors Internalin (InlA) and InlB reduces MAPK activation. Whereas inhibition of either Erk1/2 or p38 signaling significantly attenuates infection of HIBCPP cells with L. monocytogenes, simultaneous inhibition of both MAPK pathways shows an additive effect, and Erk1/2 and p38 are involved in regulation of cytokine and chemokine expression following infection. Blocking of endocytosis with the synthetic dynamin inhibitor dynasore strongly abrogates infection of HIBCPP cells with L. monocytogenes. Concurrent inhibition of MAPK signaling further reduces infection, suggesting MAPKs mediate infection with L. monocytogenes during inhibition of dynamin-mediated endocytosis.     

Syndecans promote mycobacterial internalization by lung epithelial cells

Pulmonary tuberculosis (TB) is an airborne disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Alveolar epithelial cells and macrophages are the first point of contact for Mtb in the respiratory tract. However, the mechanisms of mycobacterial attachment to, and internalization by, nonprofessional phagocytes, such as epithelial cells, remain incompletely understood. We identified syndecan 4 (Sdc4) as mycobacterial attachment receptor on alveolar epithelial cells. Sdc4 mRNA expression was increased in human and mouse alveolar epithelial cells after mycobacterial infection. Sdc4 knockdown in alveolar epithelial cells or blocking with anti‐Sdc4 antibody reduced mycobacterial attachment and internalization. At the molecular level, interactions between epithelial cells and mycobacteria involved host Sdc and the mycobacterial heparin‐binding hemagglutinin adhesin. In vivo, Sdc1/Sdc4 double‐knockout mice were more resistant to Mtb colonization of the lung. Our work reveals a role for distinct Sdcs in promoting mycobacterial entry into alveolar epithelial cells with impact on outcome of TB disease.     

Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes

Listeria monocytogenes surface proteins internalin (Inl)A and InlB interact with the junctional protein E-cadherin and the hepatocyte growth factor (HGF) receptor Met, respectively, on the surface of epithelial cells to mediate bacterial entry. Here we show that InlA triggers two successive E-cadherin post-translational modifications, i.e. the Src-mediated tyrosine phosphorylation of E-cadherin followed by its ubiquitination by the ubiquitin-ligase Hakai. E-cadherin ubiquitination induces the recruitment of clathrin that is required for optimal bacterial internalization. We also show that the initial clustering of E-cadherin at the bacterial entry site requires caveolin, a protein normally involved in clathrin-independent endocytosis. Strikingly clathrin and caveolin are also recruited at the site of entry of E-cadherin-coated sepharose beads and functional experiments demonstrate that these two proteins are required for bead entry. Together these results not only document how the endocytosis machinery is recruited and involved in the internalization of a zippering bacterium, but also strongly suggest a functional link between E-cadherin endocytosis and the formation of adherens junctions in epithelial cells.     

Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion

Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier.     

High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells

A procedure has been developed for electroporation-mediated transformation of Listeria monocytogenes with plasmid DNA. The method was optimized for intact cells of L. monocytogenes 23074 by determining the effects of field strength, cell density, and plasmid DNA topology. Transformation efficiencies were dramatically increased when cells were treated with penicillin. Optimum frequencies of transformation (4 x 10(6) transformants/microgram DNA) were obtained when cells were grown in 10 micrograms/ml of penicillin G and electroporated at a field strength of 10 kV/cm. Using this procedure, transformation of relaxed plasmid DNA from ligation reactions provided 1 x 10(4) transformants/microgram DNA, allowing direct molecular cloning of DNA into this organism.     

LC3-associated phagocytosis initiated by integrin ITGAM-ITGB2/Mac-1 enhances immunity to Listeria monocytogenes

The macroautophagic/autophagic machinery cannot only target cell-endogenous components but also intracellular pathogenic bacteria such as Listeria monocytogenes. Listeria are targeted both by canonical autophagy and by a noncanonical form of autophagy referred to as LC3-associated phagocytosis (LAP). The molecular mechanisms involved and whether these processes contribute to anti-listerial immunity or rather provide Listeria with a replicative niche for persistent infection, however, remained unknown. Recently, using an in vivo mouse infection model, we have been able to demonstrate that Listeria in tissue macrophages are targeted exclusively by LAP. Furthermore, our data show that LAP is required for killing of Listeria by macrophages and thereby contributes to anti-listerial immunity of mice, whereas canonical autophagy is completely dispensable. Moreover, we have elucidated the molecular mechanisms that trigger LAP of Listeria and identified the integrin ITGAM-ITGB2/Mac-1/CR3/integrin α_Mß₂ as the receptor that initiates LAP in response to Listeria infection.     

Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells

Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram-positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C-terminal end. By immunofluorescence and immunogold labelling, we show that in wild-type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C-terminal region of internalin is necessary for cell-surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a‘domain-swapping’strategy - replacement of the cell wall anchor of InIA by the membrane anchor of ActA - we show that the reduced ability to adhere and enter cells of strains expressing InIA-ActA correlates with a lower amount of surface-exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.     

Invasion assay of Listeria monocytogenes using Vero and Caco-2 cells

The invasion ability of Listeria monocytogenes into cultured cells has been used to evaluate its pathogenicity. In this study, invasive ability was investigated using Vero and Caco-2 cell lines. The form of invasion showed no morphological differences between both cell lines inoculated with L. monocytogenes L89-H2 or L96-23C1 strains when double fluorescence stained with rhodamine and FITC or with Giemsa staining. Recovery count and recovery rate of L. monocytogenes from Vero cells was related to the number of inoculated bacteria (2 x 10(5) to 2 x 10(7)/ml) in a bell-shape pattern, though the relationship was unclear in Caco-2 cells. Recovery rate of L. monocytogenes was higher in Vero cells than Caco-2 cells at a multiplicity of infection (MOI) 10, though the rates in both cells showed different stable stages over a considerably wide range of MOI. The recovery rate of all five L. monocytogenes strains from listeriosis patients was 15% at MOI 10 from infected Vero cells, while meat-derived strains showed variable rates regardless of the serovar. These results suggest that the Vero cell line is suitable for an invasion assay and that a recovery rate of 15% may be the critical limit for the expression of pathogenicity in the host.     

Bacillus anthracis internalization by human fibroblasts and epithelial cells

The current model for Bacillus anthracis dissemination in vivo focuses on macrophages as carriers. However, recent evidence suggested that other host cells may also play a role in the process. Here, we tested the possibility of B. anthracis being internalized by a human fibroblast cell line, HT1080 and an epithelial cell line, Caco-2. A combination of gentamicin protection assays, scanning and transmission electron microscopy (EM) and fluorescence microscopy was used. The results demonstrated for the first time that both spores and vegetative cells of B. anthracis Sterne strain 7702 were able to adhere to and be internalized by cultured HT1080 and Caco-2 cells. Spore adherence to and internalization by HT1080 cells were not affected by a germination inhibitor. This suggested that certain features on dormant spores were sufficient for these processes. Vegetative cell adherence to and internalization by both cell lines were growth phase-dependent. EM images suggested that vegetative cells may have the ability to escape phagocytic vacuoles. Finally, we showed that internalization of both spores and vegetative cells required active functions of the host cell cytoskeleton. These results raised the possibility that B. anthracis may disseminate in vivo by directly infecting non-phagocytic cells.