We developed a screening procedure to identify small-molecule compounds that altered infection by Listeria monocytogenes to gain insights into bacterial/host cellular processes required for intracellular pathogenesis. A small-molecule library of 480 compounds with known biological functions was screened, and 21 compounds that altered the L. monocytogenes infection of murine bone marrow-derived macrophages (BMM) were identified. The identified compounds affected various cellular functions, such as actin polymerization, kinase/phosphatase activity, calcium signaling, and apoptosis. Pimozide, an FDA-approved drug used to treat severe Tourette's syndrome and schizophrenia, was further examined and shown to decrease the bacterial uptake and vacuole escape of L. monocytogenes in BMM. The inhibitory effect of pimozide on internalization was not specific for L. monocytogenes, as the phagocytosis of other bacterial species (Bacillus subtilis, Salmonella enterica serovar Typhimurium, and Escherichia coli K12) was significantly inhibited in the presence of pimozide. The invasion and cell-to-cell spread of L. monocytogenes during the infection of nonprofessional phagocytic cells also was decreased by pimozide treatment. Although pimozide has been reported to be an antagonist of mammalian cell calcium channels, the infection of BMM in a calcium-free medium did not relieve the inhibitory effects of pimozide on L. monocytogenes infection. Our results provide a generalizable screening approach for identifying small-molecule compounds that affect cellular pathways that are required for intracellular bacterial pathogenesis. We also have identified pimozide, a clinically approved antipsychotic drug, as a compound that may be suitable for further development as a therapeutic for intracellular bacterial infections.
Listeria monocytogenes is a bacterial pathogen that replicates within the cytosol of infected host cells. The ability to rapidly escape the phagocytic vacuole is essential for efficient intracellular replication. In the murine model of infection, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for vacuolar dissolution, as LLO-deficient (ΔLLO) mutants remain trapped within vacuoles. In contrast, in many human cell types ΔLLO L. monocytogenes are capable of vacuolar escape at moderate to high frequencies. To better characterize the mechanism of LLO-independent vacuolar escape in human cells, we conducted an RNA interference (RNAi) screen to identify vesicular trafficking factors that play a role in altering vacuolar escape efficiency of ΔLLO L. monocytogenes. RNAi knockdown of 18 vesicular trafficking factors resulted in increased LLO-independent vacuolar escape. Our results suggest that knockdown of one factor, RABEP1 (rabaptin-5), decreased the maturation of vacuoles containing ΔLLO L. monocytogenes. Thus, we provide evidence that increased vacuolar escape of ΔLLO L. monocytogenes in human cells correlates with slower vacuolar maturation. We also determined that increased LLO-independent dissolution of vacuoles during RABEP1 knockdown required the bacterial broad-range phospholipase PC-PLC. We hypothesize that slowing the kinetics of vacuolar maturation generates an environment conducive for vacuolar escape mediated by the bacterial phospholipases.
Listeria monocytogenes; listeriolysin O; vacuolar maturation; rabaptin-5; PC-PLC
Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs).
In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome.
Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria.
Macrophage activation often contributes to the strong immune response elicited upon infection. The ability of macrophages to become activated was discovered when sub-lethal primary infections of mice with the bacterium Listeria monocytogenes provided protection against secondary infections through nonhumoral immunity. L. monocytogenes infect and propagate in macrophages by escaping the phagosome into the cytosol, where they avoid humoral immune mediators. Activated macrophages kill L. monocytogenes by blocking phagosomal escape. The timing of the antimicrobial activities within the phagosome is crucial to the outcome. In non-activated macrophages, bacterial factors generally prevail, and L. monocytogenes can escape from the vacuoles and grow within cytoplasm. Activated macrophages generate reactive oxygen or nitrogen intermediates early after bacterial uptake, which prevent the bacteria from escaping vacuoles into cytoplasm. The heterogeneity in the interactions between L. monocytogenes and the macrophage indicate a complex relationship between the host and the pathogen governed by chemistries that promote and inhibit escape from vacuoles. This review examines the mechanisms used by activated and non-activated macrophages to kill microbes, and how those mechanisms are employed against L. monocytogenes.
listeriolysin O; ROI; RNI; NOS2; phagosome maturation; cholesterol-dependent cytolysin; interferon-gamma; Rab5; lysosome
Listeria monocytogenes evades the antimicrobial mechanisms of macrophages by escaping from the phagosome into the cytosolic space via a unique cytolysin that targets the phagosomal membrane, listeriolysin O (LLO), encoded by hly. Gamma interferon (IFN-γ), which is known to play a pivotal role in the induction of Th1-dependent protective immunity in mice, appears to be produced, depending on the bacterial virulence factor. To determine whether the LLO molecule (the major virulence factor of L. monocytogenes) is indispensable or the escape of bacteria from the phagosome is sufficient to induce IFN-γ production, we first constructed an hly-deleted mutant of L. monocytogenes and then established isogenic L. monocytogenes mutants expressing LLO or ivanolysin O (ILO), encoded by ilo from Listeria ivanovii. LLO-expressing L. monocytogenes was highly capable of inducing IFN-γ production and Listeria-specific protective immunity, while the hly-deleted mutant was not. In contrast, the level of IFN-γ induced by ILO-expressing L. monocytogenes was significantly lower both in vitro and in vivo, despite the ability of this strain to escape the phagosome and the intracellular multiplication at a level equivalent to that of LLO-expressing L. monocytogenes. Only a negligible level of protective immunity was induced in mice against challenge with LLO- and ILO-expressing L. monocytogenes. These results clearly show that escape of the bacterium from the phagosome is a prerequisite but is not sufficient for the IFN-γ-dependent Th1 response against L. monocytogenes, and some distinct molecular nature of LLO is indispensable for the final induction of IFN-γ that is essentially required to generate a Th1-dependent immune response.
Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.
Curcumin, a principal component of turmeric, acts as an immunomodulator regulating the host defenses in response to a diseased condition. The role of curcumin in controlling certain infectious diseases is highly controversial. It is known to alleviate symptoms of Helicobacter pylori infection and exacerbate that of Leishmania infection. We have evaluated the role of curcumin in modulating the fate of various intracellular bacterial pathogens. We show that pretreatment of macrophages with curcumin attenuates the infections caused by Shigella flexneri (clinical isolates) and Listeria monocytogenes and aggravates those caused by Salmonella enterica serovar Typhi CT18 (a clinical isolate), Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica. Thus, the antimicrobial nature of curcumin is not a general phenomenon. It modulated the intracellular survival of cytosolic (S. flexneri and L. monocytogenes) and vacuolar (Salmonella spp., Y. enterocolitica, and S. aureus) bacteria in distinct ways. Through colocalization experiments, we demonstrated that curcumin prevented the active phagosomal escape of cytosolic pathogens and enhanced the active inhibition of lysosomal fusion by vacuolar pathogens. A chloroquine resistance assay confirmed that curcumin retarded the escape of the cytosolic pathogens, thus reducing their inter- and intracellular spread. We have demonstrated that the membrane-stabilizing activity of curcumin is crucial for its differential effect on the virulence of the bacteria.
Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection.
However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs) deficient for autophagy (atg5−/−). Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO) can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3) lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO), induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5−/− mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5−/− BMDMs.
We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs.
Macrophages from mice infected with facultative intracellular organisms such as Listeria monocytogenes and BCG have been shown to resist infection by antigenically unrelated intracellular bacterial parasites. This study compares phagocytosis, bacterial growth inhibition, and oxidation of glucose by macrophages from normal mice, mice infected with listeria or BCG, or mice immunized with killed listeria in incomplete Freund's adjuvant. Macrophages from listeria- and BCG-infected mice ingested more listeria; 67 and 57%, respectively, had three or more cell-associated bacteria versus 22% of controls (P < 0.001). Peritoneal macrophages from listeria- and BCG-infected animals significantly (P < 0.001 covariance analysis) inhibited growth of listeria in suspension, whereas control macrophages had no such inhibitory effect. The rate of oxidation of glucose-1-14C was higher in macrophages from listeria- and BCG-infected mice than from either uninfected animals or those immunized with killed listeria. During phagocytosis of killed or live bacteria, or latex particles, the rate of glucose oxidation was increased (P < 0.01). These data suggest that the cellular immunity after infection by an intracellular organism is associated with an increase in metabolic activity of macrophages, namely, an increase in the rate of glucose oxidation resulting in enhancement of phagocytosis and killing.
Listeria monocytogenes are facultative intracellular pathogenic bacteria that can infect macrophages as well as non-professional phagocytes. After entry in the host cell, the bacteria escape from the phagosome into the cytoplasm. In murine macrophages and in cell lines derived from these cells, escape of L. monocytogenes from the phagosome is absolutely dependent on listeriolysin O (LLO) and facilitated by a secreted phosphatidylinositol-specific phospholipase C (PI-PLC) Work in this laboratory has previously demonstrated a LLO and PI-PLC-dependent translocation of host PKCβ isoforms. Pharmacological inhibition of PKCβ resulted in a significant reduction in permeabilization of the phagosome, and in the number of bacteria reaching the cytosol. These findings led to the prediction that the bacterial PI-PLC promotes escape through the production of diacylglycerol leading to the activation of host PKCβ. To test this hypothesis, bone marrow-derived macrophages (BMMφ) obtained from PKCβ knockout (PKCβKO) or C57Bl/6 mice were infected with L. monocytogenes. We observed that wild type L. monocytogenes escapes from the phagosome of PKCβKO BMMφ as well as they do from C57Bl/6 BMMφ. However, in PKCβKO BMMφ, L. monocytogenes uses a PI-PLC-independent, but phosphatidylcholine-preferring PLC (PC-PLC)-dependent pathway to facilitate escape. These findings strongly support the hypothesis that PI-PLC promotes escape through mobilization of host PKCβ
Listeria monocytogenes; PKCβ; PI-PLC; listeriolysin O; PC-PLC; knockout
Listeria monocytogenes is an intracellular bacterium that secretes proteins into the cytosol of infected macrophages. Major histocompatibility complex (MHC) class I molecules bind peptides that are generated by the degradation of bacterial proteins and present them to cytolytic T lymphocytes (CTL). In this study we have investigated CTL responses in L. monocytogenes-immunized mice to peptides that (i) derive from the L. monocytogenes proteins phosphatidylinositol-specific phospholipase C, lecithinase (most active on phosphatidylcholine), metalloprotease (Mpl), PrfA, and the ORF-A product and (ii) conform to the binding motif of the H2-Kd MHC class I molecule. We identified a nonamer peptide, Mpl 84-92, that is presented to L. monocytogenes-specific CTL by H2-Kd MHC class I molecules. Unlike other motif-conforming peptides derived from the secreted Mpl of L. monocytogenes, Mpl 84-92 is bound with high affinity by H2-Kd. Mpl 84-92 is the fourth L. monocytogenes-derived peptide found to be presented to CTL by the H2-Kd molecule during infection and demonstrates the importance of high-affinity interactions between antigenic peptides and MHC class I molecules for CTL priming.
Listeria monocytogenes is a Gram-positive facultative intracellular bacterium responsible for the food borne infection listeriosis, affecting principally the immunocompromised, the old, neonates and pregnant women. Following invasion L. monocytogenes escapes the phagosome and replicates in the cytoplasm. Phagosome escape is central to L. monocytogenes virulence and is required for initiating innate host-defence responses such as the secretion of the cytokine interleukin-1. Phagosome escape of L. monocytogenes is reported to depend upon host proteins such as γ-interferon-inducible lysosomal thiol reductase and the cystic fibrosis transmembrane conductance regulator. The host cytosolic cysteine protease calpain is required in the life cycle of numerous pathogens, and previous research reports an activation of calpain by L. monocytogenes infection. Thus we sought to determine whether host calpain was required for the virulence of L. monocytogenes. Treatment of macrophages with calpain inhibitors blocked escape of L. monocytogenes from the phagosome and consequently its proliferation within the cytosol. This was independent of any direct effect on the production of bacterial virulence factors or of a bactericidal effect. Furthermore, the secretion of interleukin-1β, a host cytokine whose secretion induced by L. monocytogenes depends upon phagosome escape, was also blocked by calpain inhibition. These data indicate that L. monocytogenes co-opts host calpain to facilitate its escape from the phagosome, and more generally, that calpain may represent a cellular Achilles heel exploited by pathogens.
Listeria monocytogenes is a facultative intracellular bacterial pathogen that can infect the placenta, a chimeric organ made of maternal and fetal cells. Extravillous trophoblasts (EVT) are specialized fetal cells that invade the uterine implantation site, where they come into direct contact with maternal cells. We have shown previously that EVT are the preferred site of initial placental infection. In this report, we infected primary human EVT with L. monocytogenes. EVT eliminated ∼80% of intracellular bacteria over 24-hours. Bacteria were unable to escape into the cytoplasm and remained confined to vacuolar compartments that became acidified and co-localized with LAMP1, consistent with bacterial degradation in lysosomes. In human placental organ cultures bacterial vacuolar escape rates differed between specific trophoblast subpopulations. The most invasive EVT—those that would be in direct contact with maternal cells in vivo—had lower escape rates than trophoblasts that were surrounded by fetal cells and tissues. Our results suggest that EVT present a bottleneck in the spread of L. monocytogenes from mother to fetus by inhibiting vacuolar escape, and thus intracellular bacterial growth. However, if L. monocytogenes is able to spread beyond EVT it can find a more hospitable environment. Our results elucidate a novel aspect of the maternal-fetal barrier.
Infection of the placenta and fetus is an important cause of pregnancy complications and fetal and neonatal morbidity and mortality. Listeria monocytogenes is an intracellular bacterial pathogen that causes pregnancy-related infections in humans. The pathogenesis of listeriosis during pregnancy is poorly understood. We have previously shown that transmission of L. monocytogenes from maternal cells and tissues to fetal cells occurs in the uterine implantation site, and that a small subpopulation of specialized fetal cells called extravillous trophoblasts are the preferred initial site of infection. Here we use primary human placental organ and cell culture systems to characterize the intracellular fate of L. monocytogenes in extravillous trophoblasts. We found that these cells entrap bacteria in vacuolar compartments where they are degraded and therefore reduce bacterial dissemination into deeper structures of the placenta. Our study provides new insights into the nature of the maternal-fetal barrier. Extravillous trophoblasts that are accessible to infection with intracellular pathogens from infected maternal cells have host defense mechanisms that constitute a bottleneck in maternal-fetal transmission.
Microbial pathogens have evolved mechanisms to overcome immune responses and successfully infect their host. Here, we studied how Listeria monocytogenes evades immune detection by peptidoglycan (PGN) modification. By analyzing L. monocytogenes muropeptides, we detected O-acetylated muramic acid residues. We identified an O-acetyltransferase gene, oatA, in the L. monocytogenes genome sequence. Comparison of PGN from parental and isogenic oatA mutant strains showed that the O-acetyltransferase OatA O-acetylates Listeria PGN. We also found that PGN O-acetylation confers resistance to different types of antimicrobial compounds targeting bacterial cell wall such as lysozyme, β-lactam antibiotics, and bacteriocins and that O-acetylation is required for Listeria growth in macrophages. Moreover, oatA mutant virulence is drastically affected in mice following intravenous or oral inoculation. In addition, the oatA mutant induced early secretion of proinflammatory cytokines and chemokines in vivo. These results suggest an important role for OatA in limiting innate immune responses and promoting bacterial survival in the infected host.
CD44 has been implicated in immune and inflammatory processes. We have analyzed the role of CD44 in the outcome of Listeria monocytogenes infection in murine bone marrow-derived macrophages (BMM). Surprisingly, a dramatically decreased intracellular survival of L. monocytogenes was observed in CD44−/− BMM. CD44−/− heart or lung fibroblast cultures also showed reduced bacterial levels. Moreover, livers from CD44−/−-infected mice showed diminished levels of L. monocytogenes. In contrast, intracellular growth of Salmonella enterica serovar Typhimurium was the same in CD44−/− and control BMM. The CD44-mediated increased bacterial proliferation was not linked to altered BMM differentiation or to secretion of soluble factors. CD44 did not mediate listerial uptake, and it played no role in bacterial escape from the primary phagosome or formation of actin tails. Furthermore, CD44-enhanced listerial proliferation occurred in the absence of intracellular bacterial spreading. Interestingly, coincubation of BMM with hyaluronidase or anti-CD44 antibodies that selectively inhibit hyaluronan binding increased intracellular listerial proliferation. Treatment of cells with hyaluronan, in contrast, diminished listerial growth and induced proinflammatory transcript levels. We suggest that L. monocytogenes takes advantage of the CD44-mediated signaling to proliferate intracellularly, although binding of CD44 to certain ligands will inhibit such response.
Listeria monocytogenes, a gram-positive motile bacterium which can cause severe bacterial infection in humans, is considered to be pathogenic by virtue of its ability to resist intracellular killing. Since the mechanism of intracellular survival is poorly understood, we assessed the sensitivity of L. monocytogenes to several potent antibacterial products. Phorbol myristate acetate (PMA)-stimulated polymorphonuclear cells (PMNs) produced extracellular antibacterial products which were inhibited completely by catalase, suggesting a role for oxidative agents in this process. L. monocytogenes in logarithmic (log) growth phase resisted PMA-stimulated PMN extracellular products significantly more than L. monocytogenes in stationary (stat) growth phase or Escherichia coli (three strains) in either phase of growth. The role of oxidative agents was studied further by using xanthine oxidase-xanthine, glucose oxidase-glucose, and myeloperoxidase enzyme systems to generate hydroxyl radical (.OH), hydrogen peroxide (H2O2), and hypochlorous acid (OCl-), respectively. L. monocytogenes in log phase resisted the antibacterial products of these enzyme systems under conditions which produced superoxide (O2-) and H2O2 at concentrations similar to those produced extracellularly by PMA-stimulated PMNs, while stat-growth-phase L. monocytogenes and E. coli in either phase of growth were susceptible. Antibacterial activity could be blocked or inhibited by exogenous catalase (for all oxygen radical-generating systems), mannitol, or desferoxamine (for xanthine oxidase-xanthine) and alanine (for myeloperoxidase), suggesting that .OH and OCl- were responsible for this activity. Log-phase L. monocytogenes had 2.5-fold higher bacteria-associated catalase activity, as compared with stat-phase L. monocytogenes. These experiments, therefore, suggest that log-phase L. monocytogenes resists oxidative antibacterial agents by producing sufficient catalase to inactivate these products. This may contribute to the ability of L. monocytogenes to survive intracellularly.
The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγflox/flox). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6Chi monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.
The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allows L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst.
Flow cytometry is a potentially powerful tool for analyzing the interactions of facultative intracellular bacteria and macrophages on a cellular level, particularly when fluorochromes are used to label the bacteria. We labeled Listeria monocytogenes and Salmonella typhimurium with a lipophilic dye, PKH-2, and used flow cytometry to investigate phagocytosis by J774A.1 cells and short-term bacterial survival. Labeled and unlabeled bacteria were identical in terms of viability, growth kinetics, and survival within macrophages, although recovery per macrophage was much greater for L. monocytogenes than for S. typhimurium. Using L. monocytogenes as a prototypical facultative intracellular bacterium, we estimated bacterial survival during phagocytosis on the basis of linear fluorescence measurements of infected J774A.1 cells and recovery of L. monocytogenes from sorted cells. The lower percentage of surviving L. monocytogenes in macrophages containing higher bacterial loads indicated the accumulation of nonviable bacteria within phagocytes. Removal of the external source of viable bacteria by washes and gentamicin treatment reduced the percentage of surviving intracellular L. monocytogenes to a baseline level, and all baseline levels were similar, regardless of bacterial load. Listeria enrichment recoveries, derived from individually sorted J774A.1 cells, demonstrated the heterogeneity of macrophages in intracellular bacterial survival, especially within heavily infected cells. These results indicated that survival of L. monocytogenes was dependent on the adaptations of a small fraction of bacteria within a population of macrophages which permit intracellular growth.
Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin O was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin O-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.
Subclinical infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes results in the development of protective antilisterial immunity. L. monocytogenes can infect hepatocytes, and antilisterial cytotoxic T lymphocytes (CTL) lyse Listeria-infected hepatocytes in a major histocompatibility complex (MHC) class Ia-restricted manner. It remained to be determined whether L. monocytogenes-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis. In this study, we showed that hepatocytes express MHC class Ib molecule Qa-1b mRNA and protein. We further showed that Listeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis, since C57BL/6-derived Listeria-infected hepatocytes were lysed by BALB/c-derived antilisterial CTL. These results establish that Listeria-infected hepatocytes are susceptible to cytolysis by MHC class Ib restricted Listeria-specific CTL.
Listeria monocytogenes, a facultative intracellular pathogen, readily enters cells and multiplies in the cytosol after escaping from phagosomal vacuoles. Macrophages exposed to gamma interferon, one of the main cellular host defenses against Listeria, become nonpermissive for bacterial growth while containing Listeria in the phagosomes. Using the human myelomonocytic cell line THP-1, we show that the combination of l-monomethyl arginine and catalase restores bacterial growth without affecting the phagosomal containment of Listeria. A previous report (B. Scorneaux, Y. Ouadrhiri, G. Anzalone, and P. M. Tulkens, Antimicrob. Agents Chemother. 40:1225–1230, 1996) showed that intracellular Listeria was almost equally sensitive to ampicillin, azithromycin, and sparfloxacin in control cells but became insensitive to ampicillin and more sensitive to azithromycin and sparfloxacin in gamma interferon-treated cells. We show here that these modulations of antibiotic activity are largely counteracted by l-monomethyl arginine and catalase. In parallel, we show that gamma interferon enhances the cellular accumulation of azithromycin and sparfloxacin, an effect which is not reversed by addition of l-monomethyl arginine and catalase and which therefore cannot account for the increased activity of these antibiotics in gamma interferon-treated cells. We conclude that (i) the control exerted by gamma interferon on intracellular multiplication of Listeria in THP-1 macrophages is dependent on the production of nitric oxide and hydrogen peroxide; (ii) intracellular Listeria may become insensitive to ampicillin in macrophages exposed to gamma interferon because the increase in reactive oxygen and nitrogen intermediates already controls bacterial growth; and (iii) azithromycin and still more sparfloxacin cooperate efficiently with gamma interferon, one of the main cellular host defenses in Listeria infection.
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.
Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses.
Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of non-activated macrophages within 30 min of infection. Macrophages activated with IFN-γ, LPS, IL-6, and a neutralizing antibody against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91phox−/− and NOS2−/− mice showed that vacuolar retention required ROI and was augmented by RNI. Live-cell imaging with the fluorogenic probe dihydro-2′,4,5,6,7,7′-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the combined actions of ROI and RNI in a small space.
macrophage; nitric oxide; lipopolysaccharide; bacterial