Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes 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 allow L.?monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst.     

Drosophila S2 cells: an alternative infection model for Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that infects humans and animals. Its pathogenic strategy involves the expression of virulence proteins that mediate intracytosolic growth and cell-to-cell spread. A key virulence protein is the cholesterol-dependent cytolysin, listeriolysin O (LLO), which is largely responsible for mediating escape from the phagosome into the host cytosol. To study further the host processes exploited during L. monocytogenes infection, we sought to develop Drosophila S2 cells as a model for infection. Here, we show that S2 cells share a number of properties with mammalian cell culture models of infection. As with mouse macrophages, LLO was required for phagosomal escape from S2 cells. Furthermore, vacuolar escape was dependent on their acidification via the ATPase proton pumps, as bafilomycin A1 treatment sharply decreased escape. However, unlike in mouse macrophages, LLO mutants replicated in the phagosome of S2 cells. Drosophila cells are cholesterol auxotrophs, and exogenous cholesterol increased the infection rate of L. monocytogenes (LLO independent) and also augmented the efficiency of vacuolar escape (LLO dependent). With available genetic tools such as RNA interference, S2 cells could become an important model in the study of host-pathogen interactions.     

Dependency of caspase-1 activation induced in macrophages by Listeria monocytogenes on cytolysin, listeriolysin O, after evasion from phagosome into the cytoplasm

Listeriolysin O (LLO), an hly-encoded cytolysin from Listeria monocytogenes, plays an essential role in the entry of this pathogen into the macrophage cytoplasm and is also a key factor in inducing the production of IFN-gamma during the innate immune stage of infection. In this study, we examined the involvement of LLO in macrophage production of the IFN-gamma-inducing cytokines IL-12 and IL-18. Significant levels of IL-12 and IL-18 were produced by macrophages upon infection with wild-type L. monocytogenes, whereas an LLO-deficient mutant (the L. monocytogenes Deltahly) lacked the ability to induce IL-18 production. Complementation of Deltahly with hly completely restored the ability. However, when Deltahly was complemented with ilo encoding ivanolysin O (ILO), a cytolysin highly homologous with LLO, such a restoration was not observed, although ILO-expressing L. monocytogenes invaded and multiplied in the macrophage cytoplasm similarly as LLO-expressing L. monocytogenes. Induction of IL-18 was diminished when pretreated with a caspase-1 inhibitor or in macrophages from caspase-1-deficient mice, suggesting the activation of caspase-1 as a key event resulting in IL-18 production. Activation of caspase-1 was induced in macrophages infected with LLO-expressing L. monocytogenes but not in those with Deltahly. A complete restoration of such an activity could not be observed even after complementation with the ILO gene. These results show that the LLO molecule is involved in the activation of caspase-1, which is essential for IL-18 production in infected macrophages, and suggest that some sequence unique to LLO is indispensable for some signaling event resulting in the caspase-1 activation induced by L. monocytogenes.     

Role of bacterial hemolysin production in induction of macrophage Ia expression during infection with Listeria monocytogenes

The production of a hemolytic exotoxin (Hly) termed listeriolysin O (LLO) is a major determinant of the virulence of the Gram-positive bacterium Listeria monocytogenes. As determined by lethal inoculum size, LLO- strains of L. monocytogenes generally are several orders of magnitude less virulent than their LLO+ counterparts. The generation of protective anti-Listeria T cell immunity also has been shown to depend on the LLO phenotype of the bacteria present during primary infection, although the cellular basis of this observation is not known. The experiments described here address the role of LLO in regulation of the expression of class II MHC (Ia) molecules by murine macrophages. Because Ia expression by macrophages and other APC is thought to be a central factor in the generation of T cells specific for bacterial Ag, we have tested the hypothesis that the failure of LLO- strains to elicit anti-Listeria T cell responses might be secondary to an inability of these strains to stimulate increases in macrophage Ia levels. Our results show that the macrophage Ia response after i.p. injection of L. monocytogenes correlates strongly with the LLO phenotype of the bacteria. The presence of LLO+ organisms, even at very small numbers (as few as 10), elicits a striking increase in Ia expression by peritoneal macrophages. In contrast, even at very high numbers (up to 10(6) per mouse), LLO- bacteria fail to stimulate a strong Ia response. We also have analyzed macrophage Ia expression after injection of lysates of Escherichia coli expressing recombinant LLO protein. Similar to the results obtained with LLO+ and LLO- L. monocytogenes, we have observed Ia induction only with LLO+ lysates. Ia induction by this crude recombinant LLO preparation can be inhibited by cholesterol or heat. Furthermore, supernatants derived from cultures of LLO+ (but not LLO-) L. monocytogenes can cause Ia induction when administered via i.p. injection. Taken together, these findings suggest that the failure of macrophages to respond to LLO- organisms with an increase in Ia expression may be a major underlying cause of the failure of these bacteria to induce Listeria-specific protective T cell immunity. Furthermore, we propose that the induction of macrophage Ia expression in response to bacterial toxins such as Hly may represent one component of a set of early, innate immune mechanisms, and that this induction may provide a critical "bridge" to later, acquired, Ag-specific immune processes.     

Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection

We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes. An L. monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent promoter. Listeriolysin O (LLO) is a pore-forming cytolysin that mediates lysis of L. monocytogenes-containing phagosomes. Using hemolytic-activity assays and Western blot analysis, we demonstrated dose-dependent IPTG induction of LLO during growth in broth culture. Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG. We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth. Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line. Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent. The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed.     

Sec16p potentiates the action of COPII proteins to bud transport vesicles

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.     

Listeria monocytogenes evades killing by autophagy during colonization of host cells

Listeria monocytogenes is an intracellular pathogen that is able to colonize the cytosol of macrophages. Here we examined the interaction of this pathogen with autophagy, a host cytosolic degradative pathway that constitutes an important component of innate immunity towards microbial invaders. L. monocytogenes infection induced activation of the autophagy system in macrophages. At 1 h post infection (p.i.), a population of intracellular bacteria ( approximately 37%) colocalized with the autophagy marker LC3. These bacteria were within vacuoles and were targeted by autophagy in an LLO-dependent manner. At later stages in infection (by 4 h p.i.), the majority of L. monocytogenes escaped into the cytosol and rapidly replicated. At these times, less than 10% of intracellular bacteria colocalized with LC3. We found that ActA expression was sufficient to prevent autophagy of bacteria in the cytosol of macrophages. Surprisingly, ActA expression was not strictly necessary, indicating that other virulence factors were involved. Accordingly, we also found a role for the bacterial phospholipases, PI-PLC and PC-PLC, in autophagy evasion, as bacteria lacking phospholipase expression were targeted by autophagy at later times in infection. Together, our results demonstrate that L. monocytogenes utilizes multiple mechanisms to avoid destruction by the autophagy system during colonization of macrophages.     

The Listeria monocytogenes DnaK chaperone is required for stress tolerance and efficient phagocytosis with macrophages

Listeria monocytogenes is a facultative intracellular pathogen which can escape bactericidal mechanisms and grow within macrophages. The intracellular environment of macrophages is one of the most stressful environments encountered by an invading bacterium during the course of infection. To study the role of the major stress protein, DnaK, of L. monocytogenes in survival under intracellular stress induced by macrophage-phagocytosis as well as under extracellular environmental stresses, we cloned, sequenced, and analyzed the dnaK locus from L. monocytogenes. Then we constructed an insertional mutation in the dnaK gene by homologous recombination and characterized it. Sequencing has revealed that the dnaK locus consists of four open reading frames in the order hrcA-grpE-dnaK-dnaJ. The mutant grows neither at temperatures above 39 degrees C nor under acidic conditions e.g. pH 3.0. Using the macrophage cell line JA-4, the ability of the dnaK mutant to grow intracellularly was examined. Immediately after phagocytosis, the number of viable dnaK mutant bacteria found within macrophages was significantly lower compared to that of intracellular wild type bacteria. However, following a 1-3 h latency period, the mutant multiplied in a similar fashion to the wild type within macrophage cells. A quantitative analysis of intracellular bacteria in macrophage cells by microscope and a binding assay of bacteria to the surface of macrophages by ELISA revealed that the lower number of viable dnaK mutant in macrophages after phagocytosis is due to the low efficiency of phagocytosis resulting from the reduced binding capacity of the dnaK mutant. These results demonstrate that DnaK of L. monocytogenes is essentially required for survival under high temperatures and acidic conditions. Though it does not largely contribute to the survival of L. monocytogenes in macrophage cells, it is essential for efficient phagocytosis. This is the first evidence that DnaK is required for the efficient phagocytosis of a facultative intracellular pathogen with macrophages.     

Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence

Listeria monocytogenes is a bacterial pathogen that grows within the cytosol of infected host cells. Entry into the cytosol is largely mediated by a secreted bacterial cytolysin, listeriolysin O (LLO). In order to prevent host cell damage, the pore-forming activity of LLO is restricted to the phagosome. Compartmentalization of LLO requires a PEST-like sequence; PEST sequences can direct eukaryotic proteins for proteasomal degradation. Here we test the hypothesis that LLO's PEST-like sequence compartmentalizes pore-forming activity by targeting this bacterial protein for degradation in the host cytosol. We show that intracellular LLO was degraded in a proteasome-dependent manner, and that, prior to degradation, LLO was ubiquitinated and was phosphorylated within the PEST-like sequence. However, wild-type LLO and PEST region mutants had similarly short intracellular half-lives and both the wild-type and mutant proteins were stabilized by inhibitors of host proteasomes. Additionally, blocking host proteasomes did not cause toxicity in a wild-type infection, but enhanced the cytotoxicity of PEST region mutants. Together with the observation that PEST region mutants exhibit higher intracellular LLO levels than wild-type bacteria, these data suggest that LLO's PEST-like region does not mediate proteasomal degradation by the host, but controls LLO production in the cytosol.     

Lysosomal ubiquitin and the demise of Mycobacterium tuberculosis

The antimicrobial activity of macrophages is mediated by both oxidative and non-oxidative mechanisms. Oxidative mechanisms include the action of reactive oxygen and nitrogen intermediates on bacteria. Non-oxidative mechanisms include the maturation of the phagosome into an acidified, hydrolytically active compartment as well as the action of antimicrobial peptides. Mycobacterium tuberculosis parasitizes the host macrophage by arresting the normal maturation of its phagosome and resides in a compartment that fails to fuse with lysosomes. When bacteria are unable to regulate phagosome maturation, such as in activated macrophages, they are delivered to lysosomal compartments, where they are killed. Recent data indicate that the antimycobacterial mechanism of the lysosome is due in part to the action of ubiquitin-derived peptides.     

Avoiding death by autophagy: interactions of Listeria monocytogenes with the macrophage autophagy system

Autophagy restricts the growth of a variety of intracellular pathogens. However, cytosol-adapted pathogens have evolved ways to evade restriction by this innate immune mechanism. Listeria monocytogenes is a Gram-positive bacterial pathogen that utilizes a cholesterol-dependent pore-forming toxin, listeriolysin O (LLO), to escape from the phagosome. Autophagy targets L. monocytogenes in LLO-damaged phagosomes and also in the cytosol under some experimental conditions. However, this bacterium has evolved multiple mechanisms to evade restriction by autophagy, including actin-based motility in the cytosol and an as yet undefined mechanism mediated by bacterial phospholipases C (PLCs). A population of L. monocytogenes with inefficient LLO activity forms Spacious Listeria-containing Phagosomes (SLAPs), which are autophagosome-like compartments that do not mature, allowing slow bacterial growth within enlarged vesicles. SLAPs may represent a stalemate between bacterial LLO action and the host autophagy system, resulting in persistent infection.     

The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages

Under stress conditions, the facultative intracellular pathogen Listeria monocytogenes produces a ClpC ATPase, which is a general stress protein encoded by clpC and belonging to the HSP-100/Clp family. A ClpC-deficient mutant was obtained by gene disruption in strain LO28, which became highly susceptible to stress conditions in vitro . Intracellular growth of this mutant was restricted within macrophages, one of the major target cells of L . monocytogenes , during the infectious process. A quantitative electron microscope study showed that, contrary to wild-type bacteria that rapidly gain access to the cytoplasm of macrophages, mutant bacteria remained confined to membrane-bound phagosomes. Only a few mutant bacteria disrupted the phagosome membrane after 4 h of incubation, then polymerized actin filaments and multiplied within the cytoplasm. The ClpC ATPase, therefore, promotes early bacterial escape from the phagosome of macrophages, thus enhancing intracellular survival. The ClpC ATPase was produced in vivo during experimental infection by wild-type bacteria. The virulence of the ClpC-deficient mutant was severely attenuated in mice, with a three-log decrease in its 50% lethal dose compared with wild-type bacteria. Bacterial growth of mutant bacteria was strongly restricted in organs, presumably because of an impairment of intracellular survival in host tissues. Our results provide evidence that a general stress protein is required for the virulence of L . monocytogenes , which behaves as a virulence factor promoting intracellular survival of this pathogen.     

Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection

Listeria monocytogenes escapes from the phagosome of macrophages and replicates within the cytosolic compartment. The macrophage responds to L. monocytogenes through detection pathways located on the cell surface (TLRs) and within the cytosol (Nod-like receptors) to promote inflammatory processes aimed at clearing the pathogen. Cytosolic L. monocytogenes activates caspase 1, resulting in post-translational processing of the cytokines IL-1beta and IL-18 as well as caspase 1-dependent cell death (pyroptosis). We demonstrate that the presence of L. monocytogenes within the cytosolic compartment induces caspase 1 activation through multiple Nod-like receptors, including Ipaf and Nalp3. Flagellin expression by cytosolic L. monocytogenes was detected through Ipaf in a dose-dependent manner. Concordantly, detection of flagellin promoted bacterial clearance in a murine infection model. Finally, we provide evidence that suggests cytosolic L. monocytogenes activates caspase 1 through a third pathway, which signals through the adaptor protein ASC. Thus, L. monocytogenes activates caspase 1 in macrophages via multiple pathways, all of which detect the presence of bacteria within the cytosol.     

Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread

We investigated the role of listeriolysin O (LLO) and the bacterial phospholipases PI-PLC and PC-PLC in cell-to-cell spread of Listeria monocytogenes. We showed that LLO is essential for cell-to-cell spread in primary murine macrophages. Electron micrographs revealed that in the absence of continued LLO expression, bacteria remain trapped in secondary spreading vacuoles having either a double or single membrane. In bacteria lacking PI-PLC and PC-PLC, cessation of LLO expression after initiation of infection resulted in a significant increase in the proportion of bacteria trapped in double-membrane compartments. We propose that the bacterial phospholipases are involved in the dissolution of the inner membrane of the spreading vacuole, yet are not sufficient for disruption of the outer membrane. As a consequence, we identified LLO as a key factor in the disruption of the outer membrane. This model is consistent with the observation that LLO is dispensable for cell-to-cell spread from human macrophages into a cell type in which LLO is not required for vacuolar escape. These data suggest that during human infection, spreading of L. monocytogenes to distant organs is likely to occur even in the absence of LLO expression, and that the bacterial phospholipases may be sufficient to mediate continued cell-to-cell spread.     

Inhibition of calpain blocks the phagosomal escape of Listeria monocytogenes

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.     

Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes (Lm) evades the antimicrobial mechanisms of macrophages by escaping from vacuoles to the cytosol, through the action of the cytolysin listeriolysin O (LLO). Because of heterogeneities in the timing and efficiency of escape, important questions about the contributions of LLO to Lm vacuole identity and trafficking have been inaccessible. Expression of cyan fluorescent protein (CFP)-labelled endocytic membrane markers in macrophages along with a yellow fluorescent protein (YFP)-labelled indicator of Lm entry to the cytosol identified compartments lysed by bacteria. Lm escaped from Rab5a-negative, lysosome-associated membrane protein-1 (LAMP1)-negative, Rab7-positive, phosphatidylinositol 3-phosphate [PI(3)P]-positive vacuoles. Lm vacuoles did not label with YFP-Rab5a unless the bacteria were first opsonized with IgG. Wild-type Lm delayed vacuole fusion with LAMP1-positive lysosomes, relative to LLO-deficient Lm. Bacteria prevented from expressing LLO until their arrival into LAMP1-positive lysosomes escaped inefficiently. Thus, the LLO-dependent delay of Lm vacuole fusion with lysosomes affords Lm a competitive edge against macrophage defences by providing bacteria more time in organelles they can penetrate.     

The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum.

Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia.     

Listeriolysin O: a genuine cytolysin optimized for an intracellular parasite

Cholesterol-dependent cytolysins (CDCs)* are produced by a large number of pathogenic gram-positive bacteria. A member of this family, listeriolysin O (LLO), is produced by the intracellular pathogen Listeria monocytogenes. A unique feature of LLO is its low optimal pH activity (approximately 6) which permits escape of the bacterium from the phagosome into the host cell cytosol without damaging the plasma membrane of the infected cell. In a recent study (Glomski et al., 2002, this issue), Portnoy's group has addressed the molecular mechanism underlying the pH sensitivity of LLO. Unexpectedly, a single amino acid substitution in LLO L461T results in a molecule more active at neutral pH and promoting premature permeabilization of the infected cells, leading to attenuated virulence. This finding highlights how subtle changes in proteins can be exploited by bacterial pathogens to establish and maintain the integrity of their specific niches.