Role of FliF and FliI of Listeria monocytogenes in flagellar assembly and pathogenicity

Flagellar structures have been shown to participate in virulence in a variety of intestinal pathogens. Here, we have identified two potential flagellar genes of Listeria monocytogenes: lmo0713, encoding a protein similar to the flagellar basal body component FliF, and lmo0716, encoding a protein similar to FliI, the cognate ATPase energizing the flagellar export apparatus. Expression of fliF and fliI appears to be downregulated at 37 degrees C, like that of flaA, encoding flagellin. By constructing two chromosomal deletion mutants, we show that inactivation of either fliF or fliI (i) abolishes bacterial motility and flagella production, (ii) impairs adhesion and entry into nonphagocytic epithelial cells, and (iii) also reduces uptake by bone marrow-derived macrophages. However, the DeltafliF and DeltafliI mutations have only a minor impact on bacterial virulence in the mouse model, indicating that the flagellar secretion apparatus itself is not essential for survival in this animal model. Finally, among 100 human clinical isolates of L. monocytogenes tested, we found 20 strains still motile at 37 degrees C. Notably, all these strains adhered less efficiently than strain EGD-e to Caco-2 cells at 37 degrees C but showed no defect of intracellular multiplication. These data suggest that expression of the flagella at 37 degrees C might hinder optimal adhesion to epithelial cells but has no impact on intracytosolic survival of L. monocytogenes.     

Pathogenicity test for Listeria monocytogenes using immunocompromised mice.

The lethality of Listeria isolates was determined with normal adult mice and mice that were immunocompromised by treatment with 20 mg of carrageenan per kg. The mean 50% lethal doses (LD50s) of the pathogenic isolates were significantly lower (alpha = 0.05) in the immunocompromised mice than in the untreated mice, with an average reduction of 5.8 log10 units. In contrast, the mean LD50s of the nonpathogenic isolates were lower in the immunocompromised mice by an average of only 0.4 log10 unit, a difference that was not significant (alpha = 0.05). When immunocompromised mice were used, the LD50s of pathogenic Listeria monocytogenes isolates were lower than those of nonpathogenic L. innocua and L. seeligeri isolates by greater than or equal to 6 log10 units and lower than those of nonpathogenic L. ivanovii isolates by greater than or equal to 4 log10 units. Pathogenic L. monocytogenes isolates could be distinguished from nonpathogenic isolates by their ability to cause deaths in immunocompromised mice in 3 days at a dose of approximately 10(4) CFU per mouse. An alternative procedure using iron-overloaded mice failed to effectively differentiate pathogenic Listeria isolates.     

Pathogenicity of Listeria monocytogenes isolates in immunocompromised mice in relation to listeriolysin production

The virulence of 74 Listeria monocytogenes isolates from clinical cases and food products and of 11 isolates of other Listeria species was tested in mice immunocompromised with carrageenan. Isolates of species other than L. monocytogenes were not lethal to such mice. All 29 clinical isolates of L. monocytogenes (serotypes 1/2a, 1/2b, 4b) and 33 of 42 isolates of various serotypes isolated mainly from dairy products killed all test mice (100% lethality) at an inoculum of 10(4) cfu/mouse. All lethal strains of L. monocytogenes were haemolytic and possessed the 58-Kda band specific for listeriolysin O as demonstrated by SDS-PAGE immunoblotting. The nine avirulent strains of L. monocytogenes had detectable haemolytic activity, but in six of them this activity was significantly weaker than in virulent strains and the 58-Kda band was not detected. The other three avirulent strains were highly haemolytic and possessed the 58-Kda band, which suggests that other factor(s) could be involved in the virulence of L. monocytogenes.     

Pathogenicity and Immunogenicity of a Listeria monocytogenes Strain That Requires d-Alanine for Growth

Listeria monocytogenes is an intracellular bacterial pathogen that elicits a strong cellular immune response following infection and therefore has potential use as a vaccine vector. However, while infections by L. monocytogenes are fairly rare and can readily be controlled by a number of antibiotics, the organism can nevertheless cause meningitis and death, particularly in immunocompromised or pregnant patients. We therefore have endeavored to isolate a highly attenuated strain of this organism for use as a vaccine vector. d-Alanine is required for the synthesis of the mucopeptide component of the cell walls of virtually all bacteria and is found almost exclusively in the microbial world. We have found in L. monocytogenes two genes that control the synthesis of this compound, an alanine racemase gene (dal) and a d-amino acid aminotransferase gene (dat). By inactivating both genes, we produced an organism that could be grown in the laboratory when supplemented with d-alanine but was unable to grow outside the laboratory, particularly in the cytoplasm of eukaryotic host cells, the natural habitat of this organism during infection. In mice, the double-mutant strain was completely attenuated. Nevertheless, it showed the ability, particularly under conditions of transient suppression of the mutant phenotype, to induce cytotoxic T-lymphocyte responses and to generate protective immunity against lethal challenge by wild-type L. monocytogenes equivalent to that induced by the wild-type organism.Listeria monocytogenes is a gram-positive facultative intracellular microorganism which has been used for decades as a model pathogen for the study of cell-mediated immunity (24). Immunization of mice with a sublethal L. monocytogenes infection results in the generation of immunity which is largely major histocompatibility complex (MHC) class I mediated. Such infections generate CD8⁺ T cells, which can adoptively transfer immunity and specifically recognize and kill Listeria-infected target cells (3, 6, 17, 19).Recently, the cell biology of L. monocytogenes intracellular growth has been defined (47). Subsequent to internalization, the bacteria escape from a phagocytic vacuole and replicate in the host cell cytosol. Hence, secreted proteins of L. monocytogenes are delivered directly into the cytosol and into the MHC class I pathway of antigen processing and presentation (1, 5). Mutants of L. monocytogenes which are unable to enter the cytosol are absolutely avirulent and fail to immunize mice, and cells infected by such mutants are not recognized by L. monocytogenes-immune CD8⁺ T cells (6, 28).The natural properties of L. monocytogenes make it particularly attractive as a potential live vaccine vector for the induction of cell-mediated immunity to foreign antigens. Indeed, recombinant L. monocytogenes expressing such antigens successfully has been used to protect mice against lymphocytic choriomeningitis virus (15, 40) and influenza virus (22) infections and against lethal tumor cell challenge (32, 33). We have suggested the use of L. monocytogenes for the induction of cytolytic T cells directed against human immunodeficiency virus (HIV) antigens and have shown that strong cell-mediated immune responses against HIV-1 Gag protein can be induced in mice infected with recombinant L. monocytogenes carrying a chromosomal copy of the HIV-1 gag gene (13).Because of the potential broad use of this organism as a vaccine vector in infectious disease and cancer, the safety of L. monocytogenes becomes an important issue. While infections by L. monocytogenes are fairly rare and can readily be controlled by a number of antibiotics, the organism can nevertheless cause meningitis and death, particularly in immunocompromised or pregnant patients. An ideal vaccine strain of L. monocytogenes would be absolutely avirulent but fully immunogenic. We therefore sought to isolate a mutant which could enter the cytosol but have limited growth potential both in vivo and in the environment.d-Alanine is required for the synthesis of the mucopeptide component of the cell walls of virtually all bacteria, including L. monocytogenes (21, 23, 43), and is also found in the lipoteichoic acids of this and some other gram-positive organisms (11, 37). However, it is present in only trace quantities and fails to accumulate in vertebrates; the likely origin of these trace quantities is the breakdown products of intestinal and food bacteria (16, 20, 25, 29). We hypothesized that a strain of L. monocytogenes that is unable to synthesize this compound could be grown in the laboratory when supplemented with d-alanine but should be unable to grow outside the laboratory, particularly in the cytoplasm of eukaryotic host cells, the natural habitat of this organism during infection. The isolation of such a mutant of L. monocytogenes required the identification and inactivation of two genes, dal and dat. dal encodes alanine racemase, which catalyzes the reaction: l-alanine↔d-alanine. dat encodes d-amino acid aminotransferase, which catalyzes the reaction d-glutamic acid + pyruvate↔α-ketoglutaric acid + d-alanine. The dal dat double-mutant strain had the anticipated phenotype and in addition showed the ability to induce cytotoxic T-lymphocyte (CTL) responses and to generate protective immunity against lethal challenge by wild-type L. monocytogenes in infected mice under restricted conditions.     

Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model

Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA- and plcB-deficient L. monocytogenes strain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD(50)] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.     

Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape of L. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.     

Role of macrophage scavenger receptors in response to Listeria monocytogenes infection in mice

Type I and type II macrophage scavenger receptors (SR-A I/II) recognize a variety of polyanions including bacterial cell-wall products such as lipopolysaccharide, suggesting a role for SR-A I/II in immunity against bacterial infection. SR-A I/II-deficient (MSR-A-/-) mice were more susceptible to infection with listeriolysin-O (LLO)-producing Listeria monocytogenes. After infection, Kupffer cells in wild-type (MSR-A+/+) mice phagocytized larger numbers of Listeria than those in MSR-A-/- mice. The number and the diameter of hepatic granulomas were larger in MSR-A-/- mice than MSR-A+/+ mice. L. monocytogenes replicated at higher levels in the liver of MSR-A-/- mice compared with MSR-A+/+ mice, and macrophages from MSR-A-/- mice showed impaired ability to kill Listeria in vitro. However, macrophages from MSR-A+/+ and MSR-A-/- mice showed similar levels of listericidal activity against isogenic mutant L. monocytogenes with an inactivated LLO gene. The listerial phagocytic activities of MSR-A+/+ macrophages treated with an anti-SR-A I/II antibody (2F8) and MSR-A-/- macrophages were significantly impaired compared with untreated MSR-A+/+ macrophages, indicating that SR-A I/II function as a receptor for L. monocytogenes. Electron microscopy revealed that most L. monocytogenes had been eliminated from the lysosomes of MSR-A+/+ macrophages in vivo and in vitro. In contrast, L. monocytogenes rapidly lysed the phagosomal membrane and escaped to the cytosol in MSR-A-/- macrophages and in MSR-A+/+ macrophages treated with 2F8 before phagosome-lysosome fusion. These findings imply that SR-A I/II plays a crucial role in host defense against listerial infection not only by functioning as a receptor but also by mediating listericidal mechanisms through the regulation of LLO-dependent listerial escape from the macrophages.     

Penicillin-binding proteins in Listeria monocytogenes

The Penicillin-Binding Proteins (PBP) of Listeria monocytogenes 29-CCM-A: 454 (ATCC 15313) are described by the use of 125I-Penicillin X as radiotracer. The membranes of this tolerant bacilli contained at least five proteins with different affinities for the radiotracer or Dicloxacillin. The molecular weights of these proteins were estimated as 76, 74, 67, 66 and 47 KDa. Dicloxacillin induced the formation of straight filaments when present at sub-inhibitory concentrations, while Penicillin G did not induce any visible alteration in the morphology of this microorganism.     

Role of Listeria monocytogenes exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C in activation of human neutrophils

Polymorphonuclear leukocytes (PMN) are essential for resolution of infections with Listeria monocytogenes. The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation. Different Listeria strains, mutated in individual virulence genes, as well as purified LLO were used. Coincubation of human neutrophils with wild-type L. monocytogenes provoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis. Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD- and the avirulent strain L. innocua were used. These effects were fully reproduced by a recombinant L. innocua strain expressing LLO (INN+) and by the purified LLO molecule. LLO secretion was also required for PAF synthesis. However, wild-type L. monocytogenes was more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors. This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD- and by a recombinant L. innocua strain producing listerial PlcA. We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation. Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst. In this way, listerial exotoxins may influence the host defense against infections with L. monocytogenes.     

Role of proapoptotic BH3‐only proteins in Listeria monocytogenes infection

The ability of pathogens to influence host cell survival is a crucial virulence factor. Listeria monocytogenes (Lm) infection is known to be associated with severe apoptosis of hepatocytes and spleen cells. This impairs host defense mechanisms and thereby facilitates the spread of intracellular pathogens. The general mechanisms of apoptosis elicited by Lm infection are understood, however, the roles of BH3‐only proteins during primary Lm infection have not been examined. To explore the roles of BH3‐only proteins in Lm‐induced apoptosis, we studied Listeria infections in mice deficient in Bim, Bid, Noxa or double deficient in BimBid or BimNoxa. We found that BimNoxa double knockout mice were highly resistant to high‐dose challenge with Listeria. Decreased bacterial burden and decreased host cell apoptosis were found in the spleens of these mice. The ability of the BH3‐deficient mice to clear bacterial infection more efficiently than WT was correlated with increased concentrations of ROS, neutrophil extracellular DNA trap release and downregulation of TNF‐α. Our data show a novel pathway of infection‐induced apoptosis that enhances our understanding of the mechanism by which BH3‐only proteins control apoptotic host cell death during Listeria infection.     

Role of interleukin-6 in T-cell activation during primary and secondary infection with Listeria monocytogenes.

Injection of recombinant interleukin-6 (IL-6) into mice enhances recovery from infection with Listeria monocytogenes. In this study, the role of IL-6 during primary and secondary Listeria infection was further tested. Neutralization of IL-6 by polyclonal antibody exacerbated primary infection and significantly delayed gamma interferon production by cultured spleen cells. In contrast, administration of anti-IL-6 antibody at the time of secondary infection did not affect the recovery of mice from infection or gamma interferon production, showing that activated T cells are not dependent on IL-6.     

Ingested Listeria monocytogenes survive and multiply in protozoa

Listeria monocytogenes cells are ingested by protozoa such as Acanthamoeba sp. or Tetrahymena pyriformis. However, they are not killed, but survive within the protozoa and may multiply intracellularly. The protozoa are lysed within about 8 days, releasing viable L. monocytogenes. No co-existence was observed between L. monocytogenes and Tetrahymena. A co-culture of L. monocytogenes and Acanthamoeba sp. showed a decay of locomotive forms and release of listeria from vegetative protozoan cells whereas the bacteria were destroyed in cysts. These phenomena provide an insight into the pathogenesis of listeria infection in man and warm-blooded animals because intracellular processes occurring in protozoa after ingestion of L. monocytogenes may be similar to those observed in mammalian cells.     

Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii.

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.     

Critical Role for the Host GTPase-Activating Protein ARAP2 in InlB-Mediated Entry of Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses culminating in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. Interaction of InlB with the Met receptor elicits host downstream signaling pathways that promote F-actin cytoskeletal changes responsible for pathogen engulfment. Here we show that the mammalian signaling protein ARAP2 plays a critical role in cytoskeletal remodeling and internalization of Listeria. Depletion of ARAP2 through RNA interference (RNAi) caused a marked inhibition of InlB-mediated F-actin rearrangements and bacterial entry. ARAP2 contains multiple functional domains, including a GTPase-activating protein (GAP) domain that antagonizes the GTPase Arf6 and a domain capable of binding the GTPase RhoA. Genetic data indicated roles for both the Arf GAP and RhoA binding domains in Listeria entry. Experiments involving Arf6 RNAi or a constitutively activated allele of Arf6 demonstrated that one of the ways in which ARAP2 promotes bacterial uptake is by restraining the activity of Arf6. Conversely, Rho activity was dispensable for Listeria internalization, suggesting that the RhoA binding domain in ARAP2 acts by engaging a host ligand other than Rho proteins. Collectively, our findings indicate that ARAP2 promotes InlB-mediated entry of Listeria, in part, by antagonizing the host GTPase Arf6.Listeria monocytogenes is a Gram-positive, food-borne bacterial pathogen capable of causing gastroenteritis, meningitis, or abortions (39, 32). Listeria induces its own internalization (entry) into nonphagocytic mammalian cells, a process that likely plays an important role in traversal of the intestinal, placental, and blood-brain barriers (7, 14, 16, 23). One of the pathways of Listeria entry is mediated by interaction of the bacterial surface protein InlB with its host receptor, the Met receptor tyrosine kinase (16, 37).InlB-Met interaction triggers activation (tyrosine phosphorylation) of the Met receptor and subsequent rearrangements in the F-actin cytoskeleton of the mammalian cell (16, 29). These cytoskeletal changes remodel the host cell surface, resulting in engulfment of adherent Listeria. One of the host mammalian proteins that acts downstream of Met to promote F-actin rearrangements and bacterial entry is type IA phosphoinositide (PI) 3-kinase (8, 17, 18). This PI 3-kinase is a heterodimeric enzyme comprised of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (4, 11). p85-p110 generates phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], which are lipid second messengers that regulate a variety of biological processes, including growth, survival, and motility of mammalian cells. A plethora of downstream “target” proteins that bind PI(4,5)P₂ and/or PI(3,4,5)P₃ and mediate the biological effects of p85-p110 have been identified (4, 24). However, mammalian proteins that act downstream of type IA PI 3-kinase to control Listeria uptake have yet to found.In this work, we demonstrate that the human GTPase-activating protein (GAP) ARAP2 is required for InlB-mediated cytoskeletal changes and entry of Listeria. ARAP2 is known to bind PI(3,4,5)P₃, resulting in upregulation of a GAP domain that inactivates the mammalian GTPase Arf6 (42). We provide genetic evidence indicating that the ArfGAP domain of ARAP2 stimulates Listeria entry by antagonizing Arf6. ARAP2 has several functional domains in addition to its ArfGAP domain. One of these domains in ARAP2 interacts with the mammalian GTPase RhoA (42). Our genetic data demonstrate that this “RhoA binding” (RB) domain also plays a critical role in bacterial entry. Surprisingly, pharmacological experiments indicate that the RB domain controls Listeria uptake through an unknown mechanism that does not involve Rho proteins. Our work indicates a key role for host ARAP2 in InlB-mediated entry of Listeria. One of the likely ways that type IA PI 3-kinase controls entry of Listeria is through regulation of ARAP2.     

Induced biliary excretion of Listeria monocytogenes

Listeria monocytogenes is a ubiquitous gram-positive bacterium that can cause systemic and often life-threatening disease in immunocompromised hosts. This organism is largely an intracellular pathogen; however, we have determined that it can also grow extracellularly in animals, in the lumen of the gallbladder. The significance of growth in the gallbladder with respect to the pathogenesis and spread of listeriosis depends on the ability of the bacterium to leave this organ and be disseminated to other tissues and into the environment. Should this process be highly inefficient, growth in the gallbladder would have no impact on pathogenesis or spread, but if it occurs efficiently, bacterial growth in this organ may contribute to listeriosis and dissemination of this organism. Here, we use whole-body imaging to determine the efficacy and kinetics of food- and hormone-induced biliary excretion of L. monocytogenes from the murine gallbladder, demonstrating that transit through the bile duct into the intestine can occur within 5 min of induction of gallbladder contraction by food or cholecystokinin and that movement of bacteria through the intestinal lumen can occur very rapidly in the absence of fecal material. These studies demonstrate that L. monocytogenes bacteria replicating in the gallbladder can be expelled from the organ efficiently and that the released bacteria move into the intestinal tract, where they pass into the environment and may possibly reinfect the animal.