Macrophage intracellular signaling induced by Listeria monocytogenes

Macrophages are critical for control of Listeria monocytogenes infections; accordingly, the interactions of L. monocytogenes with these cells have been intensively studied. It has become apparent that this facultative intracellular pathogen interacts with macrophages both prior to entry and during the intracellular phase. This review covers recent work on signaling induced in macrophages by L. monocytogenes, especially intracellular signals induced by secreted proteins including listeriolysin O and two distinct phospholipases C.     

The in vitro inhibition of growth of intracellular Listeria monocytogenes by lymphocyte products

The listeria-inhibiting activity of culture supernatants from listeria-immune and nonimmune lymphocytes was assessed on listeria-infected macrophage cultures. It was found that supernatant from listeria-immune lymphocyte cultures stimulated in vitro with antigen was markedly inhibitory to the multiplication of intracellular listeria. Some inhibitory activity was also evident in supernatant from antigen-stimulated non-immune lymphocyte cultures. Supernatant from listeria-immune lymphocytes stimulated in vivo with antigen was capable of inhibiting listeria. Some inhibitory activity was still evident upon dilution of active supernatant at 1:100.     

Invasive extravillous trophoblasts restrict intracellular growth and spread of Listeria monocytogenes

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.     

Cutting edge: protective cell-mediated immunity to Listeria monocytogenes in the absence of myeloid differentiation factor 88

In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response. The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM). In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM. Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM. These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.     

Cutting edge: recombinant Listeria monocytogenes expressing a single immune-dominant peptide confers protective immunity to herpes simplex virus-1 infection

The vast majority of the world's population is infected with HSV. Although antiviral therapy can reduce the incidence of reactivation and asymptomatic viral shedding, and limit morbidity and mortality from active disease, it cannot cure infection. Therefore, the development of an effective vaccine is an important global health priority. In this study, we demonstrate that recombinant Listeria monocytogenes (Lm) expressing the H-2K(b) glycoprotein B (gB)(498-505) peptide from HSV-1 triggers a robust CD8 T cell response to this Ag resulting in protective immunity to HSV infection. Following challenge with HSV-1, immune-competent mice primed with recombinant Lm-expressing gB(498-505) Ag were protected from HSV-induced paralysis. Protection was associated with dramatic reductions in recoverable virus, and early expansion of HSV-1-specific CD8 T cells in the regional lymph nodes. Thus, recombinant Lm-expressing Ag from HSV represents a promising new class of vaccines against HSV infection.     

Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes

Lipoproteins of Gram-positive bacteria are involved in a broad range of functions such as substrate binding and transport, antibiotic resistance, cell signaling, or protein export and folding. Lipoproteins are also known to initiate both innate and adaptative immune responses. However, their role in the pathogenicity of intracellular microorganisms is yet poorly understood. In Listeria monocytogenes, a Gram-positive facultative intracellular human pathogen, surface proteins have important roles in the interactions of the microorganism with the host cells. Among the putative surface proteins of L. monocytogenes, lipoproteins constitute the largest family. Here, we addressed the role of the signal peptidase (SPase II), responsible for the maturation of lipoproteins in listerial pathogenesis. We identified a gene, lsp, encoding a SPase II in the genome of L. monocytogenes and constructed a deltalsp chromosomal deletion mutant. The mutant strain fails to process several lipoproteins demonstrating that lsp encodes a genuine SPase II. This defect is accompanied by a reduced efficiency of phagosomal escape during infection of eucaryotic cells, and leads to an attenuated virulence. We show that lsp gene expression is strongly induced when bacteria are still entrapped inside phagosomes of infected macrophages. The data presented establish, thus, that maturation of lipoproteins is critical for efficient phagosomal escape of L. monocytogenes, a process temporally controlled by the regulation of Lsp production in infected cells.     

Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a DeltaactA mutant strain, impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria colocalize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a DeltaplcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.     

Vaccination against the intracellular bacterium Listeria monocytogenes with a clonotypic antiserum

The T cell clone 26.1.1, which confers specific protection against the intracellular bacterium Listeria monocytogenes, was fused to BW 5147. The resulting T cell hybridoma, TLm1, could be stimulated to secret interleukin 2 by antigen plus accessory cells or concanavalin A. Stimulation was specific for an epitope expressed by L. monocytogenes EGD but not ATCC 19114 and was H-2I-A restricted. Antisera against TLm1 were raised in syngeneic mice and tested for their capacity to block TLm1 responses. Two antisera were identified that blocked antigen but not concanavalin A stimulation of TLm1 and did not affect antigen stimulation of similar but not identical L. monocytogenes-specific T cell hybridomas. Hence, these antisera had clonotypic activity. When these antisera were administered subcutaneously in complete Freund's adjuvant, mice were protected against a subsequent L. monocytogenes infection. Protection was antigen specific and H-2 nonrestricted. These findings suggest the feasibility of clonotypic antibodies for vaccination against intracellular bacterial infections.     

FbpA, a novel multifunctional Listeria monocytogenes virulence factor

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.     

pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes

Listeria monocytogenes grows in the cytosol of mammalian cells and spreads from cell to cell without exiting the intracellular milieu. During cell-cell spread, bacteria become transiently entrapped in double-membrane vacuoles. Escape from these vacuoles is mediated in part by a bacterial phospholipase C (PC-PLC), whose activation requires cleavage of an N-terminal peptide. PC-PLC activation occurs in the acidified vacuolar environment. In this study, the pH-dependent mechanism of PC-PLC activation was investigated by manipulating the intracellular pH of the host. PC-PLC secreted into infected cells was immunoprecipitated, and both forms of the protein were identified by SDS-PAGE fluorography. PC-PLC activation occurred at pH 7.0 and lower, but not at pH 7.3. Total amounts of PC-PLC secreted into infected cells increased several-fold over controls within 5 min of a decrease in intracellular pH, and the active form of PC-PLC was the most abundant species detected. Bacterial release of active PC-PLC was dependent on Mpl, a bacterial metalloprotease that processes the proform (proPC-PLC), and did not require de novo protein synthesis. The amount of proPC-PLC released in response to a decrease in pH was the same in wild-type and Mpl-minus-infected cells. Immunofluorescence detection of PC-PLC in infected cells was performed. When fixed and permeabilized infected cells were treated with a bacterial cell wall hydrolase, over 97% of wild-type and Mpl-minus bacteria stained positively for PC-PLC, in contrast to less than 5% in untreated cells. These results indicate that intracellular bacteria carry pools of proPC-PLC. Upon cell-cell spread, a decrease in vacuolar pH triggers Mpl activation of proPC-PLC, resulting in bacterial release of active PC-PLC.     

Label-free quantitative proteomics reveals the Steap3-Gm2a axis inhibiting the phagosomal escape of Listeria monocytogenes

As a pathogenic microorganism, Listeria monocytogenes is widely used in the research of bacterial pathogenesis and host defense. The phagosomal escape of L. monocytogenes is essential for its replication in the cytoplasm of the host. Here, we reported that the protein abundance of the Six-transmembrane epithelial antigen of the prostate 3 (Steap3) was decreased upon L. monocytogenes infection compared to uninfected cells in macrophages. However, the decreased Steap3 abundance was not regulated by the host but was caused by LLO secreted by L. monocytogenes. Functional experiments showed that deletion of Steap3 facilitated entry of L. monocytogenes from the phagosome into the cytoplasm. Then, the comprehensive proteomic analysis revealed that the deletion of Steap3 could affect the proteins abundance of the lysosomal signaling pathway in macrophages. Among these proteins affected by Steap3, we discovered that only the Ganglioside GM2 activator (Gm2a) inhibited the phagosomal escape of L. monocytogenes as Steap3. In summary, we found that the Steap3-Gm2a axis could restrict the phagosomal escape of L. monocytogenes and serve the potential molecular drug targets for antibacterial treatment.     

Listeria monocytogenes: a multifaceted model

The opportunistic intracellular pathogen Listeria monocytogenes has become a paradigm for the study of host-pathogen interactions and bacterial adaptation to mammalian hosts. Analysis of L. monocytogenes infection has provided considerable insight into how bacteria invade cells, move intracellularly, and disseminate in tissues, as well as tools to address fundamental processes in cell biology. Moreover, the vast amount of knowledge that has been gathered through in-depth comparative genomic analyses and in vivo studies makes L. monocytogenes one of the most well-studied bacterial pathogens.     

Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes

The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32-50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wild-type protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.     

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

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

Cutting edge: paradigm revisited: antibody provides resistance to Listeria infection.

Listeriolysin O (LLO) is a secreted pore-forming toxin of the facultative intracellular bacterium Listeria monocytogenes. We assessed the ability of a murine anti-LLO mAb to affect the course of infection in mice challenged with Listeria. This mAb was previously shown to be capable of neutralizing LLO-mediated pore formation in vitro, and here we show that the passive administration of this Ab to mice before infection provides increased resistance. Mice treated with the mAb were protected from a lethal challenge with virulent Listeria and showed a significant reduction in Listeria burden during the first hours to days postinfection. These effects of the Ab were independent of host B or T cells, since treatment with the mAb provided enhanced resistance to SCID mice. The titer of anti-LLO Abs during the regular infection of mice with Listeria was found to be low to negative.