Epigallocatechin gallate inhibits intracellular survival of Listeria monocytogenes in macrophages

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-microbial and anti-tumor compound. The effects of EGCg on host defense mechanisms against Listeria monocytogenes infection were examined in vitro using mouse peritoneal exudate cells. The study showed that EGCg inhibited the intracellular growth of L. monocytogenes in macrophages. The enhancement of in vitro anti-L. monocytogenes activity by EGCg is not due to the modulation of reactive oxygen intermediates or the production of reactive nitrogen intermediates but due to the inhibition of its escaping from the phagosome into cytosolic space. Anti-L. monocytogenes of EGCg is through the inhibition of hemolytic and cholesterol-binding activity of listeriolysin O, which usually disrupts the phagosomal membrane in the escaping phase of L. monocytogenes.     

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.     

Upregulation of vascular endothelial growth factor by heat-killed Listeria monocytogenes in macrophages

We investigated the effect of heat-killed Listeria monocytogenes (HKLM) on the expression of vascular endothelial growth factor (VEGF) in RAW264.7 macrophage-like cells. The expression of VEGF was induced in RAW264.7 cells treated with HKLM. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, inhibited the induction of VEGF mRNA by HKLM. Induction of VEGF by HKLM was partially inhibited by treatment of cells with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, or a neutralizing antibody against tumor necrosis factor-alpha (TNF-alpha). In addition, HKLM induced phosphorylation of p38 MAPK. These results suggest that p38 MAPK and TNF-alpha are involved in the VEGF expression induced by HKLM in RAW264.7 cells. We confirmed that increased VEGF expression is immunohistochemically detected in splenic macrophages of mice infected with L. monocytogenes (L. monocytogenes). VEGF is thought to be involved in inflammatory reactions induced by L. monocytogenes infection.     

A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB

The multicopy sRNA LhrC of the intracellular pathogen Listeria monocytogenes has been shown to be induced under infection-relevant conditions, but its physiological role and mechanism of action is not understood. In an attempt to pinpoint the exact terms of LhrC expression, cell envelope stress could be defined as a specific inducer of LhrC. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. lapB mRNA, encoding a cell wall associated protein that was recently identified as an important virulence factor, was disclosed to be directly bound by LhrC leading to an impediment of its translation. Although LhrC binds to Hfq, it does not require the RNA chaperone for stability or lapB mRNA interaction. The mechanism of LhrC-lapB mRNA binding was shown to involve three redundant CU-rich sites and a structural rearrangement in the sRNA. This study represents an extensive depiction of a so far uncharacterized multicopy sRNA and reveals interesting new aspects concerning its regulation, virulence association and mechanism of target binding.     

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.     

Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between -1.6 and 14.5 degrees C. The mean minimum temperature for L. monocytogenes was +1.7 +/- 0.5 degrees C. The growth of non-haemolytic listerias was unobservable at +1.7 +/- 0.5 degrees C. The L. monocytogenes strains grew at about 0.6 degrees C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0 +/- 0.3 degrees C) than the other common serovar 4b (+1.3 +/- 0.4 degrees C). The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

Low-level iron-dependent mutants of Listeria monocytogenes and their virulence in macrophages

Listeria monocytogenes is an opportunistic intracellular pathogen capable of growth that requires iron for growth within phagocytic cells and virulence expression. In the presence of an appropriate concentration tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, this inhibition can be relieved by addition of dopamine, norepinephrine, or ferric citrate. By selection on streptonigrin medium supplemented with tropolone and norepinephrine, we have obtained two spontaneous mutants, Lm-8 and Lm-15, with the same iron dependence but lower iron dependence than the wild-type Lm-B38. The association between iron requirement and virulence of the two mutants and the wild type was studied in the J774 macrophage cell line. One hour after phagocytosis by the J774 macrophage cell line, the two mutants and the parental strain displayed no difference in the number of phagocytosed bacteria. Twenty-four hours after phagocytosis, the number of bacteria within the surviving macrophages was identical for the wild strain and the two clones. However, only 40% of macrophage cells infected with Lm-8 and 90% of those infected with Lm-15 were alive after 24 h in comparison with macrophage cells infected with the parental strain Lm-B38. These data demonstrate that there is no direct correlation between iron requirement and virulence of L. monocytogenes in the J774 macrophage cell line.     

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.     

Dynamic modeling of Listeria monocytogenes growth in pasteurized milk

AIMS: The development and validation of a dynamic model for predicting Listeria monocytogenes growth in pasteurized milk stored at both static and dynamic temperature conditions. METHODS AND RESULTS: Growth of inoculated L. monocytogenes in a commercial pasteurized whole milk product was monitored at various isothermal conditions from 1.5 to 16 degrees C. The kinetic parameters of the pathogen were modelled as a function of temperature using a square root type model, which was further validated using data from 92 published growth curves from eight different milk products. Compared to four published models for L. monocytogenes growth, the model developed in this study performed better, with a per cent discrepancy and bias of 49.1 and -1.01%, respectively. The performance of the model in predicting growth at dynamic temperature conditions was evaluated at four different fluctuating temperature scenarios with periodic temperature changes from -2 to 16 degrees C. The prediction of growth at dynamic storage temperature was based on the square root model in conjunction with the differential equations of the Baranyi and Roberts model, which were numerically integrated with respect to time. The per cent relative errors between the observed and the predicted growth of L. monocytogenes were less than 10% for all temperature scenarios tested. CONCLUSIONS: Available models from experiments conducted in laboratory media may result in significant overestimation of L. monocytogenes growth in pasteurized milk because they do not take into account factors such as milk composition (e.g. natural antimicrobial compounds present in milk) and the interactions of the pathogen with the natural microflora. The product-targeted model developed in the present study showed a high performance in predicting growth of L. monocytogenes in pasteurized milk under both static and dynamic temperature conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature fluctuations often occur during the transportation and storage of pasteurized milk. A high performance, dynamic model for the growth of L. monocytogenes can be a useful tool for effective management and optimization of product safety and can lead to more realistic estimations of pasteurized-milk related safety risks.     

Oxidative and phagocytic functions of macrophages during infections induced in mice by Mycobacterium intracellulare and Listeria monocytogenes

The oxidative metabolism (chemiluminescence and H2O2 release) and phagocytic activity of mouse peritoneal macrophages during chronic infections induced by Mycobacterium intracellulare and more acute infections due to Listeria monocytogenes were studied. In M. intracellulare infections, macrophage chemiluminescence in response to phorbol myristate acetate (PMA) was greatest at around 2 weeks, with a 1 week lag phase after infection, while the PMA-triggered H2O2 release was markedly enhanced even 1 d after challenge, and remained high thereafter for up to 10 weeks. The pattern of changes in the phagocytic activity of host macrophages in response to latex beads during this infection resembled the pattern seen with macrophage H2O2 release. In the L. monocytogenes infections, the PMA-triggered chemiluminescence of the host macrophages increased 4 d (in a sublethal infection) and 2 d (in a lethal infection) after bacterial challenge, whereas the PMA-triggered H2O2 release was markedly enhanced as early as 1 d after infection and the elevated level persisted until either the bacteria were eliminated or the animals died. The patterns of changes in phagocytic activity of the host macrophages during L. monocytogenes infection at sublethal and lethal doses differed. In the former, phagocytosis was most active in the early phase of infection, with a peak around day 2, followed by a rapid decrease; in the latter, the phagocytic ability increased more slowly, and remained elevated until the animals died. The results suggest that the macrophages induced by M. intracellulare are in a more activated state than are those induced by L. monocytogenes.     

A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes

We have examined the effect of covalently crosslinked profilin–actin (PxA), which closely matches the biochemical properties of ordinary profilin–actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of P_H133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin–actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin–actin is important for actin-based processes, and show that it has a key function in Listeria motility.     

The growth of Listeria monocytogenes in cheese packed under a modified atmosphere

The effect of modified atmosphere Packaging (MAP) on the growth of Listeria monocytogenes in mould ripened cheeses was studied at refrigeration temperatures (2-8.3 degrees C) over a storage period of 6 weeks. Control experiments in cling film with no atmospheric modification produced a lag time before growth of up to 1 week and rapid subsequent growth. MAP with a CO2 concentration of less than 20% allowed growth to occur but when O2 was incorporated; the lag time was reduced from 3 to 2 weeks and subsequent growth was also faster, producing an increase in cell numbers of 1.4 log cycles over the incubation period. N2-MAP in the absence of O2 increased the lag time to 3 weeks and slowed growth, while the inclusion of CO2 extended the lag to 3 weeks and slowed subsequent growth even more. In MAP with 80:10:10 (v/v/v) N2:CO2:O2, there was a lag period of 2-3 weeks before growth of L. monocytogenes occurred, while the total viable aerobic count (TVAC) decreased by 2-3 log cycles and the total Lactobacillus count showed little change. It was concluded that MAP was not suitable for preventing the growth of L. monocytogenes in such cheeses.     

Cutting edge: a novel nonoxidative phagosomal mechanism exerted by cathepsin-D controls Listeria monocytogenes intracellular growth

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells is a key issue in understanding the pathogenesis of this food-borne pathogen, which can cause diseases ranging from gastroenteritis to meningitis and abortion. In this study, we show that the lysosomal aspartyl-protease cathepsin-D (Ctsd) is of considerable importance for nonoxidative listericidal defense mechanisms. We observed enhanced susceptibility to L. monocytogenes infection of fibroblasts and bone-marrow macrophages and increased intraphagosomal viability of bacteria in fibroblasts isolated from Ctsd-deficient mice compared with wild type. These findings are further supported by prolonged survival of L. monocytogenes in Ctsd-deficient mice after infection. Transient transfection of Ctsd in wild-type cells was sufficient to revert these wild-type phagosomes back to microbicidal compartments. Based on infection experiments with mutant bacteria, in vitro degradation, and immunoprecipitation experiments, we suggest that a major target of cathepsin D is the main virulence factor listeriolysin O.     

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.