A P60 mutant of Listeria monocytogenes is impaired in its ability to cause infection in intragastrically inoculated mice

A spontaneous P60 mutant of Listeria monocytogenes was less able to cause systemic infection in A/J mice, following intragastric inoculation, than the parental wild type strain (SLCC 5764, serotype 1/2a). Significantly fewer CFU were recovered from internal organs (spleen, liver, gall bladder) and from the cecum of mice inoculated intragastrically with the P60 mutant than mice inoculated with wild type L. monocytogenes. The P60 mutant also exhibited a diminished ability to invade and multiply within Caco-2 intestinal epithelial cells. These findings indicate that P60 is required for maximal virulence of L. monocytogenes in the gastrointestinal tract of mice.     

Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread

To dissect the regulatory and structural requirements for Listeria monocytogenes intracellular growth and cell-to-cell spread, we designed a protocol based on transposon mutagenesis and the isolation of mutants which form small plaques in monolayers of mouse L2 cell fibroblasts. Two different transposable elements were used to generate libraries of insertion mutants: Tn916 and a derivative of Tn917-lac, Tn917-LTV3. Ten classes of mutants were isolated and evaluated for growth and cell-to-cell spread in J774 mouse macrophagelike cells, Henle 407 human epithelial cells, and mouse bone marrow-derived macrophages. Mutants were also evaluated for secretion of hemolysin and phospholipase (assayed by egg yolk opacity) and association with F-actin in the cytoplasm of cells, using NBD-phallacidin staining. The ten classes of mutants included (i) mutants showing abortive intracellular and extracellular growth; (ii) mutants showing abortive intracellular growth; (iii) rough mutants; (iv) mutants showing greatly reduced hemolysin and phospholipase secretion but showing normal growth in cells and little or no association with F-actin; (v) mutants with mutations mapping to an open reading frame (ORF) adjacent to hlyA and referred to as ORF U, lacking phospholipase activity, and with 50% normal hemolysin activity; (vi) mutants with reduced secretion of both hemolysin and phospholipase; (vii) nonhemolytic mutants with mutations mapping to the structural gene, hlyA; (viii) mutants with 25% normal hemolysin secretion and absolutely no association with F-actin; (ix) mutants with mutations mapping to ORF U, lacking phospholipase activity, and with normal hemolysin activity; and (x) mutants showing a mixed-plaque morphology but normal for all other parameters.     

Sodium bicarbonate enhances the severity of infection in neutropenic mice orally inoculated with Listeria monocytogenes EGD

Epidemiological studies have suggested an association between antacid therapy and development of listeriosis in humans. In this study we used a neutropenic mouse model to demonstrate that oral administration of sodium bicarbonate shortly before intragastric (i.g.) inoculation with Listeria monocytogenes EGD (serotype 1/2a) significantly increased the severity of the resulting systemic infection. An explanation for this observation is provided by evidence that L. monocytogenes EGD is rapidly inactivated in synthetic gastric fluid at pH below 5. A second strain of L. monocytogenes (CM [serotype 1/2b]) exhibited little ability to cause systemic infection following i.g. inoculation and was not significantly enhanced by administration of sodium bicarbonate. Strain CM was readily inactivated in synthetic gastric fluid even at pH 7. These data suggest that gastric acidity and enzymes provide some innate defense against gastrointestinal listeriosis in neutropenic mice.     

Hemolysin is required for extraintestinal dissemination of Listeria monocytogenes in intragastrically inoculated mice.

In this study we demonstrated that a hemolytic strain of Listeria monocytogenes, but not a nonhemolytic mutant derived from it, translocated in substantial numbers to the mesenteric lymph nodes, spleen, and liver after intragastric inoculation of mice. Growth at 4 degrees C prior to inoculation did not increase the virulence of the nonhemolytic mutant. These results indicate that hemolytic activity is required for the virulence of L. monocytogenes via the gastrointestinal tract, as has been shown previously for parenteral challenge.     

Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua.

Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. The species secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by the plcA gene. A plcA gene from L. monocytogenes was cloned downstream of a gram-positive promoter in the plasmid pWS2-2. To determine what effect plcA would have on intracellular survival when introduced into Listeria innocua, a species that does not growth intracellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/pWS2-2 was confirmed on a brain heart infusion-phosphatidylinositol agar plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured in the macrophage-like cell line J774 by Giemsa staining and viable count determinations at specific time points following infection. The J774 cells infected with wild-type L. innocua showed a falling viable count through 8 h postinfection. Although J774 cells infected with L. innocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h postinfection before a subsequent decline again at 16 h postinfection. Giemsa staining revealed fewer than 6 bacteria in individual macrophage cells at 2 h postinfection, and yet approximately 15% of the J774 cells had 6 to 12 bacteria localized to one area of the macrophage cell after 6 h; moreover, electron micrographs showed that the L. innocua/pWS2-2 cells were replicating inside the phagosome of the host cell. Furthermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the compartments were acidified. These results demonstrate that L. innocua cells transformed with the plasmid-borne plcA gene, and expressing functional PI-PLC, are able to grow intracellularly in what appear to be phagolysosomes, although between 3 and 6 h is needed for this to manifest itself. Intracellular growth specifically in L. innocua may be a secondary function associated with the plcA gene product. The addition of this one gene, plcA, to a species of Listeria that in the wild-type state does not replicate intracellularly apparently can now allow some of the bacteria to transiently multiply inside the phagosomes of host macrophage cells.     

Transforming growth factor beta is protective in host resistance against Listeria monocytogenes infection in mice.

The role of transforming growth factor beta (TGF-beta) in host resistance against Listeria monocytogenes infection was studied with mice. The constitutive expression of TGF-beta 1 mRNA was observed in the spleens and livers of mice before and after infection. Injecting the mice with anti-TGF-beta 1 peptide serum resulted in diminished antilisterial resistance, whereas the administration of human platelet-derived TGF-beta 1 enhanced the resistance. Moreover, mice were protected against lethal infection when treated with TGF-beta 1. These results suggest the TGF-beta 1 might be involved in antilisterial resistance. On the other hand, injecting the mice with TGF-beta 1 resulted in a decrease in the titers of endogenous gamma interferon, tumor necrosis factor alpha, and interleukin-6, which are crucial in antilisterial resistance, in sera and in extracts of spleen and liver. Thus, a complicated mechanism might be involved in the role of TGF-beta 1 in host resistance against L. monocytogenes infection.     

Cytokine profile and natural killer cell activity in Listeria monocytogenes infected mice treated orally with Petiveria alliacea extract

In this work, we investigated the effects of Petiveria alliacea extract on the production of Th1-type and Th2-type cytokines and on NK cells activity in normal and Listeria monocytogenes infected mice. Our results demonstrated that in normal/non-infected mice P. alliacea administration led to increased levels of Interleukin-2 (IL-2). The infection alone enhanced INF-γ levels and NK cell activity at 48 and 72 hours of infection. The treatment with five consecutive doses of 1000mg/kg/day of P. alliacea extract, given previously to infection, led to further increases in IL-2 levels, in relation to normal/non-infected/P. alliacea treated controls, and in INF-γ levels at 72h of infection, compared to infected mice. On the other hand, the production of IL-4 and IL-10 were not altered either by the infection or by the treatment with P. alliacea extract. NK cells activity increased at 48h and 72h following the inoculation of the bacteria. When mice were treated with P. alliacea previously to infection, NK activity was higher than that observed at 48h, 72h and 120h of infection in the infected animal. Based on these findings we suggest that P. alliacea up-regulates anti-bacterial immune response by enhancing both Th1 function and the activity of NK cells.     

NaCl consumption is attenuated in female KCNE1 null mutant mice.

The role of potassium channels in the regulation of NaCl intake has not been investigated previously. One potassium channel, KCNQ1, and its regulator, KCNE1, are expressed in salivary glands and kidneys, and KCNE1 null mutant mice are deficient in KCNQ1 potassium currents. To understand the role of the KCNQ1/KCNE1 channel complex in NaCl taste and intake, we compared the NaCl consumption of KCNE1 +/+ (129/Sv), KCNE1 +/-, and KCNE1 -/- mice using two-bottle intake tests and lick rate tests. Although KCNE1 +/+ and KCNE1 +/- mice exhibited consumption patterns for 75-150 mM NaCl solutions considered typical for 129/Sv mice, the KCNE1 -/- null mutant 129/Sv mice were indifferent to or rejected them. This effect was observed in female mice only, required prior exposure to NaCl solutions, and the extent of rejection was greater after prior exposure to 150 mM NaCl solution than 75 mM NaCl solution. No differences were observed in the avidity for KCl solutions or in lick rates of naive mice for 150 or 300 mM NaCl solutions. These results demonstrate that a single potassium channel gene can influence voluntary NaCl intake. We speculate that disruption of the KCNE1 gene impairs sodium metabolism in female mice drinking high levels of 150 mM NaCl, which causes malaise that becomes associated with NaCl taste, and as a consequence, reduced preference for NaCl.     

Expression of truncated Internalin A is involved in impaired internalization of some Listeria monocytogenes isolates carried asymptomatically by humans

Fourteen human carriage Listeria monocytogenes isolates were compared to sporadic and epidemic-associated human strains in order to ascertain the pathogenic behavior of these unrecognized asymptomatic strains. Experimental infection of 14-day-old chick embryos revealed that the majority of the carriage strains were attenuated for virulence. Of the 10 attenuated carriage strains, 5 were affected in their invasion capacities in vitro. Western blot analysis with antibody directed against InlA, the surface protein implicated in the internalization in host cells, allowed correlation between the ability of the carriage strains to enter Caco-2 cells and InlA expression. Indeed, these five carriage strains produced truncated forms of InlA. Four of the five truncated forms of InlA had an apparent molecular mass of 47 kDa. In order to assess the existence of a genetic lineage, partial sequences of inlA gene of these four strains were compared and revealed that they had a high degree of sequence conservation at the gene (99.86%) and amino acid (100%) levels. Comparison of their nucleotide sequences with that of the corresponding segment of inlA from EGD-e and Scott A strains, taken as epidemic references, showed more divergence. Taken together, these observations suggest the presence of specific traits that characterize L. monocytogenes strains isolated during asymptomatic carriage. Some of these traits could provide some explanations about the determinants that make them unable to cause systemic human infection.     

Mutants of Listeria monocytogenes defective in In vitro invasion and cell-to-cell spreading still invade and proliferate in hepatocytes of neutropenic mice

Listeria monocytogenes mutants defective in the actA gene, the plcB gene, and the inlA and inlB genes were less virulent when injected intravenously into BALB/c mice. The growth of these strains as well as of the virulent wild-type strains was increased by treating mice with a neutrophil-specific depleting monoclonal antibody, RB6-8C5. Histologic examination of the livers of the treated animals showed intrahepatocytic proliferation of the listeriae in all cases. Our data show that more than one pathway exists that allows L. monocytogenes to invade parenchymal cells. One pathway most likely involves the actA and plcB gene products, and a second one probably involves the internalins.     

Aberrant macrophage and neutrophil population dynamics and impaired Th1 response to Listeria monocytogenes in colony-stimulating factor 1-deficient mice

Listeria monocytogenes, a facultative intracellular bacterium, has been used extensively to study innate immune responses. Macrophages act as hosts for this bacterium as well as a major defense against it. Using mice homozygous for a null mutation (Csf1(op)) in the gene for the mononuclear phagocytic growth factor colony-stimulating factor 1 (CSF-1), we have demonstrated that CSF-1-regulated macrophages were essential to defend against a listerial infection. In the absence of CSF-1, monocytes were not recruited to the sites of infection due to the lack of synthesis of the macrophage chemoattractant chemokine MCP-1. In addition, there was no burst of interleukin-10 (IL-10) synthesis that has been shown to result in the egress of neutrophils from sites of infection. Consequently, neutrophils were not replaced by macrophages, and numerous neutrophil-filled microabscesses developed, followed by tissue destruction and death of the mice. In the CSF-1 nullizygous mice compared to wild-type mice, there was also a very low synthesis of gamma interferon (IFN-gamma), resulting in reduced macrophage activation. However, the concentrations of the IFN-gamma-inducing cytokines IL-12 and IL-18 at this bacterial load were similar in these mutant mice. In contrast, IL-6 concentrations were dramatically reduced. Administration of IL-6 to Csf1(op)/Csf1(op) mice significantly increased the synthesis of IFN-gamma and reduced the bacterial burden to a greater extent than treatment with IFN-gamma alone. These data indicate that IL-6 occupies a central role in the CSF-1-regulated macrophage response to L. monocytogenes.     

A prfA transposon mutant of Listeria monocytogenes F2365, a serotype 4b strain, is able to survive in the gastrointestinal tract but does not cause systemic infection of the spleens and livers of intragastrically inoculated mice

prfA is a member of the Crp/Fnr family of global regulatory genes in Listeria monocytogenes that has been shown previously to regulate several key virulence determinants both in vitro and in parenterally inoculated laboratory rodents. However, the role of prfA in the ability of L. monocytogenes to cause infection via the gastrointestinal (GI) tract has not been clearly established. In this study, we used a prfA transposon mutant of L. monocytogenes F2365, a serotype 4b strain, to assess the role of prfA in the pathogenesis of gastrointestinal listeriosis in mice. We found that the prfA mutant was able to survive in the GI tract (i.e., cecum) of mice, albeit in numbers somewhat less than those of the wild-type parent strain of L. monocytogenes. However, mice inoculated with the prfA mutant did not exhibit systemic infection of the spleen and liver, as was noted for mice inoculated with the wild-type parent strain. Survival of the prfA mutant in synthetic gastric fluid at pH 2.5 or 5 was somewhat reduced compared to that of the wild-type strain, as was its ability to invade and multiply within differentiated human intestinal epithelial cells (Caco-2 cells). Prior infection with the prfA mutant gave mice some protection against a subsequent challenge with virulent L. monocytogenes, although much less than that gained by prior gastrointestinal infection with the wild-type parent strain. These findings indicate that the global regulatory gene prfA is dispensable for colonization of the GI tract in mice but not for systemic infection.     

Lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus

Lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (PEDV) strain CV-777 and challenged 21 days later with the virulent isolate of the same virus. A lymphoproliferative assay was developed in which mononuclear cells isolated from lymphoid tissues at different postinoculation and postchallenge days underwent a secondary in vitro stimulation with semipurified antigen obtained from PEDV-infected cell cultures. Vigorous lymphocyte proliferative responses were detected in the pigs inoculated with the virulent PEDV at postinoculation days 4-21, especially in the mesenteric lymph nodes and the blood; however, in the spleen this response was lower and less regular. The pigs inoculated with the attenuated virus showed a less intense response, the higher lymphocyte proliferation also corresponded to the mononuclear cells from mesenteric lymph nodes. Lymphocyte proliferation responses showed high correlations with protection against homologous challenge with virulent PEDV, and this correlation was higher in the gut associated lymphoid tissues (mesenteric lymph nodes). The cell proliferation response detected in blood mirrored that detected in the mesenteric lymph nodes, and showed also good correlation with protection. The results confirm that T-cell-helper function, assessed by lymphocyte proliferation responses, contributes to establishing a protective immune response against PEDV infections.     

The defined attenuated Listeria monocytogenes Δmpl2 mutant is an effective oral vaccine carrier to trigger a long-lasting immune response against a mouse fibrosarcoma

Listeria monocytogenes has been proposed as a carrier to elicit major histocompatibility complex class-I restricted immune responses able to protect against tumor challenge. In this study the properties of the attenuated L. monocytogenes Δmpl2 mutant has been evaluated in vivo against a highly aggressive mouse fibrosarcoma which expresses β-galactosidase (β-gal) as a tumor-associated antigen (TAA). Immunization with the vaccine prototypes resulted in both elicitation of specific antibodies and generation of cytotoxic lymphocytes (CTL). Oral vaccination protected 55–64% of the immunized animals from tumor take (p < 0.01) and strongly reduced the average size of the tumor in the other 34–45% (p < 0.01). Vaccinated mice developed a long-lasting response, which resulted in 100% protection from a subsequent tumor challenge. Substitution of the whole TAA by its CTL-defined immunodominant epitope resulted in 43% protection, suggesting a contribution of the humoral response to the observed antitumor effect. No statistically significant differences were observed in the antitumor response when mice were immunized with strains expressing the immunodominant TAA epitope in the context of carrier proteins which were either exported or restricted to the bacterial cytoplasm. This suggests that the topology of the recombinant antigen does not play a major role in the outcome of the protective response.     

Analysis of macrophage bactericidal function in genetically resistant and susceptible mice by using the temperature-sensitive mutant of Listeria monocytogenes.

Innate resistance to infection by Listeria monocytogenes is genetically controlled and is critically dependent on prompt macrophage recruitment to the sites of infection. Experiments reported here were designed to examine whether there was an additional, qualitative difference between the intrinsic bactericidal activity of the inflammatory macrophages of genetically resistant (C57BL/6J) and susceptible (A/J) hosts. To critically evaluate the bactericidal (rather than bacteriostatic) function of the macrophage, a temperature-sensitive (ts) mutant of L. monocytogenes was developed. Mutagenesis was induced with nitrosoguanidine, and the ts mutants were isolated following enrichment with penicillin-gentamicin combinations. The ts mutants were found to carry the cell surface and biochemical characteristics of the original wild-type strain of L. monocytogenes. Inflammatory peritoneal macrophages from resistant C57BL/6J mice were found to have enhanced listericidal activity when compared with inflammatory macrophages from susceptible A/J mice. However, further analysis of the macrophage populations revealed that this seemingly qualitative advantage was due to the relatively greater proportion of inflammatory macrophages present in the inflammatory exudates of resistant C57BL/6J mice. When homogeneous populations of pure inflammatory macrophages were compared, no interstrain differences in their listericidal activity in vitro were seen. These results suggest that the susceptibility of A/J strain mice to L. monocytogenes is not due to an intrinsic deficiency of the listericidal activity of the inflammatory macrophage. The slight increase in bactericidal activity of macrophages from resistant mice that was reported by others (C. J. Czuprynski, B. P. Canono, P. M. Henson, and P. A. Campbell, Immunology 55:511-518, 1985) is caused by the difference in the relative percentage of resident cells present in the peritoneal exudates from resistant and susceptible mice.     

小檗碱和育亨宾对LPS诱导的小鼠肠道损伤和肠上皮细胞增殖抑制的影响

目的:观察小檗碱(Ber)和育亨宾(Y)对LPS诱导的小鼠肠道损伤和肠上皮细胞增殖抑制的影响。方法:雄性BALB/c小鼠随机分为对照组(control)、脂多糖组(LPS)、小檗碱组(Ber+LPS)、小檗碱与育亨宾合剂组(Ber+Y+LPS)、育亨宾组(Y+LPS)、小檗碱组(Ber)、小檗碱和育亨宾合剂组(Ber+Y)及育亨宾组(Y)。分别予以双蒸水、Ber(50mg/kg)、Ber(50mg/kg)+Y(2mg/kg)、Y(2mg/kg)灌胃,1time/d,连续3d,于实验第3d灌胃后1h,腹腔注射生理盐水或LPS(18mg/kg,0.2mL/10g)。观察各组小鼠肠组织形态学、肠黏膜重量和绒毛高度的改变;酶联免疫吸附实验(ELISA)测定肠组织二胺氧化酶(DAO)含量;免疫组化染色方法分析各组肠组织增殖细胞核抗原(PCNA)的表达。结果:LPS组小鼠肠腔显示炎性渗出、出血;肠损伤评分明显高于对照组;LPS组肠黏膜重量、肠绒毛高度、肠组织DAO含量、肠上皮细胞PCNA表达明显低于对照组。与LPS组比较,Ber组、Ber+Y组上述改变明显减轻,但两组间未见明显差异;育亨宾对LPS引起的上述指标变化无明显抑制作用。结论:Ber通过非α2肾上腺素能受体依赖的途径减轻LPS引起的肠上皮细胞增殖抑制和肠道损伤。     

In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2.

Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin O was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin O-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.     

OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival

We identified a new oligopeptide permease operon in the pathogen Listeria monocytogenes. This opp operon consists of five genes (oppA, oppB, oppC, oppD, and oppF) and displays the same genetic organization as those of several bacterial species. The first gene of this operon, oppA, encodes a 62-kDa protein sharing 33% identity with OppA of Bacillus subtilis and is expressed predominantly during exponential growth. The function of oppA was studied by constructing an oppA deletion mutant. The phenotype analysis of this mutant revealed that OppA mediates the transport of oligopeptides and is required for bacterial growth at low temperature. The wild-type phenotype was restored by complementing the mutant with oppA. We also found that OppA is involved in intracellular survival in macrophages and in bacterial growth in organs of mice infected with L. monocytogenes, although the level of virulence was not altered in the mutant. These results show the major role of OppA in the uptake of oligopeptides and the pleiotropic effects of this oligopeptide-binding protein on the behavior of this pathogen in the environment and in its host.