Virulent Listeria Monocytogenes Research Articles

Development and verification of crossing priming amplification on rapid detection of virulent and viable L. monocytogenes: In-depth analysis on the target and further application on food screening

Virulent Listeria monocytogenes is responsible for listeriosis, a highly fatal foodborne illness that impairs human health. One of the major causes of its pathogenicity is the carriage of virulence factor, listeriolysin O, encoded by hylA gene. Herein, the specificity and conservativity of hylA gene sequences were analyzed, with most conservative region selected. Then, a crossing priming amplification (CPA) assay targeting hylA conservative region was established under an isothermal condition (63 °C) to identify virulent L. monocytogenes within 60 min. Forty-four reference strains including two standard L. monocytogenes strains and forty-two strains of other species were selected to assess sensitivity and specificity. The CPA assay showed strong specificity and sensitivity (genomic DNA at 5.66 pg/μL). The practicality of the CPA assay was further confirmed in four artificially contaminated rice-flour products with limit of detection at 104 CFU/mL. In addition, a PMA-CPA assay was developed to direct detect L. monocytogenes viable cells with rice-flour products as a model system. In conclusion, the developed CPA method has advantages of high sensitivity, specificity and quick measurement in the detection of virulent L. monocytogenes, showing considerably feasible promise for future food safety.

Prevalence and Antimicrobial Resistance of Virulent Listeria monocytogenes and Cronobacter sakazakii in Dairy Cattle, the Environment, and Dried Milk with the In Vitro Application of Natural Alternative Control.

This study aims to detect the prevalence and antimicrobial resistance of Listeria monocytogenes and Cronobacter sakazakii in three dairy households and dried milk from different suppliers, and evaluate the antimicrobial effect of rose water, rose, and orange essential oils. In total, 360 samples were collected from cattle, the environment, and dried milk (n = 30). Antimicrobial activity was evaluated with twofold microtube dilution and the time-kill method. L. monocytogenes was identified in all households (13.3%) with a prevalence in the range of 5.8–17.5%, while C. sakazakii was identified in one household (5.3%). The former and latter pathogens were highly isolated from the feces at 20% and 2.5% and bedding at 12.5% and 1.6%, respectively. L. monocytogenes was isolated only from milk at 7.5%, but C. sakazakii was not detected in either milk or dried milk. L. monocytogenes strains were screened for virulence genes (iap, hylA, and actA). All strains were positive for the iap gene, while for hlyA and actA, the percentages were (35.4% 16.6%, respectively). L. monocytogenes strains showed high resistance against sulfamethoxazole–trimethoprim (100%), followed by gentamicin, penicillin, and imipenem (95.8%, 95.8%, and 91.6%, respectively). All C. sakazakii strains were susceptible to all tested antibiotics. The bactericidal activity of orange oil was the strongest, appeared after 1 h for both pathogens, followed by rose oil and then rose water.

Core fucosylation of N-linked glycans: a novel player in memory CD8+ T cell differentiation following acute viral infection

Abstract Core fucosylation (CF) of N-linked glycans is indispensable for normal physiology, but whether CF regulates any aspect of T cell activation, differentiation, or function remains unknown. Fucosyltransferase 8 (Fut8) is the only enzyme in mammals that catalyzes CF and following activation, both expression of Fut8 and CF synthesis increases on CD8+ T cells. To test the hypothesis that CF of N-glycans regulates memory T cell differentiation following acute viral infection, we generated mice with a floxed Fut8 gene. WT (CD4-Cre−) and Fut8−/− (CD4-Cre+) mice were infected with LCMV Armstrong (LCMV-Arm) and the differentiation of LCMV-specific CD8+ T cells was analyzed by measuring the surface expression of killer-cell lectin like receptor G1 (KLRG1) and the α chain of interleukin 7 receptor (CD127) to identify short-lived effector and memory precursor T cells, respectively. LCMV-specific CD8+ T cells from both WT and Fut8−/− followed the same pattern of expansion and contraction without any significant difference in the overall frequency of LCMV-specific CD8+ T cells. However, Fut8−/− retained a higher percentage of CD127+KLRG1− and a lower percentage of KLRG1+CD127− CD8 T cells throughout the course of infection suggesting CF influences the progression to long-term memory. At day 42 post infection, mice were infected with virulent Listeria monocytogenes expressing the LCMV epitope GP33–41. Both WT and Fut8−/− GP33-specific CD8+ T cells expanded equally. However, Fut8−/− retained a higher frequency of GP33-specific CD8+ T cells and did not undergo contraction until >3 weeks post-2ry challenge. Taken together, our findings suggest that CF of N-linked glycans regulates memory CD8+ T cell differentiation following acute viral infection. Supported by grants from NIH (R21 AI159401)

Anti-infective activity of Cratylia argentea lectin (CFL) against experimental infection with virulent Listeria monocytogenes in Swiss mice

BackgroundThe lectin from Cratylia argentea (CFL) is able to modulate the immune system response and is thus a potential phytotherapeutic substance. Hypothesis/PurposeIn this study, we investigated the role of CFL on control of bacterial infection caused by Listeria monocytogenes, the causative agent of human listeriosis. Study designSwiss mice were infected with L. monocytogenes and then treated with CFL. MethodsAdult Swiss mice weighing with 30–40 g were infected intraperitoneally with a bacterial suspension (0.2 ml; 1 × 107 CFU/ml). After 30 min, the mice were treated with CFL intravenously at concentrations of 0.1 or 10 mg/kg. Control mice received phosphate-buffered saline (PBS). The animals were euthanized 24 h after infection. ResultsWe observed that i.v. administration of CFL to Swiss mice did not cause acute toxicity, and reduced the leukocyte counts in the bloodstream 24 h after infection with virulent L. monocytogenes. There was a reduction in the bacterial burden within peritoneal macrophages after infection in CFL-treated mice. Accordingly, the bacterial counts in the bloodstream, spleen and liver also decreased in comparison with the PBS group. Histological damage in the spleen and liver was lower in mice that received CFL treatment. In vitro antimicrobial assays demonstrated that CFL does not inhibit the growth of L. monocytogenes. The mRNA expression of the anti-inflammatory cytokine IL-10 was enhanced with CFL treatment after infection. ConclusionThe lectin from C. argentea (CFL) has immunomodulatory and anti-infective properties of pharmacological interest for control of infectious diseases.

Anti-inflammatory activity of caffeine (1,3,7-trimethylxanthine) after experimental challenge with virulent Listeria monocytogenes in Swiss mice

BackgroundImmunomodulatory therapies are claimed to enhance antimicrobial immunity and counterbalance antimicrobial resistance mechanisms of pathogenic bacteria. PurposeTo investigate whether caffeine can be useful for control of inflammation derived from experimental systemic infection with Listeria monocytogenes. MethodsPeritoneal macrophages (pMØ) from Swiss mice were cultured with caffeine in 96-well plates, and then infected with virulent L. monocytogenes 619. In another experiment, the pMØ were first infected with the bacterium and then treated with caffeine. Swiss mice were inoculated intraperitoneally with L. monocytogenes and then treated intravenously with caffeine (0.05; 0.5 or 5 mg/Kg). ResultsCaffeine did not exert direct antibacterial activity in vitro against L. monocytogenes. Macrophages exposed to caffeine before or after infection with L. monocytogenes had increased cell viability, although the intracellular bacterial loads were similar to the control groups. Caffeine treatments of Swiss mice reduced leukocyte infiltration into the peritoneal cavity after L. monocytogenes infection. However, the bacterial burden was reduced in the spleen and liver. The mRNA expressions of IL-1β, IL-6 and the enzyme inducible nitric oxide synthase (iNOS) were reduced whereas IL-10 was increased. ConclusionCaffeine has an anti-infectious potential and ameliorated infection-derived inflammation following experimental infection with L. monocytogenes.

Control of Virulent Listeria monocytogenes Originating from Dairy Products and Cattle Environment Using Marine Algal Extracts, Silver Nanoparticles Thereof, and Quaternary Disinfectants.

IntroductionListeria monocytogenes is an important foodborne pathogen of public- and animal-health concern globally. The persistence of L. monocytogenes in the dairy-processing environment has multifactorial causes, including lack of hygiene, inefficient cleaning, and improper disinfection practices.Materials and MethodsA total of 300 dairy-product and environmental samples were collected from dairy-cattle facilities and local dairy shops and vendors in Qena, Egypt. Samples were screened for the incidence of Listeria spp. and to detect virulence determinants and disinfectant-resistance genes. Three marine algal species — Caulerpa racemosa, Jania rubens, and Padina pavonica — were collected from Hurghada on the Red Sea coast. Algal extracts were screened using gas chromatography–mass spectrometry. The antimicrobial activity of some marine algal extracts, nanoparticles derived therefrom, and some disinfectants against L. monocytogenes strains were assessed in vitro using agar-well diffusion and liquid-broth methods. The impact of P. pavonica extract on the growth and survival of virulent L. monocytogenes in cheese and whey were clarified.Results and DiscussionThe incidence of L. monocytogenes in dairy products and environmental samples was 15.5% and 19%, respectively. The most common toxigenic gene profile found among the isolates was hlyA+–inlA+–prfA+. The sensitivity pattern of L. monocytogenes strains to disinfectant containing alkyl (C12–16) dimethyl BAC was high compared to other tested quaternary ammonium compounds (QAC) disinfectants tested, which showed lower log reductions against resistant strains. The QAC disinfectant–resistance gene qacH was detected in 40% of the isolates. Potent bactericidal activity of a petroleum ether extract of P. pavonica and silver nanoparticles of P. pavonica were obtained against the virulent L. monocytogenes strain. The population of L. monocytogenes in cheese curd and whey after 14 days was reduced at a rate of 9 log CFU/g and 8 log CFU/mL, respectively due to the effect of P. pavonica extract. After 28 days of storage, L. monocytogenes was completely inactivated in those dairy products.ConclusionP. pavonica extract showed promising antimicrobial properties, calling for further comprehensive studies prior to it being applied in the food industry to enhance the safety, quality, and shelf life of products and protect public health.

Microbial Exposure Enhances Immunity to Pathogens Recognized by TLR2 but Increases Susceptibility to Cytokine Storm through TLR4 Sensitization

Microbial exposures can define an individual's basal immune state. Cohousing specific pathogen-free (SPF) mice with pet store mice, which harbor numerous infectious microbes, results in global changes to theimmune system, including increased circulating phagocytes and elevated inflammatory cytokines. How these differences in the basal immune state influence the acute response to systemic infection is unclear. Cohoused mice exhibit enhanced protection from virulent Listeria monocytogenes (LM) infection, but increased morbidity and mortality to polymicrobial sepsis. Cohoused mice have more TLR2+ and TLR4+ phagocytes, enhancing recognition of microbes through pattern-recognition receptors. However, the response to a TLR2 ligand is muted in cohoused mice, whereas the response to a TLR4 ligand is greatly amplified, suggesting a basis for the distinct response to Listeria monocytogenes and sepsis. Our data illustrate how microbial exposure can enhance the immune response to unrelated challenges but also increase the risk of immunopathology from a severe cytokine storm.

YopE specific CD8+ T cells provide protection against systemic and mucosal Yersinia pseudotuberculosis infection.

Prior studies indicated that CD8+ T cells responding to a surrogate single antigen expressed by Y. pseudotuberculosis, ovalbumin, were insufficient to protect against yersiniosis. Herein we tested the hypothesis that CD8+ T cells reactive to the natural Yersinia antigen YopE would be more effective at providing mucosal protection. We first confirmed that immunization with the attenuated ksgA- strain of Y. pseudotuberculosis generated YopE-specific CD8+ T cells. These T cells were protective against challenge with virulent Listeria monocytogenes expressing secreted YopE. Mice immunized with an attenuated L. monocytogenes YopE+ strain generated large numbers of functional YopE-specific CD8+ T cells, and initially controlled a systemic challenge with virulent Y. pseudotuberculosis, yet eventually succumbed to yersiniosis. Mice vaccinated with a YopE peptide and cholera toxin vaccine generated robust T cell responses, providing protection to 60% of the mice challenged mucosally but failed to show complete protection against systemic infection with virulent Y. pseudotuberculosis. These studies demonstrate that vaccination with recombinant YopE vaccines can generate YopE-specific CD8+ T cells, that can provide significant mucosal protection but these cells are insufficient to provide sterilizing immunity against systemic Y. pseudotuberculosis infection. Our studies have implications for Yersinia vaccine development studies.

Biomarker Tools to Design Clinical Vaccines Determined from a Study of Annual Listeriosis Incidence in Northern Spain.

Two regions of northern Spain, Gipuzkoa, and Cantabria present high annual incidence of listeriosis (1.86 and 1.71 cases per 100,000 inhabitants, respectively). We report that the high annual incidences are a consequence of infection with highly virulent Listeria monocytogenes isolates linked to fatal outcomes in elderly patients with cancer. In addition, listeriosis patients with cancer present low IL-17A/IL-6 ratios and significantly reduced levels of anti-GAPDH1–22 antibodies, identified as two novel biomarkers of poor prognosis. Analysis of these biomarkers may aid in reducing the incidence of listeriosis. Moreover, GAPDH1–22-activated monocyte-derived dendritic cells of listeriosis patients with cancer seem useful tools to prepare clinical vaccines as they produce mainly Th1 cytokines.

Characterization of virulent Listeria monocytogenes isolates recovered from ready-to-eat meat products and consumers in Cairo, Egypt

Aim: This study aimed to investigate the occurrence of some virulence genes distributed in Listeria monocytogenes isolated from ready-to-eat (RTE) meat products and consumers in Cairo province, Egypt. Materials and Methods: A total of 120 beef luncheon, chicken luncheon and frankfurter beef (40 samples, each) were collected from 10 different local shops situated in Al-salam city, Cairo province, Egypt. Stool samples were collected from 40 people who had the habit of consuming RTE meat. The suspected L. monocytogenes isolates were subjected to a multiplex polymerase chain reaction (PCR) for rapid speciation and virulence determination using primers specific for inIA, inIC, and inIJ genes. Results: Culture examination of all samples on Oxford media revealed presence of colonies characteristic to L. monocytogenes in 6 beef luncheon (15%), 4 chicken luncheon (10%), 1 frankfurter beef (2.5%) and 1 human stool (2.5%) samples. Species identity of L. monocytogenes was verified through the amplification of a 800 bp fragment with inIA primers in 2 out of 6 culture isolates from beef luncheon (5%), and 1 out 4 culture isolates from chicken luncheon (2.5%) samples. Statistical analysis revealed no significant difference between the occurrence of L. monocytogenes in different food samples examined (p>0.05). The virulence of these strains was ascertained by the presence of 517 bp and 238 bp fragments of inIC and inIJ genes, respectively in the isolates that contained the 800 bp fragment. The culture isolates obtained from one frankfurter beef sample, and one human stool sample were found negative by multiplex PCR for the presence of L. monocytogenes and its virulence specific genes. Conclusion: It could be concluded that L. monocytogenes are circulating in beef and chicken luncheon sold in Cairo, Egypt. Multiplex PCR is reliable for confirmation of L. monocytogenes. This study suggests the implementation of hygienic measures at all levels from production to consumption in order to improve food safety. Furthermore, authors recommended consumption of beef frankfurter or any RTE meat sold in their original intact packing due to low level of contamination.

Polyphasic characterization and genetic relatedness of low-virulence and virulent Listeria monocytogenes isolates

BackgroundCurrently, food regulatory authorities consider all Listeria monocytogenes isolates as equally virulent. However, an increasing number of studies demonstrate extensive variations in virulence and pathogenicity of L. monocytogenes strains. Up to now, there is no comprehensive overview of the population genetic structure of L. monocytogenes taking into account virulence level. We have previously demonstrated that different low-virulence strains exhibit the same mutations in virulence genes suggesting that they could have common evolutionary pathways. New low-virulence strains were identified and assigned to phenotypic and genotypic Groups using cluster analysis. Pulsed-field gel electrophoresis, virulence gene sequencing and multi-locus sequence typing analyses were performed to study the genetic relatedness and the population structure between the studied low-virulence isolates and virulent strains.ResultsThese methods showed that low-virulence strains are widely distributed in the two major lineages, but some are also clustered according to their genetic mutations. These analyses showed that low-virulence strains initially grouped according to their lineage, then to their serotypes and after which, they lost their virulence suggesting a relatively recent emergence.ConclusionsLoss of virulence in lineage II strains was related to point mutation in a few virulence genes (prfA, inlA, inlB, plcA). These strains thus form a tightly clustered, monophyletic group with limited diversity. In contrast, low-virulence strains of lineage I were more dispersed among the virulence strains and the origin of their loss of virulence has not been identified yet, even if some strains exhibited different mutations in prfA or inlA.

IFN-γ triggers CCR2-independent monocyte entry into the brain during systemic infection by virulent Listeria monocytogenes

Listeria monocytogenes (Lm) is a bacterial pathogen that infects the brain via parasitized monocytes. CCR2 is important for monocyte migration into the brain after it is infected, but the degree of CCR2 involvement in monocyte migration to the CNS during systemic infection is less clear. Our recent data demonstrate that systemic infection with non-neuroinvasive ΔactA Lm mutants triggers IFN-γ-dependent brain influxes of Ly-6Chigh monocytes. Studies presented here tested the extent to which CCR2 and IFN-γ are essential for monocyte migration to the brain during systemic infection with virulent Lm. For this, we assessed expression of monocyte-attracting chemokines in brains of normal and IFN-γ mice during infection and tested the degree to which brain influxes of Ly-6Chigh monocytes were inhibited in chemokine- and chemokine receptor-deficient mice. In normal mice, systemic infection induced up-regulation of CCR2-binding (CCL2, CCL7, CCL8, CCL12) and CXCR3-binding chemokines (CXCL9, CXCL10). IFN-γ mice had negligible mRNA and protein expression of CXCR3-binding chemokines, whereas expression of CCR2-binding chemokines was reduced, but remained significant. In addition, infection-triggered monocyte influxes were significantly reduced in IFN-γ mice. Remarkably, brain monocyte influxes were normal during infection of CXCR3-, CCL2-, CCR1-, CCR5-, and CX3CR1-deficient mice. Influxes were transiently reduced in CCR2−/− mice, corresponding with retention of monocytes in the bone marrow but this was eventually overcome during infection. These data show that IFN-γ is critical for triggering brain influxes of Ly-6Chigh monocytes during systemic infection with virulent Lm. This initial burst of monocyte migration is largely independent of individual chemokine receptors.

Variations in the nanomechanical properties of virulent and avirulent Listeria monocytogenes

Atomic force microscopy (AFM) was used to quantify both the nanomechanical properties of pathogenic (ATCC 51776 & EGDe) and non-pathogenic (ATCC 15313 & HCC25) Listeria monocytogenes strains and the conformational properties of their surface biopolymers. The nanomechanical properties of the various L. monocytogenes strains were quantified in terms of Young's moduli of cells. To estimate Young's moduli, the classic Hertz model of contact mechanics and a modified version of it that takes into account substrate effects were used to fit the AFM nanoindentation-force measurements collected while pushing onto the bacterial surface biopolymer brush. When compared, the classic Hertz model always predicted higher Young's moduli values of bacterial cell elasticity compared to the modified Hertz model. On average, the modified Hertz model showed that virulent strains are approximately twice as rigid (88.1 ± 14.5 KPa) as the avirulent strains (47.3 ± 7.6 kPa). To quantify the conformational properties of L. monocytogenes' strains surface biopolymers, two models were used. First, the entropic-based, statistical mechanical, random walk formulation, the wormlike chain (WLC) model was used to estimate the elastic properties of the bacterial surface molecules. The WLC model results indicated that the virulent strains are characterized by a more flexible surface biopolymers as indicated by shorter persistence lengths (L(p) = 0.21 ± 0.08 nm) compared to the avirulent strains (L(p) = 0.24 ± 0.14 nm). Second, a steric model developed to describe the repulsive forces measured between the AFM tip and bacterial surface biopolymers indicated that the virulent strains are characterized by crowded and longer biopolymer brushes compared to those of the avirulent strains. Finally, scaling relationships developed for grafted polyelectrolyte brushes indicated L. monocytogenes strains' biopolymer brushes are charged. Collectively, our data indicate that the conformational properties of the bacterial surface biopolymers and their surface densities play an important role in controlling the overall bacterial cell elasticity.

An oralSalmonellavaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.

Listeriolysin O-Deficient Listeria monocytogenes as a Vaccine Delivery Vehicle: Antigen-Specific CD8 T Cell Priming and Protective Immunity

Strains of Listeria monocytogenes (LM) that are deficient in the virulence factor listeriolysin O (LLO) are highly attenuated and are thought not to elicit protective immunity. This failure has been attributed to the inability of the bacterium to enter the host cell cytosol and access MHC class I Ag processing machinery. We reexamined this issue using recombinant strains of LM that are deficient in LLO but express an additional CD8 T cell epitope derived from lymphocytic choriomeningitis virus. After infection with LLO-deficient strains, we find sizable priming of epitope-specific CD8 T cells and the development of a functional memory cell population. Mice primed with the LLO-deficient LM strain are equally resistant against high-dose challenge with virulent LM as mice primed with wild-type virulent bacteria and also resist heterologous challenge with lymphocytic choriomeningitis virus. Interestingly, priming with a low dose of LLO-deficient LM, which occurred in environment of reduced inflammation (IFN-gamma), allowed rapid amplification of Ag-specific CD8 T cells by booster immunization, despite an undetectable primary response. We conclude that the generation of protective immunity by LLO-deficient strains of LM does in fact occur and that this highly attenuated LM strain may be a useful platform for vaccine delivery.

Production, characterisation and potential application of a novel monoclonal antibody for rapid identification of virulent Listeria monocytogenes

A panel of hybridomas was produced using intact Listeria monocytogenes serotype 1/2a cells as the immunogen. An IgG2a monoclonal antibody (mAb) ‘mAb2B3’ was isolated that reacted with L. monocytogenes but not with a representative panel of related Listeria spp. and non- Listeria spp. Binding activity was greatest against L. monocytogenes serotype 1/2a and was significantly enhanced when cells were prepared in Listeria enrichment broth (LEB). The reactive epitope was deduced, by immunoblot analysis, to be a surface localised protein of approximately 80 kilodaltons (kDa), putatively assumed to be internalin A (InlA). Recombinant InlA protein was subsequently expressed in Escherischia coli. When crude E. coli cell lysates were subjected to immunoblot analysis, it was demonstrated that the mAb bound specifically to the heterologously expressed recombinant InlA protein, thus confirming the specificity of the mAb. The mAb was further evaluated in a series of enzyme-linked-immunosorbent assay (ELISA)-based formats and in a surface plasmon resonance (SPR)-based biosensor platform. Both configurations were capable of differential identification of virulent L. monocytogenes at concentrations greater than or equal to 1 × 10 7 cells/ml. Notwithstanding the apparent insensitivity, the results indicate that InlA could be exploited as a marker for highly specific confirmatory identification of pathogenic L. monocytogenes following primary enrichment of suspect food samples, using the anti-InlA antibody ‘mAb2B3’, described herein.

A PrfA‐regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes

The ability to colonize the gall bladder has recently been shown to be an important feature of virulent Listeria monocytogenes (J. Hardy, K. P. Francis, M. DeBoer, P. Chu, K. Gibbs, C. H. Contag. Science 303: 851-853, 2004). We suggest that the cytotoxic effects of bile may be increased upon release from the gall bladder into the upper small intestine, and report the identification of a novel bile exclusion system which plays an essential role in intestinal colonization and virulence of L. monocytogenes. In silico analysis of the L. monocytogenes EGDe genome revealed a two-gene operon (formerly opuB) exhibiting significant sequence similarity to members of the betaine carnitine choline transporter (BCCT) family. The operon, herein designated bilE (bile Exclusion) is preceded by consensus sigmaA- and sigmaB-dependent promoter-binding sites and is transcriptionally upregulated at elevated osmolarities and reduced temperatures (stresses known to induce sigB). Furthermore, a significant reduction in the level of bilE transcription was observed in the absence of sigmaB. In addition, we demonstrate an important role for PrfA, the master regulator of virulence potential in L. monocytogenes, in coordinating bilE expression. Computational structural analysis suggests that, rather than functioning as a compatible solute uptake system as was previously believed, BilE is more likely to be an exclusion system, a conclusion substantiated by radiolabelled bile accumulation studies. In addition, functionally inactivating BilE resulted in a five-log reduction in the ability of the bacterium to tolerate lethal concentrations of bovine bile (oxgall) and also significantly increased sensitivity to physiological concentrations of human bile, a phenotype which translates to a significant reduction in virulence potential when administered to a murine model by the oral route. Thus, this novel bile exclusion locus bilE, coordinately regulated by sigmaB and PrfA, represents a new and important virulence factor in L. monocytogenes.

Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes.

Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.

Deficient anti-listerial immunity in the absence of perforin can be restored by increasing memory CD8+ T cell numbers.

Compared with wild-type (WT) mice, Listeria monocytogenes (LM)-vaccinated perforin-deficient (PKO) mice have elevated levels of CD8(+) T cell memory, but exhibit reduced levels of protection against virulent LM. In this study, Ag-specific CD8(+) T cells from LM-vaccinated WT and PKO mice were used in adoptive transfer assays to determine the contribution of perforin-dependent cytolysis in protective immunity to LM. Perforin deficiency resulted in an approximately 5-fold reduction in the per-cell protective capacity of Ag-specific memory CD8(+) T cells that was not caused by differences in memory cell quality as measured by CD62L/CD27 expression, TCR repertoire use, functional avidity, differences in expansion of Ag-specific cells upon infection, or maintenance of memory levels over time. However, perforin-deficient CD8(+) T cells exhibited reduced in vivo cytotoxic function compared to WT CD8(+) T cells. Consistent with the existence of perforin-independent effector pathways, double-vaccinated PKO mice were as resistant to challenge with LM as single-vaccinated WT mice. Thus, increasing the number of memory CD8(+) T cells can overcome diminished per-cell protective immunity in the absence of perforin.

IL-12-assisted immunization generates CD4 + T cell-mediated immunity to Listeria monocytogenes

Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity. We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12. Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4 + T cells are selectively generated using this adjuvant system. We have also evaluated the importance of endogenous production of IFN-γ and IL-12 for the efficacy of IL-12-assisted immunization. IFN-γ-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses. In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T H1 response. IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4 + effector cells can be analyzed.