Use of monoclonal antibodies that recognize p60 for identification of Listeria monocytogenes

Listeria monocytogenes causes major food-borne outbreaks of disease worldwide. Specific identification of this microorganism is of utmost importance to public health and industry. Listeria species are known to secrete a 60-kDa protein collectively termed p60, which is encoded by the iap (invasion-associated protein) gene and secreted in large quantities into the growth media. p60 is a highly immunogenic murein hydrolase that is essential for cell division. Due to these properties, p60 is an ideal diagnostic target for the development of immunological detection systems for L. monocytogenes. We report here two independent lines of monoclonal antibody (MAb): p6007, which specifically recognizes L. monocytogenes p60, and p6017, which reacts with a wide range of Listeria p60 proteins. By combining these antibodies with a polyclonal antibody, we developed efficient sandwich enzyme-linked immunosorbent assay (ELISA) systems which can specifically identify L. monocytogenes or generally detect Listeria species. Since an excess amount of the peptide corresponding to PepA or PepD did not interfere with the ELISA, and direct ELISAs were unable to detect both peptides, we concluded that the epitope presumed to be recognized by p6007 or p6017 could be distinguished from PepA and PepD as described by Bubert et al. (Appl. Environ. Microbiol. 60:3120-3127, 1997). To our best knowledge, this is the first example of an immunological identification system that uses p60-recognizing MAbs.     

A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources

An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10⁸ cfu/ml into a suspension of Ped-2E9 (10⁶/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test, Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 μm filter.     

Prevalence,identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia

Objectives

We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates.

Material and methods

Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus.

Results

The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases.

Conclusion

We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources.     

Protein tyrosine kinase inhibitors block the entries of Listeria monocytogenes and Listeria ivanovii into epithelial cells

The internalization of Listeria by intestinal epithelial cells is still poorly understood, however it is becoming apparent that microorganisms have developed the ability to interact with host cell receptor molecules to induce their own internalization. In this report we show that inhibition of cell tyrosine phosphorylation by protein tyrosine kinase (PTK) inhibitors blocks L. monocytogenes entry into both finite and immortalized intestinal cell lines. Some differences were observed between the Listeria species. L. monocytogenes entry was inhibited by between 10- to 100-fold by PTK inhibitors competing with the tyrosine residue binding of the kinase as erbstatin or by PTK inhibitors competing with the binding of ATP to the enzyme as genistein and some tyrphostins. On the other hand, L. ivanovii entry was inhibited by erbstatin as observed with L. monocytogenes but poorly by genistein and tyrphostins. The use of these several PTK inhibitors shows that even though both L. monocytogenes and L. ivanovii entered intestinal and other call lines by stimulating PTK, it seems that L. monocytogenes stimulated a different PTK than L. ivanovii. According to the fact that the number of PTK receptors increases on immortalized cells, the higher L. monocytogenes internalization observed with immortalized cell lines could be related to a higher PTK receptor number on these calls compared to finite cell lines,     

Recombinant interleukin-12 enhances resistance of mice to Listeria monocytogenes infection

The effect of recombinant murine IL-12 (rIL-12) or anti-IL-12 antibody administration on resistance to murine listeriosis was investigated. Mice given a single 0.5 μg dose of rIL-12 had 1.5 log₁₀ fewer listeriae in their spleens and livers as compared with control infected mice 3 days after L. monocytogenes challenge. Conversely, administration of anti-IL-12 IgG caused an equivalent increase in the cfu of L. monocytogenes recovered from the spleens and livers as compared to control mice. This is the first report of such a protective effect from a single dose of rIL-12. Treatment of uninfected mice with rIL-12 induced IFN-γ mRNA production in their livers. Infection of mice with L. monocytogenes caused a similar increase in IFN-γ mRNA levels that was not increased further by concurrent treatment with rIL-12. Treatment of mice with an anti-IFN-γ MAb eliminated the protective effect of IL-12 on Listeria infection. Expression of TNF-γ, IL-10 and IL-12p40 mRNA in L. monocytogenes-infected mice were not significantly altered by administration of either anti-IL-12 IgG or rIL-12. rIL-12 administration was associated with increased serum AST levels, a measure of liver damage, 1 day after treatment in L. monocytogenes-infected mice. In addition, rIL-12 administration was associated with the increased presence of small inflammatory foci and necrotic hepatocytes in both infected and uninfected mice, suggesting a proinflammatory role for IL-12 in the liver.     

Listeria monocytogenes-Infected Hepatocytes Are Targets of Major Histocompatibility Complex Class Ib-Restricted Antilisterial Cytotoxic T Lymphocytes

Subclinical infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes results in the development of protective antilisterial immunity. L. monocytogenes can infect hepatocytes, and antilisterial cytotoxic T lymphocytes (CTL) lyse Listeria-infected hepatocytes in a major histocompatibility complex (MHC) class Ia-restricted manner. It remained to be determined whether L. monocytogenes-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis. In this study, we showed that hepatocytes express MHC class Ib molecule Qa-1^b mRNA and protein. We further showed that Listeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis, since C57BL/6-derived Listeria-infected hepatocytes were lysed by BALB/c-derived antilisterial CTL. These results establish that Listeria-infected hepatocytes are susceptible to cytolysis by MHC class Ib restricted Listeria-specific CTL.Injection of BALB/c mice with a sublethal dose of virulent Listeria monocytogenes leads to an initial wave of unhindered intracellular replication of the microorganism within the first 24 to 48 h (16). The spleen and liver are the principal target organs for the experimental infection. Hepatocytes are the principal cell type harboring the replicating bacterial pathogen within the liver (8, 18). By 72 h following injection, the numbers of CFU in these organs begin to decline, and by 6 days following injection, L. monocytogenes CFU typically are not detectable (14, 15). The decline and eventual clearance of L. monocytogenes have been attributed to stimulation of Listeria-specific CD4⁺ and CD8⁺ T-cell subsets (11, 16). Adoptive transfer studies have shown that the Listeria-specific CD4⁺ T-cell subset mediates delayed-type hypersensitivity reactivity while the Listeria-specific CD8⁺ T-cell subset is responsible for protective immunity against a lethal challenge dose of virulent L. monocytogenes (1, 2).In vitro studies have shown that the CD8⁺ T-cell subset is cytolytic, and targets cells such as J774 macrophage-like cells, bone marrow-derived macrophages, and fibroblasts that have been infected with viable, hemolysin-secreting strains of L. monocytogenes are lysed by Listeria-immune CD8⁺ T cells (3). In addition, gamma interferon is released from Listeria-specific CD8⁺ T cells only following interaction with target cells infected with viable, hemolysin-secreting strains of L. monocytogenes (7). Target cells infected with non-hemolysin-secreting strains of L. monocytogenes or pulsed with nonviable bacteria such as heat-killed L. monocytogenes are not lysed by CD8⁺ Listeria-immune cells (4). The CD8⁺ T-cell subset is major histocompatibility complex (MHC) class I restricted, and both classical (MHC class Ia) and nonclassical (MHC class Ib) elements have been described as restriction molecules (3). In BALB/c mice, MHC class Ia responses are restricted to H2-K^d-presented peptides. MHC class Ib molecules that are restricting elements for antilisterial cytotoxic T lymphocytes (CTL) include Qa-1^b, which is T region encoded (6), and M3, which is M region encoded (12, 17).A role for MHC class Ib-restricted CD8⁺ T cells in antilisterial immunity is indicated from recent studies which showed that short-lived antilisterial protection can be adoptively transferred to MHC class Ia-disparate, MHC class Ib-syngeneic recipients (10, 13). MHC class Ib-restricted CTL appear to be biologically relevant, since MHC class Ib-restricted CTL activity can be detected in vivo when CFU are declining and the disappearance of effector function of this population correlates with clearance of L. monocytogenes from the immunized animals (5). It has been clearly established that L. monocytogenes-infected macrophages present MHC class Ia- and Ib-associated peptide targets to antilisterial CTL (3). It has recently been established that Listeria-infected hepatocytes are susceptible to MHC class Ia-restricted lysis by Listeria-specific CD8⁺ T cells obtained from mice immunized 11 days previously (9). This same CD8⁺ T-cell population failed to exhibit MHC class Ib-restricted cytolytic activity. This finding suggests that Listeria-infected hepatocytes may not present MHC class Ib-associated targets to Listeria-specific CTL. The study presented in this report was initiated to address specifically whether hepatocytes express MHC class Ib molecules and to determine if Listeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis by utilizing a population of Listeria-specific effector cells with known MHC class Ib-restricted CTL activity (3). The data presented show that hepatocytes express MHC class Ib molecules and that L. monocytogenes-infected hepatocytes are lysed by MHC class Ib-restricted Listeria-specific CTL.     

Existing Antilisterial Immunity Does Not Inhibit the Development of a Listeria monocytogenes-Specific Primary Cytotoxic T-Lymphocyte Response

Infection of BALB/c mice with Listeria monocytogenes stimulates an antilisterial immune response evident by the appearance of H2-K^d-restricted CD8⁺ cytotoxic T lymphocytes (CTLs) specific for the nanomer peptides amino acids (aa) 91 to 99 of listeriolysin O (LLO 91–99) and aa 217 to 225 of the p60 molecule (p60 217–225). We have introduced point mutations at anchor residues within LLO 91–99 (92F) or p60 217–225 (218F), and BALB/c mice infected with L. monocytogenes strains containing these point mutations do not develop CTLs specific for LLO 91–99 or p60 217–225, respectively. We have used these strains to test whether primary CTL responses against L. monocytogenes-derived determinants can be stimulated within an environment of existing antilisterial immunity. We found that the development of a primary L. monocytogenes-specific CTL response is not altered by existing immunity to L. monocytogenes. For example, primary immunization with the p60 218F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of p60 217–225-specific CTLs at primary response levels and LLO 91–99-specific effectors at levels consistent with a memory CTL response. Similarly, primary immunization with the 92F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of LLO 91–99-specific CTLs at primary response levels and p60 217–225-specific effectors at levels consistent with a memory CTL response. These results provide additional support for the use of L. monocytogenes as a recombinant vaccine vector and show that antivector immunity does not inhibit the development of a primary CTL response when the epitope is delivered by L. monocytogenes as the vaccine strain.     

Hot water extracts of Chlorella vulgaris reduce opportunistic infection with Listeria monocytogenes in C57BL/6 mice infected with LP-BM5 murine leukemia viruses

The bacterial elimination after infection with Listeria monocytogenes was impaired in mice with murine acquired immunodeficiency syndrome (MAIDS) by infection with LP-BM5 murine leukemia virus. Oral administration of hot water extracts of Chlorella vulgaris (CVE) restored the capacity of MAIDS mice to eliminate L. monocytogenes in association with improvement of the deteriorated immune response to L. monocytogenes. DTH response to Listeria in CVE-treated MAIDS mice was significantly higher than that of MAIDS mice after Listeria infection in association with increases in number of CD4⁺CD8⁻ and CD4⁻CD8⁺ αβ T-cells in the infected sites. CVE might be effective in the treatment of opportunistic infection in retrovirus-induced immunodeficient patients.     

Penetration ofListeria monocytogenesin mice infected by the oral route

In this study, it is suggested that the Peyer's patches are the most important point of entry ofListeria monocytogenesin the host after subclinical infection by the oral route. Microbiological, histopathological and ultrastructural evidence of infection was obtained in mice inoculated with a sublethal dose of 10⁹cfu. No mortality was observed.L. monocytogeneswas isolated from the mesenteric lymph nodes from 6 hours post infection (hpi) through day 7 p.i. and from the liver and spleen from 24 h p.i. until days 5 and 7 p.i. respectively. Lesions were mainly restricted to the dome area of Peyer's patches and consisted of a purulent to pyogranulomatous inflammatory reaction. Scarce and minor lesions were also observed in the mesenteric lymph nodes and liver.L. monocytogeneswas detected by immunohistochemistry in the Peyer's patches from 12 h p.i. to day 6 p.i. Ultrastructural study of Peyer's patches showed that the majority ofListeriacells were free within the cytoplasm of neutrophils and macrophages, not surrounded by a phagosomal membrane, and some of them were dividing.     

Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen

The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1, Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogen Listeria monocytogenes. Within a few hours of systemic infection, the massive proliferation of L. monocytogenes in Perforin-2^−/− mice leads to a rapid appearance of acute disease symptoms. We go on to show in cultured Perforin-2^−/− cells that the vacuole-to-cytosol transitioning of L. monocytogenes is greatly accelerated. Unexpectedly, we found that in Perforin-2^−/− macrophages, Listeria-containing vacuoles quickly (≤15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation of L. monocytogenes to its replicative niche in the cytosol. This hypothesis was supported by our finding that a L. monocytogenes strain expressing virulence factors at a constitutively high level replicated equally well in Perforin-2^+/+ and Perforin-2^−/− macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification of Listeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.     

Cytolysin-dependent escape of the bacterium from the phagosome is required but not sufficient for induction of the Th1 immune response against Listeria monocytogenes infection: distinct role of Listeriolysin O determined by cytolysin gene replacement

Listeria monocytogenes evades the antimicrobial mechanisms of macrophages by escaping from the phagosome into the cytosolic space via a unique cytolysin that targets the phagosomal membrane, listeriolysin O (LLO), encoded by hly. Gamma interferon (IFN-γ), which is known to play a pivotal role in the induction of Th1-dependent protective immunity in mice, appears to be produced, depending on the bacterial virulence factor. To determine whether the LLO molecule (the major virulence factor of L. monocytogenes) is indispensable or the escape of bacteria from the phagosome is sufficient to induce IFN-γ production, we first constructed an hly-deleted mutant of L. monocytogenes and then established isogenic L. monocytogenes mutants expressing LLO or ivanolysin O (ILO), encoded by ilo from Listeria ivanovii. LLO-expressing L. monocytogenes was highly capable of inducing IFN-γ production and Listeria-specific protective immunity, while the hly-deleted mutant was not. In contrast, the level of IFN-γ induced by ILO-expressing L. monocytogenes was significantly lower both in vitro and in vivo, despite the ability of this strain to escape the phagosome and the intracellular multiplication at a level equivalent to that of LLO-expressing L. monocytogenes. Only a negligible level of protective immunity was induced in mice against challenge with LLO- and ILO-expressing L. monocytogenes. These results clearly show that escape of the bacterium from the phagosome is a prerequisite but is not sufficient for the IFN-γ-dependent Th1 response against L. monocytogenes, and some distinct molecular nature of LLO is indispensable for the final induction of IFN-γ that is essentially required to generate a Th1-dependent immune response.     

Entry of Listeria monocytogenes into Neurons Occurs by Cell-to-Cell Spread: an In Vitro Study

Listeria monocytogenes is an intracellular pathogen that causes severe central nervous system infection in humans and animals. The ability of this bacterium to penetrate nerve cells was investigated by using rat spinal cell cultures. Entry into distinct cell types, i.e., glial cells and neurons, was monitored by a differential immunofluorescence technique with antibodies against cell type-specific markers and the bacterial pathogen. L. monocytogenes was detected predominantly within macrophages constituting the microglia. Astrocytes and oligodendrocytes, the major components of macroglia, were infected to a lesser extent. Surprisingly, Listeria innocua, a noninvasive and nonpathogenic species, also has the capacity to enter into these three types of glial cells. Entry into neurons was a very rare event. In contrast, we found that L. monocytogenes could efficiently invade neurons when these latter cells were cocultivated with Listeria-infected mouse macrophages. In this case, infection of neurons occurs by cell-to-cell spread via an actA-dependent mechanism. These data support the notion that infected phagocytes can be vectors by which L. monocytogenes gains access to privileged niches such as the central nervous system.     

Phosphatidylcholine-Specific Phospholipase C from Listeria monocytogenes Is an Important Virulence Factor in Murine Cerebral Listeriosis

Meningoencephalitis is a serious and often fatal complication of Listeria monocytogenes infection. The aim of the present study was to analyze the role of internalin A (InlA) and B, which are involved in the invasion of L. monocytogenes into cultivated host tissue cells, and that of phosphatidylcholine-specific phospholipase C (PlcB), which mainly promotes the direct cell-to-cell spread of L. monocytogenes, in murine cerebral listeriosis by use of an InlA/B (ΔinlAB2)- and a PlcB (ΔplcB2)-deficient isogenic deletion mutant strain and the wild-type (WT) L. monocytogenes EGD. Listeria strains were directly applied to the brain, a technique which has been employed previously to study the pathogenesis of cerebral listeriosis (D. Schlüter, S. B. Oprisiu, S. Chahoud, D. Weiner, O. D. Wiestler, H. Hof, and M. Deckert-Schlüter, Eur. J. Immunol. 25:2384–2391, 1995). We demonstrated that PlcB, but not InlA or InlB, is an important virulence factor in cerebral listeriosis. Nonimmunized mice infected intracerebrally with the ΔplcB2 strain survived significantly longer and had a reduced intracerebral bacterial load compared to mice infected with the ΔinlAB2 strain or WT bacteria. In addition, immunization with the WT prior to intracerebral infection significantly increased the survival rate of mice challenged intracerebrally with the ΔplcB2 strain compared to that of mice infected with the WT or ΔinlAB2 strain. Histopathology revealed that the major difference between the various experimental groups was a significantly delayed intracerebral spread of the ΔplcB2 mutant strain, indicating that cell-to-cell spread is an important pathogenic feature of cerebral listeriosis. Interestingly, irrespective of the Listeria mutant used, the apoptosis of hippocampal and cerebellar neurons and an internal hydrocephalus developed in surviving mice, indicating that these complications are not dependent on the virulence factors InlA/B and PlcB. In conclusion, this study points to PlcB as a virulence factor important for the intracerebral pathogenesis of murine L. monocytogenes meningoencephalitis.     

Anti-Listeria monocytogenes immunity in μ-suppressed mice: a comparison of treatment with conventional hyperimmune rabbit anti-mouse IgM and affinity-purified,monoclonal rat anti-mouse IgM

The capacity of anti-IgM treated, B-cell-depleted mice to control infection by Listeria monocytogenes was evaluated. Suppression was achieved with a hyperimmune rabbit anti-mouse-IgM antiserum (IRS), with affinity-purified IRS (IRP), or with an affinity-purified, monoclonal, rat anti-mouse-IgM antibody (LO-MM-9). B-cell depletion in specifically treated mice was judged to be complete by the following criteria: absence of significant response to a B-cell mitogen lipopolysaccharide, absence of B-cells with detectable IgM or kappa light chain on their surface, absence of detectable IgM, and presence of free anti-IgM antibodies in serum. BALB/c mice, conventionally treated from birth with IRS, had an increased capacity to clear L. monocytogenes from the blood during the first 5 min after intravenous infection. Furthermore, control of infection seemed to be enhanced during the first 24 h but was found to be impaired when assessed 3 and 4 days after initiation of infection. These effects were, however, not IRS specific, because control mice treated with normal rabbit serum behaved comparably. Mortality caused by 2 × 10³ L. monocytogenes injected intraperitoneally into BALB/c mice susceptible to L. monocytogenes was increased more in NRSthan in IRS-treated mice when both were compared with untreated control mice. Therefore, chronic injection of IRS or NRS seemed to disturb anti-L. monocytogenes immunity, rendering an evaluation of the role of antibodies impossible. Chronic treatment of mice, born from and raised by mothers similarly treated with monoclonal, rat anti-mouse IgM (LO-MM-9), showed that initial blood clearance (at 5 min), early (1–24 h) and late (3–5 day) bactericidal anti-Listeria activity in spleens and livers and also susceptibility as monitored by mortality were comparable to those observed in untreated BALB/c. These results demonstrate formally that antibodies, whether naturally pre-existant or specifically induced, do not play a role in either non-immunological early or subsequent T-cell-dependent control of primary Listeria monocytogenes infection in mice.Abbreviations CFA Complete Freund's adjuvant - IRS Immune rabbit anti-mouse IgM serum - IRP Purified IRS - NRS Normal rabbit serum - NRP Purified NRS - L.m. Listeria monocytogenes - LO-MM-9 Monoclonal rat anti-mouse IgM antibody - i.v. Intravenous(ly) - i.p. Intraperitoneal(ly)     

Nitric oxide mediates immunosuppression induced byListeria monocytogenesinfection: quantitative studies

Our laboratory has shown that immunization of mice with an attenuated strain ofSalmonella typhimuriuminduces profound suppression in the capacity of splenocytes to mount anin vitroantibody plaque-forming cell (PFC) response to sheep red blood cells (SRBC) and to proliferate in response to mitogens.In vitroaddition of N^G-monomethyl-L-arginine (NMMA), an inhibitor of nitric oxide (NO) synthase, to cell cultures fromSalmonella-immunized mice completely blocked suppression of the PFC responses, implicating that NO is the suppressor factor. The present study quantified the role of nitric oxide in immunosuppression induced byListeria monocytogenes, a gram positive intracellular pathogen of macrophages.Listeriainfection resulted in suppression of the PFC assay at inoculating doses of greater than 6.5×10³colony forming units, with no suppression observed at lower doses. Suppression correlated with increased nitrite production. Addition of NMMA to spleen cell cultures taken fromListeria-infected mice completely blocked suppression of the PFC response, and returned nitrite production to baseline levels. In regard toListeria-induced suppression of responses to the mitogen, Concanavalin A (Con A), the parameters were different from those observed for the PFC response. There was a direct correlation between the log₁₀of the inoculating dose ofListeriaand degree of immunosuppression, with suppression observed at doses as low as 1×10³cells. Addition of NMMA to the Con A-stimulated cultures resulted in reduced nitrite levels, but only partial restoration of the proliferative responses. Co-culture of splenocytes fromListeriainoculated mice with normal splenocytes in media with NMMA and reduced levels of L-arginine resulted in complete reversal of suppressed responses to Con A. Similar differences in ease of reversing suppression of the PFC response, as compared with responses to Con A, were previously noted using cells taken fromSalmonella-infected mice. The present results show that a gram positive intracellular pathogen of macrophages,L. monocytogenes, induces immunosuppression in mouse spleen cells by a nitric oxide mediated mechanism that closely parallels that induced by the gram negative pathogen,S. typhimurium.     

Subversion of antimicrobial calprotectin (S100A8/S100A9 complex) in the cytoplasm of TR146 epithelial cells after invasion by Listeria monocytogenes

Expressed by squamous mucosal keratinocytes, calprotectin is a complex of two EF-hand calcium-binding proteins of the S100 subfamily (S100A8 and S100A9) with significant antimicrobial activity. Calprotectin-expressing cells resist invasion by Porphyromonas gingivalis, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium (S. typhimurium). To understand the interactions between calprotectin and invasive bacteria, we studied the distribution of calprotectin in the cytoplasm of TR146 epithelial cells. In response to L. monocytogenes, calprotectin mobilized from a diffuse cytoplasmic distribution to a filamentous pattern and colocalized with the microtubule network. Listeria more frequently invaded cells with mobilized calprotectin. Calprotectin mobilization was listeriolysin O-dependent and required calcium (extracellular and intracellular) and an intact microtubule network. In the presence of preformed microtubules in vitro, the anti-Listeria activity of calprotectin was abrogated. To facilitate intraepithelial survival, therefore, Listeria mobilizes calprotectin to colocalize with cytoplasmic microtubules, subverting anti-Listeria activity and autonomous cellular immunity.     

A gerbil model for rhombencephalitis due toListeria monocytogenes

Rhombencephalitis due toListeria monocytogenesis a frequent complication of human listeriosis, inducing a high mortality and severe neurological sequelae despite antibiotic therapy. However, there is no animal model which consistently reproduces clinical rhombencephalitis. Here, we present a model ofListeriarhombencephalitis in gerbils. Animals were inoculated in the middle ears with a low infective dose ofL. monocytogenes, thus creating prolonged otitis media with persistent bacteremia. Gerbils developed a severe rhombencephalitis with circling syndrome, paresia, ataxia, rolling movements. The invasion of the central nervous system was visualized on living animals by resonance magnetic imaging and characterized by bacterial growth in the brain, reaching about 10⁷bacteria in the rhombencephalum by day 12 of infection. The histological lesions were mainly located in the brainstem, and consisted in coalescent, necrotic abscesses with perivascular sheaths, mimicking those observed in human rhombencephalitis. Bacteria were detected by electronmicroscopy inside infectious foci, either free in necrotic material or inside inflammatory cells, mainly polymorphonuclear cells. This gerbil model ofListeriarhombencephalitis will be useful to study the molecular mechanisms allowing bacteria to cross the blood–brain barrier, and to evaluate the intracerebral efficacy of antibiotics.     

A glance at Listeria and Salmonella cell invasion: different strategies to promote host actin polymerization

The facultative intracellular bacterial pathogens Listeria monocytogenes and Salmonella enterica have evolved multiple strategies to invade a large panel of mammalian cells. These pathogens use the host cell actin system for invasion and became a paradigm for the study of host-pathogen interactions and bacterial adaptation to mammalian hosts. The key signaling component that these pathogens use to orchestrate actin remodeling is the Arp2/3 complex, which is related to polymerization of actin filaments. These bacterial pathogens are able to trigger distinct invasion mechanisms. On the one hand, L. monocytogenes invade a host cell in a way dependent on the specific interactions between bacterial and host cell proteins, which in turn activate the host cell actin polymerizing machinery that culminates with bacterial internalization. Also, Listeria escapes from the newly formed parasitophorous vacuole and moves among adjacent cells by triggering actin polymerization. On the other hand, Salmonella invades a host cell by delivering into the cytoplasm virulence factors which directly interact with host regulators of actin polymerization which leads to bacterial uptake. Moreover, Salmonella avoids vacuole lyses and modulates the early and late endosomal markers presented in the vacuole membrane. This mini-review focuses on the different pathways that L. monocytogenes and S. enterica activate to modulate the actin cytoskeleton in order to invade, to form the parasitophorous vacuole, and to migrate inside host cells.     

Optical biosensors with an integrated Mach-Zehnder Interferometer for detection of Listeria monocytogenes

In this work, we have demonstrated an efficient optical immunoassay technique for the detection of a food-borne pathogen, Listeria monocytogenes, using a Mach-Zehnder Interferometer (MZI) configuration. We have investigated ten different MZI configurations with angular and Sbend Y-junction geometries. An efficient Hydrofluoric acid (HF) based technique was used for rapid and specific binding of L. monocytogenes to the sensor arm of the MZI biosensor. The MZI biosensor was able to detect L. monocytogenes at concentrations of the order of 10⁵ CFU/ml, which is lower than the infection dose for healthy human beings. SEM analysis and light intensity measurements showed the biosensor is highly selective to L. monocytogenes over other microbial species (such as Escherichia coli). Finally, a novel calibration scheme of the MZI biosensor was developed from experimental data that can be used for determining unknown concentrations of L. monocytogenes.     

Listeria monocytogenes Uses Listeria Adhesion Protein (LAP) To Promote Bacterial Transepithelial Translocation and Induces Expression of LAP Receptor Hsp60

Listeria monocytogenes interaction with the intestinal epithelium is a key step in the infection process. We demonstrated that Listeria adhesion protein (LAP) promotes adhesion to intestinal epithelial cells and facilitates extraintestinal dissemination in vivo. The LAP receptor is a stress response protein, Hsp60, but the precise role for the LAP-Hsp60 interaction during Listeria infection is unknown. Here we investigated the influence of physiological stressors and Listeria infection on host Hsp60 expression and LAP-mediated bacterial adhesion, invasion, and transepithelial translocation in an enterocyte-like Caco-2 cell model. Stressors such as heat (41°C), tumor necrosis factor alpha (TNF-α) (100 U), and L. monocytogenes infection (10⁴ to 10⁶ CFU/ml) significantly (P < 0.05) increased plasma membrane and intracellular Hsp60 levels in Caco-2 cells and consequently enhanced LAP-mediated L. monocytogenes adhesion but not invasion of Caco-2 cells. In transepithelial translocation experiments, the wild type (WT) exhibited 2.7-fold more translocation through Caco-2 monolayers than a lap mutant, suggesting that LAP is involved in transepithelial translocation, potentially via a paracellular route. Short hairpin RNA (shRNA) suppression of Hsp60 in Caco-2 cells reduced WT adhesion and translocation 4.5- and 3-fold, respectively, while adhesion remained unchanged for the lap mutant. Conversely, overexpression of Hsp60 in Caco-2 cells enhanced WT adhesion and transepithelial translocation, but not those of the lap mutant. Furthermore, initial infection with a low dosage (10⁶ CFU/ml) of L. monocytogenes increased plasma membrane and intracellular expression of Hsp60 significantly, which rendered Caco-2 cells more susceptible to subsequent LAP-mediated adhesion and translocation. These data provide insight into the role of LAP as a virulence factor during intestinal epithelial infection and pose new questions regarding the dynamics between the host stress response and pathogen infection.Listeria monocytogenes is a food-borne pathogen which causes severe opportunistic illness in humans by crossing the intestinal epithelial barrier to gain access to deeper tissues (21, 56). Physiologically stressed individuals, including pregnant women and those who are immunocompromised, are at greatest risk for listeriosis. In these hosts, Listeria is able to cross the blood-brain barrier to affect the central nervous system and the feto-placental barrier to infect the fetus in pregnant women, which may cause spontaneous abortion or stillbirth.Since it is a food-borne pathogen, the initial interaction of L. monocytogenes with the intestinal epithelium is crucial for establishing infection and promoting bacterial spread to extraintestinal sites. Adhesion is mediated by bacterial factors, including fibronectin binding protein (FbpA), ActA, Ami, CtaP, and LapB. FbpA binds fibronectin in the intestinal epithelium and on hepatocytes (17). ActA, a protein required for actin-based motility during intracellular infection, also promotes adhesion, via host cell proteoglycans (1). Ami, an autolysin amidase, contributes to adhesion via interaction with an unknown host receptor (41). CtaP, a cysteine transport-associated protein, is also involved in adhesion to host cells (61), and LapB, a newly identified PrfA-regulated virulence protein, is involved in both adhesion to and invasion of host cells (49).Members of the internalin (Inl) family of proteins mediate adhesion to and invasion of a variety of host cell types. Following oral infection, InlB, InlC, and InlJ mediate binding to the human intestinal mucin Muc2 (38), and InlJ also adheres to some cell types, including intestinal epithelial cells (51). InlA drives invasion of intestinal epithelial cells via interaction with the host receptor E-cadherin, a major component of adherens junctions (40), while InlB promotes deeper infection by binding to the receptor c-Met on cells of the endothelium and on hepatocytes (14). InlA-facilitated invasion is associated with systemic spread of L. monocytogenes, as InlA deletion mutants exhibit reduced translocation to extraintestinal sites in guinea pigs (36) and in transgenic mice expressing human E-cadherin (37). Despite the clear role of InlA in mediating epithelial invasion, in vivo studies demonstrate animal mortality following oral infection with InlA mutants or in animals lacking an InlA-specific E-cadherin molecule (3, 8, 16, 30). Such reports have identified additional virulence factors which promote intestinal pathogenesis of L. monocytogenes, independent of InlA. For example, the peptidoglycan hydrolase Auto aids in epithelial cell invasion (7), and virulence invasion protein (Vip) mediates invasion of intestinal epithelial cells by binding to the host receptor Gp96 (8). In transgenic mice expressing human E-cadherin, a Δvip strain exhibited reduced translocation to the mesenteric lymph nodes, liver, and spleen, to levels comparable to those of an ΔinlA mutant. These studies indicate that transit of Listeria across the tight intestinal barrier depends on the concerted action of multiple virulence factors, by mechanisms which are yet unclear.Our lab has identified Listeria adhesion protein (LAP), a 104-kDa alcohol acetaldehyde dehydrogenase (lmo1634), as a putative adhesion factor which promotes binding to cell lines of intestinal origin (29, 31, 32, 46) and promotes translocation to the liver and spleen following oral infection of mice (6, 31). LAP is present on the bacterial cell wall and is secreted by the SecA2 system (6). The secreted form of LAP, in conjunction with the cell wall-localized form, promotes full LAP-mediated interaction with host cells, possibly by reassociating with the bacterial cell wall (6, 29).We previously identified human heat shock protein 60 (Hsp60) as the epithelial receptor for LAP, and we demonstrated reduced LAP-mediated adhesion in Caco-2 cells following treatment of the Caco-2 cell surface with an Hsp60-specific antibody (58). Despite our previous indication that LAP is important for pathogenesis during the intestinal stage of Listeria infection (31), the exact contributions of LAP and Hsp60 to intestinal pathogenesis are unclear.Although Hsp60 performs chaperone functions primarily within the cell cytoplasm and mitochondrial matrix (28), the presence of it and other heat shock proteins has been found on the cytoplasmic membranes of various mammalian cells, and surface localization of these chaperones is now generally accepted (5, 8, 55). Others have also identified heat shock proteins as pathogen ligands: Staphylococcus aureus FbpA associates with Hsp60 to mediate cell invasion (19), the hepatitis B virus HBx protein forms complexes with host Hsp60 and Hsp70 during infection (63), and Brucella abortus exploits host Hsp70 for invasion of placental trophoblasts and induction of abortion in pregnant mice (59). The use of a host Hsp as a pathogen receptor is an intriguing phenomenon, because Hsp expression is elevated in response to physiological stressors, which include changes in temperature as well as bacterial and viral infections (4, 39, 45). Despite mounting evidence that certain pathogens use heat shock proteins as receptors, little information exists on the potential relationship between infection, the heat shock protein response, and subsequent implications for host-pathogen interaction.Our objectives in this study were to determine how LAP and Hsp60 mediate interaction of Listeria with intestinal epithelial cells and to evaluate the influence of Listeria infection on host Hsp60 expression. Here we demonstrate that the interaction of LAP and host Hsp60 promotes adherence to and translocation across intestinal epithelial monolayers. We also provide evidence that low levels of L. monocytogenes infection increase expression of host Hsp60, which may in turn lead to greater LAP-mediated association of Listeria with intestinal epithelial cells. This study reveals a novel mechanism by which Listeria may interact with the intestinal epithelial barrier and provides early evidence of how infection-induced expression of host heat shock proteins may promote host-pathogen interaction.