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Introduction: Listeria monocytogenes is known as a potentially pathogen, which can cause perinatal listeriosis, results in abortion, stillbirth or premature birth. Different factors are involved in the pathogenesis, including internalin B, which mediates the internalization of bacteria into a broad range of cell lines. However, it is not clear if expression of this protein is the same between different isolates. Methods: This study was conducted with the aim to compare L. monocytogenes isolated from pregnant women (n= 7) with different pregnancy outcomes (including healthy birth, still birth or blindness) and with abortion (n= 1), regarding expression level of inlB (internalin B encoding gene) and cell invasion. Results: Despite overexpression of inlB in most of the isolates, there was heterogeneity in the expression level between different isolates and no significant association with cell invasion and/ or pregnancy outcome was found (P> 0.05). About half of the studied isolates were able to invade HepG2 cells, while invasion to HeLa cells (cervix carcinoma cell line) was found only in one isolate. However, the difference was not significant. Likewise, no meaningful association was found between cell invasion and pregnancy outcomes. Conclusion: The expression level of inlB and cell invasion, cannot explain the difference of isolates in pathogenicity. Further studies are needed to identify bacterial and possibly host determinants.

Listeria monocytogenes (L. monocytogenes) is a foodborne intracellular pathogen that causes serious disease in both humans and animals. InlB is the major internalin protein of L. monocytogenes, which anchors to the bacterial surface and mediates its invasion into various host cells. Recent studies have shown that galactosylation of the cell wall polymer wall teichoic acid (WTA) is essential for InlB anchoring on the cell surface of L. monocytogenes serotype 4b strains. Galactosylation of WTA is exerted by the coordinated action of several glycosyltransferases, including GalU, GalE, GtcA, GttA, and GttB. Among these glycosyltransferases, GttA and GttB are specific to serotype 4b strains, whereas GalE, GalU, and GtcA are conserved across all serotypes. The role of GalE in InlB anchoring and L. monocytogenes pathogenicity remains unclear. In this study, we deleted the galE gene, which is involved in galactosylation, from L. monocytogenes strain ScottA. We found that galE deletion reduced InlB anchoring, weakened bacterial adhesion and invasion of Caco-2 cells (human colorectal adenocarcinoma cells) and MGC803 cells (human gastric carcinoma cells), increased phagocytosis but decreased proliferation in RAW264.7 cells (mouse mononuclear macrophage leukaemia cells), and decreased bacteria load, mortality, and tissue damage in infected mice. Taken together, galE deletion significantly reduced the anchoring of InlB and weakened the pathogenicity of L. monocytogenes. This finding provides new insights into the correlation between cell wall modification and pathogenicity of L. monocytogenes.

Background: Traditional plants, their parts, and phytochemicals obtained from them are beneficial for human beings. They are used as potent antimicrobials, but very little research is conducted on the use of traditional medicine against food-borne infection. Different berry plants are rich in phenolic compounds and conventionally known to have many properties such as antioxidants, anti-carcinogenic, anti-inflammatory, anti-bacterial, and anti-diabetics. However, only limited polyphenols are known for their antilisterial effect. The present study aimed to explore the antimicrobial efficacy of phenolic compounds of berries for the treatment of food-borne infection caused by the bacteria Listeria monocytogenes. Materials and Methods: Molecular docking studies employing the SwissDOCK server were performed to evaluate the antimicrobial activity of phenolic compounds obtained from different varieties of berries. Internalin B (InlB), a virulence protein of L. monocytogenes was selected as a target. The absorption, distribution, metabolism, excretion, and toxicity profiling of each test ligand was done through the SwissADME tool. Results: Among all the test ligands, p-coumaric acid, epicatechins, chlorogenic acid, and quercetin showed better binding efficiency with the target protein InlB. The binding energy obtained for quercetin, p-coumaric acid, chlorogenic acid, and epicatechins was-8.93,-8.23,-8.18,-7.58, kcal/mol, respectively. Quercetin and p-coumaric acid were forming 4 H-bonds, whereas chlorogenic acid and epicatechins were forming 3-H bonds inside the binding pocket. Conclusion: In a nutshell, analyses indicated that identified ligands have the potential to block the virulent protein InlB of L. monocytogenes and help combat Listeria infection. These phenolic compounds could be a substitute for synthetic antimicrobials and can be used in food preservation and combat food-borne diseases. However, future in-depth in vitro and in vivo analysis is needed to get more information on these four phenolic ligands of berries.

Surface-enhanced Raman scattering (SERS) is an effective method of optical vibrational spectroscopy, which allows highly sensitive detection of molecules at very low concentrations due to amplification of electromagnetic fields formed by excitation of localized surface plasmons on the surface of noble metal nanostructures. In the presented work a method of manufacturing composite nanostructures of silicon nanowires decorated with silver (AgSiNWs) has been developed. The SERS activity of AgSiNWs for protein detection was investigated using human serum albumin as an example. For the first time, the possibility of rapid diagnosis of internalin B (InlB) protein of pathogenic bacteria Listeria monocytogenes by SERS using the obtained nanostructures was shown. In the spectra of InlB adsorbed on AgSiNWs at different concentrations, distinct peaks corresponding to Raman scattering on protein molecules are observed. Based on the experimental data obtained, the InlB detection limit is calculated to be 4.8 × 10–9 M. The results presented demonstrate the high potential of synthesized composite nanostructures for diagnostics of various proteins by SERS. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24–100.0) and specificity (CI 88.78–100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.

The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto‐oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single‐particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B‐bound MET. We show that internalin B‐bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B‐bound receptors than for resting MET. Results of single‐particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.

The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto-oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single-particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B-bound MET. We show that internalin B-bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B-bound receptors than for resting MET. Results of single-particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect. [ABSTRACT FROM AUTHOR]

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen. The InlB protein is required for L. monocytogenes active invasion into epithelial cells. InlB interacts with the surface tyrosine kinase receptor c-Met. Activation of c-Met by its natural ligand, hepatocyte growth factor, induces intracellular signaling pathways and causes cell proliferation and/or migration. Interactions of InlB with c-Met in the course of infection activates the receptor and results in cytoskeleton rearrangement and bacterial uptake. In this study, we investigated the potential of the purified InlB protein as the c-Met ligand causing cell proliferation. Three distinct InlB variants have been compared. These variants were previously described in L. monocytogenes strains isolated from different sources. The variants were shown to be different in the induction of HEp-2 cell proliferation. In addition, the InlB variants differed in their potential to induce cytoskeleton rearrangements. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was to assess the potential risk posed to the human population by the presence of Listeria monocytogenes serotype 1/2c in food based on the characterization of virulence factors of Listeria involved in the invasion of host cells and sensitivity to antimicrobial agents. Methods and results In addition to sequencing of the inlA and inlB genes, the presence of genes lapB, aut, fbpA, ami, vip and llsX was tested. A premature stop codon ( PMSC) in the inlA gene was detected in all tested strains of serotype 1/2c and, concurrently, two novel PMSC mutation types were identified. However, neither PMSC in the inlB gene nor deletion of the lapB, aut, fbpA, ami and vip genes were found in any of the strains. The presence of the llsX gene was not confirmed. Even though all L. monocytogenes strains showed sensitivity to the tested antimicrobials on the basis of their phenotype, sequencing revealed the presence of IS 1542 insertion in the inlA gene, indicating the possibility of sharing of mobile genetic elements associated with antimicrobial resistance among strains. Conclusions Other than the presence of PMSCs in the inlA gene, no PMSC in inlB or deletion of other factors linked to the invasiveness of listeria were detected. Tested strains showed sensitivity to antibiotics used in the therapy of listeriosis. Significance and Impact of the Study Strains of L. monocytogenes serotype 1/2c typically carry a PMSC in the inlA gene, but these strains still represent a potential threat to public health. The possibility of transfer of IS 1542, associated with resistance to vancomycin, between enterococci and Listeria spp. was revealed. [ABSTRACT FROM AUTHOR]

Glycoprotein Ibα (GpIbα), a family of LRR (leucine-rich repeat) proteins, is a membrane protein on the platelet, and plays an important role in atherothrombotic events. The complex formation of GpIbα with the von Willebrand Factor (vWF) has been revealed to lead to acute coronary syndrome (ACS) or stroke. A considerable attention has been paid to understand the biological functions of GpIbα and its regulation. However, difficulty with the soluble expression of human GpIbα in bacteria has hampered the relevant research. Herein, we present a soluble expression of GpIbα in Escherichiacoli by replacing the N-terminal capping domain of GpIbα with that of Internalin B using a computational approach. The resulting protein was expressed as a soluble form in E. coli, maintaining its structural feature and binding property for vWF. The present approach can be broadly used for the soluble expression of human LRR proteins in E. coli. [ABSTRACT FROM AUTHOR]

Abstract: A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology. [Copyright &y& Elsevier]

The physiological relevance of contacts in crystal lattices often remains elusive. This was also the case for the complex between the invasion protein internalin B (InlB) from Listeria monocytogenes and its host cell receptor, the human receptor tyrosine kinase (RTK) MET. InlB is a MET agonist and induces bacterial host cell invasion. Activation of RTKs generally involves ligand-induced dimerization of the receptor ectodomain. The two currently available crystal structures of the InlB:MET complex show the same arrangement of InlB and MET in a 1:1 complex, but different dimeric 2:2 assemblies. Only one of these 2:2 assemblies is predicted to be stable by a computational procedure. This assembly is mainly stabilized by a contact between the Cap domain of InlB from one and the Sema domain of MET from another 1:1 complex. Here, we probe the physiological relevance of this interaction. We generated variants of the leucine-rich repeat (LRR) protein InlB by inserting an additional repeat between the first and the second LRR. This should allow formation of the 1:1 complex but disrupt the potential 2:2 complex involving the Cap-Sema contact due to steric distortions. A crystal structure of one of the engineered proteins showed that it folded properly. Binding affinity to MET was comparable to that of wild-type InlB. The InlB variant induced MET phosphorylation and cell scatter like wild-type InlB. These results suggest that the Cap-Sema interaction is not physiologically relevant and support the previously proposed assembly, in which a 2:2 InlB:MET complex is built around a ligand dimer. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host’s E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain ( ΔinlA) carrying a deletion in the gene coding for inlA. The ΔinlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-β3- and anti-β1-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-β2- and anti-β4-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the β3- and β1-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes ΔinlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that β3- and β1-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells. [ABSTRACT FROM AUTHOR]

Abstract: The performance of a low conductive growth medium (LCGM) (conductivity of <1300 μS) was evaluated for its ability to support growth of food borne bacterial pathogens including Listeria monocytogenes and to determine the expression of the two key virulence proteins in L. monocytogenes for possible applications in an impedance-based microfluidic biochip detection platform. Growth of Listeria was monitored spectrophotometrically and the lag phase, generation time, growth rate and maximum population density were determined using the Gompertz equation. LCGM had a lag phase of 2.3 h and showed a higher cell density compared to Luria Bertini (LB) broth. Length of lag phase was highly dependent on initial inoculum concentrations. The changes in conductivity with respect to growth in the low conductive medium were monitored using a conductivity probe. L. monocytogenes growth could be detected within 2 h (0.1 mS) in LCGM and within 6 h in LB. The performance of the media was also evaluated for the recovery of Listeria cells exposed to various stresses as 42 °C for 1, 2 or 6 h, an osmotic stress in 10.5% NaCl, an acidic stress at pH 2, 3 or 5 and a combined stress of 10.5% NaCl, pH 5 and 1 h exposure at 42 °C. The recovery rate was comparable with that of Tryptic soy broth containing yeast extract (TSBYE). L. monocytogenes in LCGM supported the expression of two key virulence markers, actin polymerization protein (ActA) and internalin B (InlB), which could be detected using specific antibodies. In general LCGM also supported the growth of several other bacterial species suggesting its implication in microbial quality monitoring of products. In conclusion, LCGM is a sensitive low conductive medium that supports the growth as well as the expression of virulence markers for potential applications in sensitive detection of L. monocytogenes or other food borne pathogens in impedance-based sensor platform. [Copyright &y& Elsevier]

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log 10 6.37 cfu ml -1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log 10 8.0 cfu ml -1 ) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml -1 ), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples. [ABSTRACT FROM AUTHOR]

Abstract Objectives To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985. Methods The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model. Results In HeLa and JEG-3 cells, the F2365 InlB+ strain expressing InlB was ≈9-fold and ≈1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlB+ were significantly higher compared to the F2365 strain (≈1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlB+ strain (Mann-Whitney test). Conclusions InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak. [ABSTRACT FROM AUTHOR]