Listeria Adhesion Protein Research Articles

Anchorless Bacterial Moonlighting Metabolic Enzymes Modulate the Immune System and Contribute to Pathogenesis.

Moonlighting proteins (MPs), characterized by their ability to perform multiple physiologically unrelated functions without alterations to their primary structures, represent a fascinating class of biomolecules with significant implications for host-pathogen interactions. This Review highlights the emerging importance of metabolic moonlighting proteins (MetMPs) in bacterial pathogenesis, focusing on their non-canonical secretion and unconventional surface anchoring mechanisms. Despite lacking typical signal peptides and anchoring motifs, MetMPs such as acetaldehyde alcohol dehydrogenase (AdhE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are secreted and localized to the bacterial surface under stress conditions, facilitating host colonization and immune evasion. The secretion of MetMPs, often observed during conditions such as resource scarcity or infection, suggests a complex regulation akin to the overexpression of heat shock proteins in response to environmental stresses. This Review proposes two potential pathways for MetMP secretion: membrane damage-induced permeability and co-transportation with traditionally secreted proteins, highlighting a remarkable bacterial adaptability. Biophysically, surface anchoring of MetMPs is driven by electrostatic interactions, bypassing the need for conventional anchoring sequences. This mechanism is exemplified by the interaction between the bifunctional enzyme AdhE (known as Listeria adhesion protein, LAP) and the internalin B (InlB) in Listeria monocytogenes, which is mediated by charged residues facilitating adhesion to host tissues. Furthermore, MetMPs play critical roles in iron homeostasis, immune modulation, and evasion, underscoring their multifaceted roles in bacterial pathogenicity. The intricate dynamics of MetMP secretion and anchoring underline the need for further research to unravel the molecular mechanisms underpinning these processes, offering potential new targets for therapeutic intervention against bacterial infections.

Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Listeria adhesion protein-expressing bioengineered probiotics prevent fetoplacental transmission of Listeria monocytogenes in a pregnant Guinea pig model

Pregnancy is a high-risk factor for foodborne pathogen Listeria monocytogenes (Lm), which causes abortion, premature birth, or stillbirth. The primary route of Lm transmission is oral hence intestinal epithelial barrier crossing is a prerequisite for systemic spread. Intestinal barrier crossing, in part, is attributed to the interaction of Listeria adhesion protein (LAP) with its cognate receptor, Hsp60. In a recent study, we showed that oral-dosing of bioengineered Lactobacillus caseiprobiotic (BLP) expressing the LAP protected nonpregnant mice from lethal infection; however, its ability to prevent listeriosis during pregnancy is not known. Therefore, we investigated whether BLP could prevent fetoplacental transmission of Lm in a pregnant guinea pig model. After 14 consecutive days on probiotic (~109 CFU/ml in drinking water), pregnant guinea pigs (gestational days 24–28) were orally challenged with Lm (9 × 108–2.5 × 109 CFU/animal) and were euthanized 72 h post-infection. Maternal mesenteric lymph node (MLN), liver, spleen, lungs, blood, and placenta, and fetal liver were analyzed for the presence/absence of Lm. All tissues/organs from Lm-challenged naïve dams and fetuses were Lm positive. Similar tissue distribution was also seen in guinea pigs that received wild-type Lactobacillus casei (LbcWT). Remarkably, Lm was absent in the maternal blood, kidney, lungs, and placenta, and fetal liver from the BLP-fed group even though the Lm was present in the maternal liver, spleen, and MLN. BLP feeding also suppressed Lm-induced inflammatory response in mothers. These data highlight the potential for the prevention of fetoplacental transmission of Lm by LAP-expressing BLP during pregnancy.

Receptor-targeted engineered probiotics mitigate lethal Listeria infection

Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillusprobiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3+T cells, CD11c+ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.

Magnesium ion disrupts LAP surface re‐association of Listeria monocytogenes by dissociation of InlB

Listeria monocytogenes crosses intestinal epithelial barrier for the systemic spread, which is facilitated in part, by Listeria adhesion protein (LAP). LAP, a housekeeping enzyme called acetaldehyde alcohol dehydrogenase, is present in both pathogenic and nonpathogenic Listeria. LAP is secreted with the aid of SecA2 translocon, and its’ re‐association on the surface of pathogenic Listeria i.e., L. monocytogenes is critical for its function. However, the cell wall component(s) involved in LAP interaction is unknown, which was investigated.Using L. monocytogenes F4244 (WT), an isogenic lap− strain and purified recombinant LAP in the Caco‐2 cell adhesion assay and Western blot, we examined LAP re‐association on lap− cells. Extraneous added LAP restored adhesion capacity of lap−, but not of L. innocua to Caco‐2 cells, suggesting the presence of LAP‐interacting receptor(s) only on pathogenic bacteria.To locate LAP interaction partners, co‐immunoprecipitation of WT cell wall fraction using anti‐LAP mAb was analyzed by SDS‐PAGE. Selected protein bands were subjected to MALDI‐TOF protein identification showing involvement of internalin B (InlB).Next, we investigated the effect of MgCl2 pretreatment on LAP re‐association on the cell surface, cell‐free supernatant, and consequent bacterial adhesion. Mg2+ is a wall teichoic acid (WTA) charge neutralizing agent that reduced levels of InlB binding on the WT cell wall, resulting in surface disassociation of LAP. However, only partial surface reduction of LAP was observed in ΔinlB strain suggesting more than one ligand is involved.In conclusion, our results indicate that both InlB and WTA may participate in LAP re‐association on L. monocytogenes cell surface, which is essential for its pathogenic activities.Support or Funding InformationThe technical assistance of Uma Aryal, Valerie Ryan, and Aaron Gall and funding from USDA‐ARS project number 59‐8072‐6‐001 through the Center for Food Safety Engineering at Purdue University are acknowledged.LAP Surface Ligand Fishing ExpeditionAs the experiment scheme shown (A), several Co‐immunoprecipated candidates were visualized by Sliver staining. Red arrow point to a potential receptor around 70 kDa (B). Abundance of each identified protein was graphed according to relative protein abundance (C). Internalin B is the first protein present only in pathogenic strain.Figure 1MgCl2 Treatment Affects LAP Dissociation and Bacterial AdhesionWB protein analysis in Supernatant and Cell Wall Fraction (A), growth curve (B) and adhesion (C) of LmWT grow with additional MgCl2 in TSBYE.Figure 2

Crossing the Intestinal Barrier via Listeria Adhesion Protein and Internalin A.

The intestinal epithelial cell lining provides the first line of defense, yet foodborne pathogens such as Listeria monocytogenes can overcome this barrier; however, the underlying mechanism is not well understood. Though the host M cells in Peyer's patch and the bacterial invasion protein internalin A (InlA) are involved, L. monocytogenes can cross the gut barrier in their absence. The interaction of Listeria adhesion protein (LAP) with the host cell receptor (heat shock protein 60) disrupts the epithelial barrier, promoting bacterial translocation. InlA aids L. monocytogenes transcytosis via interaction with the E-cadherin receptor, which is facilitated by epithelial cell extrusion and goblet cell exocytosis; however, LAP-induced cell junction opening may be an alternative bacterial strategy for InlA access to E-cadherin and its translocation. Here, we summarize the strategies that L. monocytogenes employs to circumvent the intestinal epithelial barrier and compare and contrast these strategies with other enteric bacterial pathogens. Additionally, we provide implications of recent findings for food safety regulations.

Listeria monocytogenes Relies on the Heme-Regulated Transporter hrtAB to Resist Heme Toxicity and Uses Heme as a Signal to Induce Transcription of lmo1634, Encoding Listeria Adhesion Protein.

For pathogenic bacteria, host-derived heme represents an important metabolic cofactor and a source for iron. However, high levels of heme are toxic to bacteria. We have previously shown that excess heme has a growth-inhibitory effect on the Gram-positive foodborne pathogen Listeria monocytogenes, and we have learned that the LhrC1-5 family of small RNAs, together with the two-component system (TCS) LisRK, play a role in the adaptation of L. monocytogenes to heme stress conditions. However, a broader knowledge on how this pathogen responds to heme toxicity is still lacking. Here, we analyzed the global transcriptomic response of L. monocytogenes to heme stress. We found that the response of L. monocytogenes to excess heme is multifaceted, involving various strategies acting to minimize the toxic effects of heme. For example, heme exposure triggers the SOS response that deals with DNA damage. In parallel, L. monocytogenes shuts down the transcription of genes involved in heme/iron uptake and utilization. Furthermore, heme stress resulted in a massive increase in the transcription of a putative heme detoxification system, hrtAB, which is highly conserved in Gram-positive bacteria. As expected, we found that the TCS HssRS is required for heme-mediated induction of hrtAB and that a functional heme efflux system is essential for L. monocytogenes to resist heme toxicity. Curiously, the most highly up-regulated gene upon heme stress was lmo1634, encoding the Listeria adhesion protein, LAP, which acts to promote the translocation of L. monocytogenes across the intestinal barrier. Additionally, LAP is predicted to act as a bifunctional acetaldehyde-CoA/alcohol dehydrogenase. Surprisingly, a mutant lacking lmo1634 grows well under heme stress conditions, showing that LAP is not required for L. monocytogenes to resist heme toxicity. Likewise, a functional ResDE TCS, which contributes to heme-mediated expression of lmo1634, is not required for the adaptation of L. monocytogenes to heme stress conditions. Collectively, this study provides novel insights into the strategies employed by L. monocytogenes to resist heme toxicity. Our findings indicate that L. monocytogenes is using heme as a host-derived signaling molecule to control the expression of its virulence genes, as exemplified by lmo1634.

Antilisterial and Antibiofilm Activities of Pediocin and LAP Functionalized Gold Nanoparticles

In this study, we synthesized and assessed the antilisterial and antibiofilm properties of a novel gold nanocomposite, functionalized with antimicrobial peptide, Pediocin AcH and Listeria adhesion protein (LAP) for targeted inactivation of L. monocytogenes. The gold nanoparticle (GNP) and the gold nanocomposites (GNP-Pediocin-LAP) were characterized using spectroscopic and transmission electron microscopy (TEM) and their effect on human enterocyte-like Caco-2 cells were assessed by lactate dehydrogenase (LDH)-based cytotoxicity and inhibition of Listeria adhesion assay. The antilisterial and antibiofilm activities of nanocomposites on L. monocytogenes were determined by a plating method. TEM image analysis indicated that the size of GNP and the gold nanoconjugates to be about 20 nm and 40 nm, respectively; and spectroscopy indicated the successful loading of proteins onto citrate-stabilized GNPs. Gold nanocomposites were non-toxic and significantly reduced L. monocytogenes adhesion to Caco-2 cells. Relative to the GNP-Pediocin conjugate, the GNP-Pediocin-LAP conjugate showed 11.1% higher zone of inhibition in agar diffusion assay, and higher reduction (1.5 log10 CFU/mL) in L. monocytogenes counts. Same preparation also showed 24% and 31% more reduction in Listeria counts in biofilms after 24 h and 48 h of incubation, respectively. Nanocomposites were also highly effective in decontamination of L. monocytogenes on a miniature industrial conveyor system. Altogether, co-action of Pediocin and the LAP functionalized on GNP (GNP-Pediocin-LAP), demonstrated higher antilisterial and antibiofilm activities compared to the Pediocin functionalized GNP or Pediocin alone suggesting GNP can provide a platform to load multiple proteins for surface decontamination of L. monocytogenes in industrial settings.

Tunicamycin Mediated Inhibition of Wall Teichoic Acid Affects Staphylococcus aureus and Listeria monocytogenes Cell Morphology, Biofilm Formation and Virulence.

The emergence of bacterial resistance to therapeutic antibiotics limits options for treatment of common microbial diseases. Subinhibitory antibiotics dosing, often aid in the emergence of resistance, but its impact on pathogen’s physiology and pathogenesis is not well understood. Here we investigated the effect of tunicamycin, a cell wall teichoic acid (WTA) biosynthesis inhibiting antibiotic at the subinhibitory dosage on Staphylococcus aureus and Listeria monocytogenes physiology, antibiotic cross-resistance, biofilm-formation, and virulence. Minimum inhibitory concentration (MIC) of tunicamycin to S. aureus and L. monocytogenes was 20–40 μg/ml and 2.5–5 μg/ml, respectively, and the subinhibitory concentration was 2.5–5 μg/ml and 0.31–0.62 μg/ml, respectively. Tunicamycin pre-exposure reduced cellular WTA levels by 18–20% and affected bacterial cell wall ultrastructure, cell membrane permeability, morphology, laser-induced colony scatter signature, and bacterial ability to form biofilms. It also induced a moderate level of cross-resistance to tetracycline, ampicillin, erythromycin, and meropenem for S. aureus, and ampicillin, erythromycin, vancomycin, and meropenem for L. monocytogenes. Pre-treatment of bacterial cells with subinhibitory concentrations of tunicamycin also significantly reduced bacterial adhesion to and invasion into an enterocyte-like Caco-2 cell line, which is supported by reduced expression of key virulence factors, Internalin B (InlB) and Listeria adhesion protein (LAP) in L. monocytogenes, and a S. aureus surface protein A (SasA) in S. aureus. Tunicamycin-treated bacteria or the bacterial WTA preparation suppressed NF-κB and inflammatory cytokine production (TNFα, and IL-6) from murine macrophage cell line (RAW 264.7) indicating the reduced WTA level possibly attenuates an inflammatory response. These results suggest that at the subinhibitory dosage, tunicamycin-mediated inhibition of WTA biosynthesis interferes with cell wall structure, pathogens infectivity and inflammatory response, and ability to form biofilms but promotes the development of antibiotic cross-resistance.

Listeria Adhesion Protein Induces Intestinal Epithelial Barrier Dysfunction for Bacterial Translocation

Intestinal epithelial cells are the first line of defense against enteric pathogens, yet bacterial pathogens, such as Listeria monocytogenes, can breach this barrier. We show that Listeria adhesion protein (LAP) induces intestinal epithelial barrier dysfunction to promote bacterial translocation. These disruptions are attributed to the production of pro-inflammatory cytokines TNF-α and IL-6, which is observed in mice challenged with WT and isogenic strains lacking the surface invasion protein Internalin A (ΔinlA), but not a lap- mutant. Additionally, upon engagement of its surface receptor Hsp60, LAP activates canonical NF-κB signaling, facilitating myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via cellular redistribution of the epithelial junctional proteins claudin-1, occludin, and E-cadherin. Pharmacological inhibition of MLCK or NF-κB in cells or genetic ablation of MLCK in mice prevents mislocalization of junctional proteins and L.monocytogenes translocation. Thus, L.monocytogenes uses LAP to exploit epithelial defenses and cross the intestinal epithelial barrier.

Listeria monocytogenes remodels the cell surface in the blood-stage.

After crossing the intestinal barrier, the bacterial pathogen Listeria monocytogenes disseminates via the blood to the liver, spleen, brain and placenta. Transcriptomic studies have shown that L. monocytogenes changes expression of many genes during this blood-stage. However, no comparable data at the protein level are known. As main interactors with the environment, we focused in surface proteins produced by L. monocytogenes in an ex vivo bovine blood model. Bacteria exposed to blood alter selectively the amount of several surface proteins compared with bacteria grown in laboratory media. Increased levels were detected for Lmo0514 and Internalin A, two surface proteins covalently bound to peptidoglycan, and the moonlighting protein alcohol-acetaldehyde dehydrogenase, also known as Lap for 'Listeria adhesion protein'. Lmo0514, induced by L. monocytogenes inside epithelial cells, is required for survival in plasma and for virulence in mice at early infection stages. Lmo0514 is also important to cope with low pH stress. By contrast, L. monocytogenes down-regulates other surface proteins following exposure to blood and plasma such as Internalin I. These data provide evidence for remodelling of the L. monocytogenes cell surface during the blood-stage, which it could facilitate pathogen dissemination to deep organs.

The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication

Lmof2365_2117 is a Listeria monocytogenes putative cell wall surface anchor protein with a conserved domain found in collagen binding proteins. We constructed a deletion mutation in lmof2365_2117 in serotype 4b strain F2365, evaluated its virulence, and determined its ability to adhere and invade colonic epithelial cells and macrophages. In A/J mice, colonization of liver was significantly higher for F2365 than for F2365Δ2117. The ability of F2365Δ2117 to adhere to Caco-2 cells was significantly lower than F2365. The mutant also showed impaired ability to replicate in intestinal epithelial cell and murine macrophages relative to wild type F2365. Lmof2365_2117 contributed to L. monocytogenes attachment to catfish fillets. Because of its role in adherence to Caco-2 cells, we designated Lmof2365_2117 Listeria adhesion protein B (LapB). The carboxy terminus of LapB is similar to a domain in collagen binding proteins, but our results show that L. monocytogenes does not bind collagen.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food.

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 104 CFU/mL.

Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier

BackgroundListeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown.MethodsAmounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells.ResultsWestern blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates.ConclusionThis study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro.

Background Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.Methodology/Principal FindingsThe objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (LbpLAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to LbpLAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, LbpLAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. LbpLAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, LbpLAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.Conclusions/SignificanceWild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, LbpLAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.

N-terminal Gly(224)-Gly(411) domain in Listeria adhesion protein interacts with host receptor Hsp60.

BackgroundListeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.MethodsUsing a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met1–Pro223) and N2 (Gly224–Gly411), and the ADH region contains C1 (Gly412–Val648) and C2 (Pro649–Val866). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.ResultsThe N2 subdomain exhibited the greatest affinity for Hsp60 with a K D of 9.50±2.6 nM. The K D of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.ConclusionThese data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies.

Human heat-shock protein 60 receptor-coated paramagnetic beads show improved capture of Listeria monocytogenes in the presence of other Listeria in food

To investigate the suitability of human Hsp60, a receptor for Listeria adhesion protein (LAP), on paramagnetic beads (PMB) to capture Listeria monocytogenes from food in the presence of other Listeria to facilitate rapid and specific detection of this pathogen. Commercially available streptavidin-coated PMBs were linked with biotinylated Hsp60 (PMB-Hsp60), and the bacterial capture efficiency from pure culture and meat samples was determined. Capture rate was also compared with the monoclonal antibody (MAb)-C11E9-coated beads (PMB-C11E9) and the commercial Dynabeads anti-Listeria. Captured cells were detected and quantified by plating on selective medium, quantitative real-time PCR (qPCR) and a light-scattering sensor. Overall, all ligand-coated beads had similar capture efficiency (varied from 1·8 to 9·2%) for L. monocytogenes under the conditions employed, and the minimum cell number required to achieve such capture was 10³ CFU ml⁻¹. PMB-Hsp60 had significantly greater capture efficiency for pathogenic Listeria (P < 0·0001) than the nonpathogenic Listeria. In contrast, PMB-C11E9 and Dynabeads anti-Listeria had similar capture efficiency for both. The efficacy of all PMBs to capture L. monocytogenes in the presence of Listeria innocua from food matrices was compared. Although Dynabeads anti-Listeria had the overall best capture efficiency, PMB-Hsp60 was able to selectively capture L. monocytogenes even in the presence of 10-100-fold more L. innocua cells from enriched meat samples. Data show that the human cell receptor, Hsp60, is suitable for the capture of pathogenic Listeria on PMB in the presence of other Listeria in food. As pathogen interaction with host cells is highly specific, host cell receptors could be used as alternate capture molecules on PMB to aid in specific detection of pathogens.

LAP, an alcohol acetaldehyde dehydrogenase enzyme in Listeria, promotes bacterial adhesion to enterocyte-like Caco-2 cells only in pathogenic species

Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase (lmo1634), interacts with host-cell receptor Hsp60 to promote bacterial adhesion during the intestinal phase of Listeria monocytogenes infection. The LAP homologue is present in pathogens (L. monocytogenes, L. ivanovii) and non-pathogens (L. innocua, L. welshimeri, L. seeligeri); however, its role in non-pathogens is unknown. Sequence analysis revealed 98 % amino acid similarity in LAP from all Listeria species. The N-terminus contains acetaldehyde dehydrogenase (ALDH) and the C-terminus an alcohol dehydrogenase (ADH). Recombinant LAP from L. monocytogenes, L. ivanovii, L. innocua and L. welshimeri exhibited ALDH and ADH activities, and displayed strong binding affinity (K(D) 2-31 nM) towards Hsp60. Flow cytometry, ELISA and immunoelectron microscopy revealed more surface-associated LAP in pathogens than non-pathogens. Pathogens exhibited significantly higher adhesion (P<0.05) to Caco-2 cells than non-pathogens; however, pretreatment of bacteria with Hsp60 caused 47-92 % reduction in adhesion only in pathogens. These data suggest that biochemical properties of LAP from pathogenic Listeria are similar to those of the protein from non-pathogens in many respects, such as substrate specificity, immunogenicity, and binding affinity to Hsp60. However, protein fractionation analysis of extracts from pathogenic and non-pathogenic Listeria species revealed that LAP was greatly reduced in intracellular and cell-surface protein fractions, and undetectable in the extracellular milieu of non-pathogens even though the lap transcript levels were similar for both. Furthermore, a LAP preparation from L. monocytogenes restored adhesion in a lap mutant (KB208) of L. monocytogenes but not in L. innocua, indicating possible lack of surface reassociation of LAP molecules in this bacterium. Taken together, these data suggest that LAP expression level, cell-surface localization, secretion and reassociation are responsible for LAP-mediated pathogenicity and possibly evolved to adapt to a parasitic life cycle in the host.

Expression of LAP, a SecA2-dependent secretory protein, is induced under anaerobic environment

Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase homolog ( lmo1634) in Listeria monocytogenes, promotes bacterial adhesion to intestinal epithelial cells in vitro. Investigation of the effect of anaerobiosis, an intrinsic gastrointestinal condition, on LAP expression and LAP-mediated infection should elucidate its significance during intestinal infection. The influence of anaerobiosis on LAP expression was determined by growing L. monocytogenes wild type (WT), and lap-deficient (KB208) and -complemented (CKB208) strains anaerobically and monitoring LAP in secreted, cell wall, and whole-cell protein fractions. The effect of anaerobiosis on LAP-mediated infection was evaluated in cell culture adhesion assays and mouse infection models. Additionally, the role of secretory system SecA2 in LAP secretion was investigated. Anaerobic growth induced significant increases in level of lap transcript and protein secretion, and secretion was SecA2-dependent. Anaerobiosis facilitated greater LAP-mediated adhesion of L. monocytogenes to cultured intestinal cells. Oral administration of WT, KB208 and CKB208 to mice confirmed that LAP is essential for full virulence, and anaerobically-grown WT exhibited greater translocation to liver and spleen relative to aerobically-grown organisms. LAP, a SecA2-dependent secreted virulence factor, plays an important role during intestinal infection, particularly when L. monocytogenes is subjected to an anaerobic environment.