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 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 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

N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60

Listeria 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.Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1)-Pro(223)) and N2 (Gly(224)-Gly(411)), and the ADH region contains C1 (Gly(412)-Val(648)) and C2 (Pro(649)-Val(866)). 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.The 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.These 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

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 FINDINGS: The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp(LAP)) 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 Lbp(LAP) 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, Lbp(LAP) pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp(LAP) also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp(LAP) cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. CONCLUSIONS/SIGNIFICANCE: Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp(LAP) 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

Investigating the antimicrobial potential of trans-cinnamaldehyde for controlling Cronobacter sakazakii infections

Cronobacter sakazakii is an emerging pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of the pathogen. In this dissertation, the efficacy of trans-cinnamaldehyde (TC), an ingredient in cinnamon, was investigated for inactivating C. sakazakii in infant formula, decreasing its stress resistance to environmental stresses, inhibiting and inactivating biofilm formation and reducing virulence in vitro. In addition, proteomics was used to elucidate the antimicrobial mechanism of action of TC at the molecular level. TC significantly (P≤0.05) reduced C. sakazakii populations in reconstituted infant formula compared to control. Resistance to acid, heat, osmotic and desiccation in C. sakazakii was significantly reduced by exposure to TC. Additionally TC at sub-inhibitory concentrations reduced the ability of C. sakazakii to form biofilms while higher concentrations of TC inactivated fully formed mature biofilms. TC reduced virulence in C. sakazakii by decreasing its motility, invasion of intestinal epithelial and brain cells, survival within macrophages, induction of nitric oxide synthesis and endotoxin production. Real time qPCR results revealed that TC downregulated the expression of genes essential for stress resistance, biofilm formation and virulence in C. sakazakii. The proteomics data revealed that TC exerts antimicrobial effects by multiple mechanisms, including disruption of carbohydrate, amino acid and lipid metabolism. The efficacy of TC in attenuating stress tolerance, biofilm formation and major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of human diseases caused by this pathogen. Additionally the proteomics study provides us with novel targets for controlling C. sakazakii infections.

Sub-Inhibitory Concentrations of Trans-Cinnamaldehyde Attenuate Virulence in Cronobacter sakazakii in Vitro

Cronobacter sakazakii is a foodborne pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of C. sakazakii. In this study, we investigated the efficacy of sub-inhibitory concentrations (SIC) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii virulence in vitro using cell culture, microscopy and gene expression assays. TC significantly (p ≤ 0.05) suppressed C. sakazakii adhesion to and invasion of human and rat intestinal epithelial cells, and human brain microvascular endothelial cells. In addition, TC inhibited C. sakazakii survival and replication in human macrophages. We also observed that TC reduced the ability of C. sakazakii to cause cell death in rat intestinal cells, by inhibiting nitric oxide production. Results from gene expression studies revealed that TC significantly downregulated the virulence genes critical for motility, host tissue adhesion and invasion, macrophage survival, and LPS (Lipopolysaccharide) synthesis in C. sakazakii. The efficacy of TC in attenuating these major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of infection caused by this pathogen

Effect of Octenidine Hydrochloride on Planktonic Cells and Biofilms of Listeria monocytogenes▿

Listeria monocytogenes is a food-borne pathogen capable of forming biofilms and persisting in food processing environments for extended periods of time, thereby potentially contaminating foods. The efficacy of octenidine hydrochloride (OH) for inactivating planktonic cells and preformed biofilms of L. monocytogenes was investigated at 37, 21, 8, and 4°C in the presence and absence of organic matter (rehydrated nonfat dry milk). OH rapidly killed planktonic cells and biofilms of L. monocytogenes at all four temperatures. Moreover, OH was equally effective in killing L. monocytogenes biofilms on polystyrene and stainless steel matrices in the presence and absence of organic matter. The results underscore OH's ability to prevent establishment of L. monocytogenes biofilms by rapidly killing planktonic cells and to eliminate preformed biofilms, thus suggesting that it could be used as a disinfectant to prevent L. monocytogenes from persisting in food processing environments