Structural Basis of the Mechanisms of Action and Immunity of Lactococcin A, a Class IId Bacteriocin.

Lactococcin A (LcnA), a class IId bacteriocin, induces membrane leakage and cell death by specifically binding to the membrane receptor-mannose phosphotransferase system (man-PTS), as is the case for pediocin-like (class IIa) bacteriocins. The cognate immunity protein of bacteriocins, which protects the producer cell from its own bacteriocin, recognizes and binds to the bacteriocin-man-PTS complex, consequently blocking membrane leakage. We previously deciphered the mode of action and immunity of class IIa bacteriocins. Here, we determined the structure of the ternary complex of LcnA, LciA (i.e., the immunity protein), and its receptor, i.e., the man-PTS of Lactococcus lactis (ll-man-PTS). An external loop on the membrane-located component IIC of ll-man-PTS was found to prevent specific binding of the N-terminal region of LcnA to the site recognized by pediocin-like bacteriocins. Thus, the N-terminal ß-sheet region of LcnA recognized an adjacent site on the extracellular side of ll-man-PTS, with the LcnA C-terminal hydrophobic helix penetrating into the membrane. The cytoplasmic cleft formed within the man-PTS Core and Vmotif domains induced by embedded LcnA from the periplasmic side is adopted by the appropriate angle between helices H3 and H4 of the N terminus of LciA. The flexible C terminus of LciA then blocks membrane leakage. To summarize, our findings reveal the molecular mechanisms of action and immunity of LcnA and LciA, laying a foundation for further design of class IId bacteriocins. IMPORTANCE Class IId (lactococcin-like) bacteriocins and class IIa (pediocin-like) bacteriocins share a few similarities: (i) both induce membrane leakage and cell death by specifically binding the mannose phosphotransferase system (man-PTS) on their target cells, and (ii) cognate immunity proteins recognize and bind to the bacteriocin-man-PTS complex to block membrane leakage. However, class IId bacteriocins lack the "pediocin box" motif, which is typical of class IIa bacteriocins, and basically target only lactococcal cells; in contrast, class IIa bacteriocins target diverse bacterial cells, but not lactococcal cells. We previously solved the structure of class IIa bacteriocin-receptor-immunity ternary complex from Lactobacillus sakei. Here, we determined the structure of the ternary complex of class IId bacteriocin LcnA, its cognate immunity protein LciA, and its receptor, the man-PTS of Lactococcus lactis. By comparing the interactions between man-PTS and class IIa and class IId bacteriocins, this study affords some clues to better understand the specificity of bacteriocins targeting the mannose phosphotransferase system.

Heterologous expression of pediocin/papA in Bacillus subtilis.

Listeria monocytogenes is a food-borne pathogen. Pediocin is a group IIα bacteriocin with anti-listeria activity that is naturally produced by Pediococcus acidilactic and Lactobacillus plantarum. The pedA/papA gene encodes pediocin/plantaricin. In native hosts, the expression and secretion of active PedA/PapA protein rely on the accessory protein PedC/PapC and ABC transporter PedD/PapD on the same operon. The excretion machines were also necessary for pediocin protein expression in heterologous hosts of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vectors carrying the codon sequence of the mature PapA peptide were constructed, one with and one without a His tag. Both fragments were inserted into the plasmid pHT43 and transformed into Bacillus subtilis WB800N. The strains were induced with IPTG to secrete the fused proteins PA1 and PA2. Supernatants from both recombinant strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole dispense with removal of the leading peptide. This is the first report of active pediocin/PapA expression without the assistance of PedCD/PapCD in heterogeneous hosts. In addition, the PA1 protein can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography.

Application of yeast surface display system in expression of recombinant pediocin PA-1 in Saccharomyces cerevisiae.

Pediocin PA-1 is a bacteriocin that shows strongly anti-microbial activity against some Gram-positive pathogens such as Listeria monocytogenes, Staphylococcus aureus, and Enterococcus faecalis. With the broad inhibitory spectrum as well as high-temperature stability, pediocin has a potential application in the food preservation and pharmaceutical industry. Pediocin has been studied to express in many heterologous expression systems such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris as a free peptide. Here we showed in this study a new strategy by using yeast surface display system to produce the anchored pediocin PA-1 on the cell surface of Saccharomyces cerevisiae, which could be used directly as a pediocin resource. We had successfully constructed a recombinant S. cerevisiae W303 strain that could express pediocin PA-1 on the cell surface. The pediocin-expressing yeast could inhibit the growth of Shigella boydii and Shigella flexneri, which have never been reported before for pediocin activity. Besides, the pediocin expression level of the recombinant S. cerevisiae strain was also evaluated in three different media: synthetic defined (SD), basic medium (BM), and fermentation medium (FM). BM medium was shown to give the highest production yield of the recombinant yeast (4.75 ± 0.75 g dry cell weight per 1 L of culture) with the ratio number of the pediocin-expressing cells of 93.46 ± 2.45%. Taken together, the results clearly showed that pediocin can be displayed on yeast cell surface as anchored protein. The application of yeast cell surface system enables a new door of pediocin application on either food or feed industries. Graphical abstract.

Evaluation of antibacterial properties of lactic acid bacteria from traditionally and industrially produced fermented sausages from Germany.

With regards to the frequently reported findings of spoilage bacteria and pathogens in various foods there is a need to explore new ways to control hazards in food production and to improve consumer safety. Fermented sausages from traditional and industrial production in Germany were screened for lactic acid bacteria with antibacterial effects towards important foodborne pathogens (Escherichia coli DSM 1103, Listeria innocua DSM 20649, Listeria monocytogenes DSM 19094, Pseudomonas aeruginosa DSM 939, Staphylococcus aureus DSM 799 and Salmonella Typhimurium DSM 19587). The obtained isolates and their cell-free supernatants were tested for their antibacterial activity by agar well diffusion assay. Isolates with an inhibitory effect were examined for the underlying antibacterial mechanism. Among the 169 collected isolates, 12.4% showed antibacterial effects only against Listeria innocua DSM 20649 and Listeria monocytogenes DSM 19094. In 6.5% of the isolates, bacteriocins were responsible for the effect. On the remaining test strains, the lactic bacteria isolates exerted no antibacterial effect. Two isolates were selected based on their antibacterial potential against Listeria spp. and the thermostability of the deriving cell free supernatants, traditional product: Pediococcus pentosaceus LMQS 331.3, industrial product: Pediococcus acidilactici LMQS 154.1, were investigated further and confirmed for the presence of bacteriocin structural genes by real-time PCR. Enriched crude bacteriocin preparations were obtained by ammonium sulfate precipitation and were found to remain stable under different pH milieus and to be active towards an extended set of Listeria spp. strains. Fermented meat products from German production are a promising source for bacteriocin-producing lactic acid bacteria. Two bacteriocin-producing isolates were identified which have the potential to contribute to product and consumer safety.

Pediocin-Like Antimicrobial Peptides of Bacteria.

Bacteriocins are bacterial antimicrobial peptides that, unlike classical peptide antibiotics, are products of ribosomal synthesis and usually have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like bacteriocins (PLBs) belong to the class IIa of the bacteriocins of Gram-positive bacteria. PLBs possess high activity against pathogenic bacteria from Listeria and Enterococcus genera. Molecular target for PLBs is a membrane protein complex - bacterial mannose-phosphotransferase. PLBs can be synthesized by components of symbiotic microflora and participate in the maintenance of homeostasis in various compartments of the digestive tract and on the surface of epithelial tissues contacting the external environment. PLBs could give a rise to a new group of antibiotics of narrow spectrum of activity.

Both IIC and IID Components of Mannose Phosphotransferase System Are Involved in the Specific Recognition between Immunity Protein PedB and Bacteriocin-Receptor Complex.

Upon exposure to exogenous pediocin-like bacteriocins, immunity proteins specifically bind to the target receptor of the mannose phosphotransferase system components (man-PTS IIC and IID), therefore preventing bacterial cell death. However, the specific recognition of immunity proteins and its associated target receptors remains poorly understood. In this study, we constructed hybrid receptors to identify the domains of IIC and/or IID recognized by the immunity protein PedB, which confers immunity to pediocin PA-1. Using Lactobacillus plantarum man-PTS EII mutant W903, the IICD components of four pediocin PA-1-sensitive strains (L. plantarum WQ0815, Leuconostoc mesenteroides 05-43, Lactobacillus salivarius REN and Lactobacillus acidophilus 05-172) were respectively co-expressed with the immunity protein PedB. Well-diffusions assays showed that only the complex formed by LpIICD from L. plantarum WQ0815 with pediocin PA-1 could be recognized by PedB. In addition, a two-step PCR approach was used to construct hybrid receptors by combining LpIIC or LpIID recognized by PedB with the other three heterologous IID or IIC compounds unrecognized by PedB, respectively. The results showed that all six hybrid receptors were recognized by pediocin PA-1. However, when IIC or IID of L. plantarum WQ0815 was replaced with any corresponding IIC or IID component from L. mesenteroides 05-43, L. salivarius REN and L. acidophilus 05-172, all the hybrid receptors could not be recognized by PedB. Taken altogether, we concluded that both IIC and IID components of the mannose phosphotransferase system play an important role in the specific recognition between the bacteriocin-receptor complex and the immunity protein PedB.

Common Mechanism of Cross-Resistance Development in Pathogenic Bacteria Bacillus cereus Against Alamethicin and Pediocin Involves Alteration in Lipid Composition.

To understand the mechanism of development of cross-resistance in food pathogen Bacillus cereus against an antimicrobial peptide pediocin and antibiotic alamethicin, the present study was designed. Pediococcus pentosaceus was taken as a source of pediocin, and it was purified by ammonium sulphate precipitation followed by cation exchange chromatography with 14.01-fold purity and 14.4 % recovery. B. cereus strains alamethicin-resistant strains (IC50 3.23 µg/ml) were selected from sensitive population with IC50 2.37 µg/ml. The development of resistance in B. cereus against alamethicin was associated with decrease in alamethicin-membrane interaction observed by in vitro assay. Resistant strain of B. cereus was found to harbour one additional general lipid as compared to sensitive strain, one amino group lacking phospholipid and one amino group containing phospholipid (ACP). In addition, ACP content was increased in resistant mutant (29.7 %) as compared to sensitive strain (14.56 %). The alamethicin-resistant mutant B. cereus also showed increased IC50 (58.8 AU/ml) for pediocin as compared to sensitive strain (IC50 47.8 AU/ml). Cross-resistance to pediocin and alamethicin in resistant mutant of B. cereus suggested a common mechanism of resistance. Therefore, this understanding could result in the development of peptide which will be effective against the resistant strains that share same mechanism of resistance.

Survival and Metabolic Activity of Pediocin Producer Pediococcus acidilactici UL5: Its Impact on Intestinal Microbiota and Listeria monocytogenes in a Model of the Human Terminal Ileum.

Pediococcus acidilactici UL5 is a promising probiotic candidate due to its high survival rate under gastric and duodenal conditions and to its ability to produce the antilisterial bacteriocin pediocin PA-1. Its survival, metabolic activity, and impact on Listeria monocytogenes in a continuous stirred tank reactor containing immobilized human intestinal microbiota were studied over a period of 32 days of feeding a nutrient medium simulating ileal chyme. The impact of P. acidilactici UL5 on different bacterial groups of intestinal origin as well as its survival and its impact on L. monocytogenes were quantified using quantitative polymerase chain reaction coupling with propidium monoazide (PMA-qPCR), which was shown to detect and quantify viable bacteria only. P. acidilactici UL5 and its non-pediocin-producing mutant had no effect on the microbiota, but the producing strain induced an increase in the production of acetic and propionic acids. P. acidilactici survived but appeared to be a poor competitor with intestinal microbiota, dropping by 1.3 and 2.8 log10 after 8 h of fermentation to 104 colony-forming units (cfu) mL-1. A 1.64 log but non-significant reduction of Listeria was observed when P. acidilactici UL5 was added at 108 cfu mL-1. P. acidilactici UL5 isolated from the reactor after 3 days was still able to produce the active bacteriocin. These data demonstrate that P. acidilactici UL5 is capable of surviving transit through the ileum without losing its ability to produce pediocin PA-1 but seems to not be enough competitive with the great diversity of organisms found in the ileum.

Recombinant pediocin in Lactococcus lactis: increased production by propeptide fusion and improved potency by co-production with PedC.

We describe the impact of two propeptides and PedC on the production yield and the potency of recombinant pediocins produced in Lactococcus lactis. On the one hand, the sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the structural gene of the mature pediocin PA-1. On the other hand, the putative thiol-disulfide oxidoreductase PedC was coexpressed with pediocin. The concentration of recombinant pediocins produced in supernatants was determined by enzyme-linked immunosorbent assay. The potency of recombinant pediocins was investigated by measuring the minimal inhibitory concentration by agar well diffusion assay. The results show that propeptides SD or LEISSTCDA lead to an improved secretion of recombinant pediocins with apparently no effect on the antibacterial potency and that PedC increases the potency of recombinant pediocin. To our knowledge, this study reveals for the first time that pediocin tolerates fusions at the N-terminal end. Furthermore, it reveals that only expressing the pediocin structural gene in a heterologous host is not sufficient to get an optimal potency and requires the accessory protein PedC. In addition, it can be speculated that PedC catalyses the correct formation of disulfide bonds in pediocin.