Antilisterial Peptide Research Articles

Anti-listerial peptides from a marine Bacillus velezensis FTL7: production optimization, characterizations and molecular docking studies.

The online version contains supplementary material available at 10.1007/s13205-024-03944-5.

Heterologous Expression of the Leuconostoc Bacteriocin Leucocin C in Probiotic Yeast Saccharomyces boulardii

The yeast Saccharomyces boulardii is well known for its probiotic effects such as treating or preventing gastrointestinal diseases. Due to its ability to survive in stomach and intestine, S. boulardii could be applied as a vehicle for producing and delivering bioactive substances of interest to human gut. In this study, we cloned the gene lecC encoding the antilisterial peptide leucocin C from lactic acid bacterium Leuconostoc carnosum in S. boulardii. The constructed S. boulardii strain secreted a peptide, which had molecular weight corresponding to leucocin C in SDS-PAGE. The peptide band inhibited Listeria monocytogenes in gel overlay assay. Likewise, concentrated S. boulardii culture supernatant inhibited the growth of L. monocytogenes. The growth profile and acid tolerance of the leucocin C secreting S. boulardii were similar as those of the strain carrying the empty vector. We further demonstrated that the cells of the leucocin C producing S. boulardii efficiently killed L. monocytogenes, also without antibiotic selection pressure. These results showed that antilisterial activity could be added to the arsenal of probiotic activities of S. boulardii, demonstrating its potential as a carrier for therapeutics delivery.

PVOH/protein blend films embedded with lactic acid bacteria and their antilisterial activity in pasteurized milk

PVOH-based polymer matrices in the form of films were evaluated as carriers of living Lactococcus lactis subsp. Lactis. These lactic acid bacteria are capable of producing nisin, which is an effective antilisterial peptide. A low percentage (1:0.125 w/w) of yeast extract, gelatin, sodium caseinate, gelatin, or casein hydrolysates was incorporated in PVOH matrices with the aim of increasing the viability of bacteria in the film. The films were obtained by casting after incorporating L. lactis. Then they were evaluated for antilisterial activity in liquid medium at 37 °C for 24 h, and also at 4 °C for 21 days in order to simulate the storage of liquid foods in refrigeration conditions. The survival of the lactic acid bacteria was also evaluated at both temperatures during the experiment. L. lactis remained viable in all the films tested at 37 and 4 °C. The antimicrobial activity of the films was greater at 4 °C than at 37 °C. With regard to the effect of the film composition, the activity of the films was higher when protein hydrolysates and sodium caseinate were incorporated in the formulation. Films supplemented with protein hydrolysates or sodium caseinate inhibited growth of the pathogen during the 21 days of storage at 4 °C. At 37 °C, after 24 h the films had slowed the growth of the inoculated pathogen by between 2 and 4 log CFU/mL.Finally, as the films developed are intended to be used in the design of active packaging of foods, they were tested in pasteurized milk inoculated with 4 log CFU/mL of Listeria monocytogenes and stored at 4 °C for 21 days. The pathogen began to grow after the second day of storage with or without film, but when the films were added to the medium the growth of the pathogen was slowed down, without reaching >6 log CFU, whereas the control reached a maximum growth of 8.5 log CFU. The pH of the milk was monitored throughout the experiment, and it decreased with time. This was due to the generation of organic acids by the lactic bacteria. Buffering the food stabilized the pH without modifying the activity of the films. Thus, the current study shows that PVOH films supplemented with nutrients can act as carriers of L. lactis, and they can help to increase the safety of refrigerated dairy beverages and sauces.

Characterization of multiple antilisterial peptides produced by sakacin P-producing Lactobacillus sakei subsp. sakei 2a.

Antimicrobial compounds produced by lactic acid bacteria can be explored as natural food biopreservatives. In a previous report, the main antimicrobial compounds produced by the Brazilian meat isolate Lactobacillus sakei subsp. sakei 2a, i.e., bacteriocin sakacin P and two ribosomal peptides (P2 and P3) active against Listeria monocytogenes, were described. In this study, we report the spectrum of activity, molecular mass, structural identity and mechanism of action of additional six antilisterial peptides produced by Lb. sakei 2a, detected in a 24h-culture in MRS broth submitted to acid treatment (pH 1.5) and proper fractionation and purification steps for obtention of free and cell-bound proteins. The six peptides presented similarity to different ribosomal proteins of Lb. sakei subsp sakei 23K and the molecular masses varied from 4.6 to 11.0kDa. All peptides were capable to increase the efflux of ATP and decrease the membrane potential in Listeria monocytogenes. The activity of a pool of the obtained antilisterial compounds [enriched active fraction (EAF)] against Listeria monocytogenes in a food model (meat gravy) during refrigerated storage (4°C) for 10days was also tested and results indicated that the populations of L. monocytogenes in the food model containing the acid extract remained lower than those at time 0-day, evidencing that the acid extract of a culture of Lb. sakei 2a is a good technological alternative for the control of growth of L. monocytogenes in foods.

Isolation, Identification of Lactobacillus mucosae AN1 and its Antilisterial Peptide Purification and Characterization.

Lactobacillus mucosae strain AN1 isolated from sheep milk and characterized for its probiotic suitability. In vitro evaluation of critical gut endurance properties of this strain were assessed by different screening methods such as bile salt, gastric acid, lysozyme tolerance assays, hemolytic, cholesterol reduction properties, and HT-29 cell line adhesion assay. Antibacterial peptide from this strain was purified using ammonium sulphate precipitation, gel filtration chromatography and reverse-phase HPLC. The molecular mass of peptides was determined by Tricine-SDS-PAGE and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectroscopy (MALDI-TOF-MS). Purified peptide was named as AN1 having a molecular mass of 10.66kDa. Helical structures of peptide were determined using circular dichroism spectroscopy. Stability of peptide AN1 towards different parameters such as pH, temperature, organic solvents, proteolytic, and glycolytic enzymes was also analyzed.

Biochemical and Molecular Analysis of the Antilisterial Peptides Produced by Enterococcus hirae Strains Isolated from Raw Ewe Milk

Biochemical and Molecular Analysis of the Antilisterial Peptides Produced by Enterococcus hirae Strains Isolated from Raw Ewe Milk

The antibacterial and antibiofilm efficacies of a liposomal peptide originating from rice bran protein against Listeria monocytogenes.

With the aim of exploring a natural antilisterial peptide from food-derived origin, an antibacterial peptide named as Alpep7 was purified from the bromelain hydrolysate of rice bran protein (RBP) in this study. The resulting amino acid consequence was identified as KVDHFPL (Lys-Val-Asp-His-Phe-Pro-Leu) by ultraperformance liquid chromatography tandem matrix-assisted laser desorption/ionisation quadrupole time-of-flight mass spectrometry (MALDI Q-TOF MS). In addition, to assess the probability of the targeted delivery of liposome encapsulation of the peptide to Listeria biofilm, Alpep7-loaded liposomes were further prepared from a mixture of dipalmitoylphosphatidylcholine, stearylamine and cholesterol in a molar ratio of 10 : 3 : 2 and characterised by the analysis of particle size, zeta potential, microtopography and storage stability. The results showed that the liposomes exhibited a well-defined spherical shape, with an average diameter below 200 nm. The liposomes maintained favourable stability after storage at 4 °C for 4 weeks. Comparisons between the activities of free and liposomal Alpep7 via microbroth dilution, regrowth analysis and confocal scanning laser microscopy suggested that liposomal delivery was more effective during the initial exposure of the liposomes to the biofilms. The thermodynamic analysis indicated that the adsorption of liposomal Alpep7 to the listerial biofilm was a spontaneous, exothermic process. The results may provide a natural means for the treatment of listerial contamination and guide the potential application of liposomes for the targeted delivery of antimicrobials to pathogenic biofilms in the food industry.

Purification of Antilisterial Peptide (Subtilosin A) from Novel Bacillus tequilensis FR9 and Demonstrate Their Pathogen Invasion Protection Ability Using Human Carcinoma Cell Line.

This study focuses on isolation, screening, and characterization of novel probiotics from gastrointestinal tract of free-range chicken (Gallus gallus domesticus). Fifty seven colonies were isolated and three isolates (FR4, FR9, and FR12) were selected and identified as Lactobacillus gasseri FR4, Bacillus tequilensis FR9, and L. animalis FR12 by 16S rRNA sequencing. Three strains were able to survive in stimulated acidic and bile conditions and inhibit the growth of pathogens. Especially, FR9 exhibited maximum inhibition against Listeria monocytogenes and none of them exhibited hemolytic activity. Native-PAGE revealed the presence of low molecular weight (3.4–5.0 KDa) antimicrobial peptide. The peptide was further purified by Sephadex G-50 column and RP-HPLC using C18 column. N-terminal amino acid sequencing of antimicrobial peptide showed 100% consensus to antilisterial peptide Subtilosin A and SboA gene was amplified from FR9 genome. FR9 showed maximum aggregation activity, exopolysaccharide production (85.46 mg/L) and cholesterol assimilation (63.12 ± 0.05 μg/mL). Strong adhesion property (12.6%) and pathogen invasion protection ability was revealed by B. tequilensis FR9 towards HCT-116 human colon carcinoma cell line. This is the first study to demonstrate antilisterial Subtilosin A production of B. tequilensis. Our results indicate that B. tequilensis FR9 strain furnish the essential characteristics of a potential probiotics and might be incorporated into human and animal food supplements.

Antilisterial peptides from Spanish dry-cured hams: Purification and identification

The typical Spanish dry-cured ham has a particular sensory quality that makes it a distinctive food, highly appreciated for consumers worldwide. Its particular physicochemical properties, such as high salt content and reduced water activity contribute to their shelf-stability. However, post-processing actions carried out for the commercialization of these products such as slicing may increase the risk of development of pathogenic microorganisms as Listeria monocytogenes. During ripening, muscle proteins are hydrolyzed by muscle peptidases releasing peptides and free amino acids. Some of these peptides have been described to exert biological activities such as antioxidant and ACE-inhibition. In this study, a peptidomic strategy using mass spectrometry techniques has been used to identify and sequence those naturally generated peptides showing antilisterial activity. One hundred and five peptides have been identified in active fractions and some synthesized and their MIC calculated. Ten peptides were able to inhibit the growth of L. monocytogenes, being the pentapeptide RHGYM the most effective showing a MIC value of 6.25 mM. This study proves for the first time the potential antimicrobial action against L. monocytogenes of certain naturally generated peptides obtained from Spanish dry-cured ham.

Antilisterial Peptides Released by Enzymatic Hydrolysis from Grass Carp Proteins and Activity on Controlling L. monocytogenes Inoculated in Surimi Noodle

The primary objective of this study was to prepare the antilisterial peptides by enzymatic (pepsin, trypsin, protamex, neutrase, flavourzyme, papain, alcalase, and acid protease) hydrolysis of grass carp proteins with various degree of hydrolysis. The second objective was to evaluate the antilisterial activity of grass carp proteins hydrolysates (GCPH) at differnet levels in the surimi noodle samples with or without boiling treatment inoculated with 10(4) CFU/g of Listeria monocytogenes for storage at 4 and 25 °C up to 20 d. These results revealed that GCPH, obtained by treatment with the neutrase hydrolysates at degree of hydrolysis of 19% (NSH19), showed the highest antilisterial activity reaching up to 64.8% inhibition of bacterial growth. The antilisterial activity of NSH19 in the raw surimi noodle was stronger than that of the boiled group, and the samples treated by NSH19 at 10 g/100 g level had no-detectable numbers of L. monocytogenes for both raw and boiled noodle samples within 20 d of storage at 4 and 25 °C. The results of this study indicated that antilisterial peptides generated via neutrase from grass carp proteins can efficiently inhibit the growth of L. monocytogenes in surimi noodle, which was useful as natural preservatives for storage and distribution of meat based products. The results of this study indicated that antilisterial peptides generated via neutrase from grass carp proteins can efficiently inhibit the growth of L. monocytogenes in surimi noodle, which was useful as natural preservatives for storage and distribution of meat based products.

Detection of Listeria monocytogenes with short peptide fragments from class IIa bacteriocins as recognition elements.

We employed a direct peptide-bacteria binding assay to screen peptide fragments for high and specific binding to Listeria monocytogenes. Peptides were screened from a peptide array library synthesized on cellulose membrane. Twenty four peptide fragments (each a 14-mer) were derived from three potent anti-listerial peptides, Leucocin A, Pediocin PA1, and Curvacin A, that belong to class IIa bacteriocins. Fragment Leu10 (GEAFSAGVHRLANG), derived from the C-terminal region of Leucocin A, displayed the highest binding among all of the library fragments toward several pathogenic Gram-positive bacteria, including L. monocytogenes, Enterococcus faecalis, and Staphylococcus aureus. The specific binding of Leu10 to L. monocytogenes was further validated using microcantilever (MCL) experiments. Microcantilevers coated with gold were functionalized with peptides by chemical conjugation using a cysteamine linker to yield a peptide density of ∼4.8×10(-3) μmol/cm2 for different peptide fragments. Leu10 (14-mer) functionalized MCL was able to detect Listeria with same sensitivity as that of Leucocin A (37-mer) functionalized MCL, validating the use of short peptide fragments in bacterial detection platforms. Fragment Leu10 folded into a helical conformation in solution, like that of native Leucocin A, suggesting that both Leu10 and Leucocin A may employ a similar mechanism for binding target bacteria. The results show that peptide-conjugated microcantilevers can function as highly sensitive platforms for Listeria detection and hold potential to be developed as biosensors for pathogenic bacteria.

Characterization and Antilisterial Effect of Phosphatidylcholine Nanovesicles Containing the Antimicrobial Peptide Pediocin.

Encapsulation may provide increased stability and antimicrobial efficiency to bacteriocins. In this work, the antilisterial peptide pediocin was encapsulated in nanovesicles prepared from partially purified soybean phosphatidylcholine. The maintenance of antimicrobial activity and properties of free and encapsulated pediocin was observed during 13days at 4°C, and after this period, the encapsulated pediocin retained 50% its initial activity. The maintenance of the bioactive properties of free and encapsulated pediocin was observed against different species of Listeria, inhibiting Listeria monocytogenes, Listeria innocua and Listeria ivanovii. The size of vesicles containing pediocin was determined by dynamic light scattering as an average of 190nm, with little change throughout the observation period. Polydispersity index values were around 0.201 and are considered satisfactory, indicating an adequate size distribution of liposomes. The efficiency of encapsulation was 80%. Considering these results, the protocol used was appropriate for the encapsulation of this bacteriocin. Results demonstrate the production of stable nanoparticulate material. The maintenance of the properties of pediocin encapsulated in liposomes is fundamental to prospect the stability in different conditions of the food matrix.

Cell-mediated killing of Listeria monocytogenes by leucocin C producing Escherichia coli

Listeria monocytogenes is a foodborne pathogen causing listeriosis. Listeria in foods can be inhibited with bacteriocins or bacteriocin producing cultures. The aim of this study was to enhance the killing of L. monocytogenes by binding bacteriocin producing Escherichia coli cells to Listeria cells. Antilisterial E. coli was obtained by transferring leucocin C production from Leuconostoc carnosum 4010. For binding of E. coli cells to Listeria cells, the Listeria phage endolysin PlyP35 cell wall binding domain (CBD) was displayed on E. coli cell surface as FliC::CBD chimeric protein in flagella. CBD insertion in flagella was confirmed by Western analysis and enterokinase cleavage. By mixing isolated flagella with L. monocytogenes WSLC 1019 cells, the FliC::CBD flagella was shown to bind to Listeria cells. However, the wild type flagella also attached to Listeria cells masking putative additional binding mediated by the CBD. Yet, the cell-mediated leucocin C killing resulted in two-log reduction of Listeria, whereas the corresponding amount of leucocin C in spent culture medium could only inhibit growth without bacteriocidal effect. Cells binding Listeria and secreting antilisterial peptides may have applications in protection against listeriosis as they kill Listeria better than free antilisterial peptides.

Evidence for a chromosomally determined mesenterocin, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides OZ

Mesenterocin, a small anti-listerial peptide of 3.5 kDa produced by Leuconostoc mesenteroides subsp. mesenteroides OZ at the end of exponential growth was inactivated by proteolytic enymes, stable to cold storage (4 °C for 3 d), heat, organic solvents and surfactants, and exhibited maximum bactericidal mode of activity in the pH range 3 to 10. Although Leu. mesenteroides subsp. mesenteroides OZ harboured plasmids ranging in size from 3.6 to 9.7 kbp, no evidence was obtained indicating that mesenterocin was under the control of extrachromosomal plasmids because loss of bacteriocin production following plasmid curing experiments could not be correlated with plasmid loss and the lack of detectable plasmids suggested a chromosomal location for the genetic determinants of mesenterocin. To determine its chromosomal location, genetic determinants of the bacteriocin of Leu. mesenteroides subsp. mesenteroides OZ were tested with previously described bacteriocins of the Leuconostocs through PCR and results of PCR indicated that the gene for mesenterocin activity is located on the chromosome. No papers have been published, to the best of our knowledge, on chromosomal location of bacteriocin of Leuconostoc species. Association of meat spoilage with bacteriocin producing heterofermentative Leu. mesenteroides subsp. mesenteroides OZ is also suggested.

Defensins enable macrophages to inhibit the intracellular proliferation of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that infects a large diversity of host cells, including macrophages. To avoid the phagosome microbicidal environment, L. monocytogenes secretes a pore-forming toxin (listeriolysin O, LLO) that releases the bacterium into the cytoplasm. We hypothesized that the α-defensins (HNPs) and/or humanized θ-defensin (RC-1) peptides produced by human and non-human primate neutrophils, respectively, cooperate with macrophages to control L. monocytogenes infection. Our results establish that HNP-1 and RC-1 enable macrophages to control L. monocytogenes intracellular growth by inhibiting phagosomal escape, as a consequence, bacteria remain trapped in a LAMP-1-positive phagosome. Importantly, HNP-1 interaction with macrophages and RC-1 interaction with bacteria are required to prevent macrophage infection. In accordance with these results, RC-1 is a more potent anti-listerial peptide than HNP-1 and HNP-1 is acquired by macrophages and trafficked to the phagocytosed bacteria. Finally, HNP-1 and RC-1 antimicrobial activity is complemented by their ability to prevent LLO function through two mechanisms, blocking LLO-dependent perforation of macrophage membranes and the release of LLO from the bacteria. In conclusion, at the site of infection the cooperation between antimicrobial peptides, such as HNP-1, and macrophages likely plays a critical role in the innate immune defence against L. monocytogenes.

Production of three anti-listerial peptides by Lactobacillus curvatus in MRS broth

Three novel bioactive peptides (BAP1–3) from Lactobacillus curvatus CWBI-B28 were isolated and purified using de Man, Rogosa and Sharp (MRS) broth by a three-step protein purification protocol including ammonium sulfate precipitation, hydrophobic C18 Sep-Pak column and reverse-phase high performance liquid chromatography (RP-HPLC). This procedure allowed the recovery of chromatographically pure antimicrobial peptides with the yields of 19%, 10% and 15% of BAP1, BAP2 and BAP3, respectively. The respective apparent molecular masses as determined by tricine-SDS-polyacrilamide gel electrophoresis were 6365, 3426 and 3496. N-terminal amino acid sequencing of the BAPs and comparison of their sequences with those of international data bases indicated that BAP1 is more likely to be a casein-derived bioactive protein produced upon hydrolysis of the tryptone present in MRS broth by Lb. curvatus CWBI-B28 during active growth. However, the identity of BAP2 and BAP3 could not be determined with certainty; yet, they would be novel bacteriocins not fitting in any of the known classes of bacteriocins. Therefore, this strain would have the ability to produce intrinsic antimicrobial substances and also release bioprotective peptides from milk-proteins upon cultivation in milk or casein-containing food products due to its proteolytic activity. Thus, Lb. curratus CWBI-B28 possesses a good potential to be used in food preservation and safety.

Heterologous expression, purification and refolding of an anti-listerial peptide produced by Pediococcus acidilactici K7

The fusion protein, 6XHis-Xpress-Ped A was constructed and expressed in Escherichia coli BL21 (DE3). The presence of a 12.8 kDa recombinant protein, localized in inclusion bodies (IBs) at high concentration, was confirmed by SDS-PAGE analysis and by western blotting using anti-His antibody. The rec-pediocin was purified by Nickel-nitrilotriacetic acid beads and refolded using 5 mM of β-mercaptoethanol along with 1 M glycine. Results indicated that the refolded rec-pediocin had an early elution profile in the RP-HPLC when compared to the unfolded protein and it exhibited biological activity against Listeria monocytogenes V7 which was approximately 25 times less active compared to native counterpart. The final yield of purified rec-pediocin was 3 mg/l of the culture and is estimated to be 8-10 times higher than the purification by conventional methods.

Heterologous expression, purification and refolding of an anti-listerial peptide produced by Pediococcus acidilactici K7

Heterologous expression, purification and refolding of an anti-listerial peptide produced by Pediococcus acidilactici K7

Partial characterization of a class IIa pediocin produced by Pediococcus parvulus 133 strain isolated from meat (Mexican “chorizo”)

Pediocin-like bacteriocins are antimicrobial substances produced by some bacteria with high antilisterial activity. Several isolates of Pediococcus acidilactici and two Pediococcus parvulus strains of vegetable origin have been reported to produce this kind of peptide. This work presents the partial characterization of the bacteriocin produced by P. parvulus 133 found in meat and confirms its identity as a heat resistant, antilisterial bacteriocin. This peptide has a relatively narrow inhibitory spectrum but a high antilisterial activity. Pediocin remained active after heating to 121 °C, but its thermoresistance varied with pH. The pH selective adsorption method resulted in a 150-fold concentration of antimicrobial activity. The final extract was obtained by ultrafiltration and resulted in an additional 10-fold concentration of activity. Molecular weight was estimated as 5 kDa and isoelectric point was 8.65. The sequence of the first 17 aminoacids at the N-terminal end of the bacteriocin showed complete coincidence with that previously reported for pediocin A1 (AcH) and with an antilisterial peptide produced by Bacillus coagulans. High sequence similarity was also found with two other antilisterial bacteriocins.

Biochemical and genetic characterization of mundticin KS, an antilisterial peptide produced by Enterococcus mundtii NFRI 7393.

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.