Two-peptide Bacteriocin Research Articles

Innovative perspectives on bacteriocins: advances in classification, synthesis, mode of action, and food industry applications.

Bacteriocins, natural antimicrobial peptides produced by bacteria, present eco-friendly, non-toxic, and cost-effective alternatives to traditional chemical antimicrobial agents in the food industry. This review provides a comprehensive update on the classification of bacteriocins in food preservation. It highlights the significant industrial potential of pediocin-like and two-peptide bacteriocins, emphasizing chemical synthesis methods like Fmoc-SPPS to meet the demand for bioactive bacteriocins. The review details the mode of action, focusing on mechanisms such as transmembrane potential disruption and pH-dependent effects. Furthermore, it addresses the limitations of bacteriocins in food preservation and explores the potential of nanotechnology-based encapsulation to enhance their antimicrobial efficacy. The benefits of nanoencapsulation, including improved stability, extended antimicrobial spectrum, and enhanced functionality, are underscored. This understanding is crucial for advancing the application of bacteriocins to ensure food safety and quality.

Synthesis, antimicrobial activity, and mechanistic studies of enterocin DD14, a leaderless two-peptide bacteriocin

Bacteriocins are promising alternatives to antibiotics in the food, veterinary and medical sectors, but their study and use is often hampered by the low yields and high costs associated with their purification from naturally occurring bacteria. Chemical synthesis has emerged as a means to overcome this limitation and design more active variants. In this study, microwave-assisted solid-phase peptide synthesis was used to produce the leaderless two-peptide bacteriocin enterocin DD14 (EntDD14), composed of EntDD14A (44 amino acids) and EntDD14B (43 amino acids). The resulting synthetic peptides, syn-EntDD14A and syn-EntDD14B, were tested against Gram-positive bacteria including Listeria, Staphylococcus and Enterococcus strains. Both peptides were found to be necessary for optimal, but not synergistic, antibacterial activity and to act through a pore-forming mechanism. Both peptides exhibited moderate cytotoxicity against eukaryotic cells.

In Vitro and In Silico Characterization of N-Formylated Two-Peptide Bacteriocin from Enterococcus faecalis CAUM157 with Anti-Listeria Activity.

Enterococcus faecalis CAUM157 (KACC 81148BP), a Gram-positive bacteria isolated from raw cow's milk, was studied for its bacteriocin production. The antimicrobial activity of CAUM157 was attributed to a two-peptide class IIb bacteriocin with potent activity against food-borne pathogen Listeria monocytogenes and periodontal disease-causing pathogens (Prevotella intermedia KCTC 15693T and Fusobacterium nucleatum KCTC 2488T). M157 bacteriocins exhibit high temperature and pH stability and resist hydrolytic enzyme degradation and detergent denaturation, potentially due to their structural conformation. Based on amino acid sequence, M157A and M157B were predicted to be 5.176kDa and 5.182kDa in size, respectively. However, purified bacteriocins and chemically synthesized N-formylated M157 peptides both showed 5.204kDa (M157A) and 5.209kDa (M157B) molecular mass, confirming the formylation of the N-terminal methionine of both peptides produced by strain CAUM157. Furthermore, the strain demonstrated favorable growth and fermentation with minimal bacteriocin production when cultured in whey-based media, whereas a 1.0% tryptone or soytone supplementation resulted in higher bacteriocin production. Although Ent. faecalis CAUM157 innately harbors genes for virulence factors and antimicrobial resistance (e.g., tetracycline and erythromycin), its bacteriocin production is valuable in circumventing the need for live microorganisms, particularly in food applications for pathogen control.

Intestinal delivery of encapsulated bacteriocin peptides in cross-linked alginate microcapsules

Oral delivery of larger bioactive peptides (>20 amino acids) to the small intestine remains a challenge due to their sensitivity to proteolytic degradation and chemical denaturation during gastrointestinal transit. In this study, we investigated the capacity of crosslinked alginate microcapsules (CLAMs) formed by spray drying to protect Plantaricin EF (PlnEF) (C-EF) in gastric conditions and to dissolve and release PlnEF in the small intestine. PlnEF is an unmodified, two-peptide (PlnE: 33 amino acids; PlnF: 34 amino acids) bacteriocin produced by Lactiplantibacillus plantarum with antimicrobial and gut barrier protective properties. After 2 h incubation in simulated gastric fluid (SGF) (pH 1.5), 43.39 % ± 8.27 % intact PlnEF was liberated from the CLAMs encapsulates, as determined by an antimicrobial activity assay. Transfer of the undissolved fraction to simulated intestinal fluid (SIF) (pH 7) for another 2 h incubation resulted in an additional release of 16.13 % ± 4.33 %. No active PlnEF was found during SGF or sequential SIF incubations when pepsin (2,000 U/ml) was added to the SGF. To test PlnEF release in C-EF contained in a food matrix, C-EF was mixed in peanut butter (PB) (0.15 g C-EF in 1.5 g PB). A total of 12.52 % ± 9.09 % active PlnEF was detected after incubation of PB + C-EF in SGF without pepsin, whereas no activity was found when pepsin was included. Transfer of the remaining PB + C-EF fractions to SIF yielded the recovery of 46.67 % ± 13.09 % and 39.42 % ± 11.53 % active PlnEF in the SIF following exposure to SGF and to SGF with pepsin, respectively. Upon accounting for the undissolved fraction after SIF incubation, PlnEF was fully protected in the CLAMs-PB mixture and there was not a significant reduction in active PlnEF when pepsin was present. These results show that CLAMs alone do not guard PlnEF bacteriocin peptides from gastric conditions, however, mixing them in PB protected against proteolysis and improved intestinal release.

Genomic and Functional Evaluation of Two Lacticaseibacillus paracasei and Two Lactiplantibacillus plantarum Strains, Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis), as Probiotics for Aquaculture.

Aquaculture plays a crucial role in meeting the increasing global demand for food and protein sources. However, its expansion is followed by increasing challenges, such as infectious disease outbreaks and antibiotic misuse. The present study focuses on the genetic and functional analyses of two Lacticaseibacillus paracasei (BF3 and RT4) and two Lactiplantibacillus plantarum (BF12 and WT12) strains isolated from a rotifer cultivation tank used for turbot larviculture. Whole-genome sequencing (WGS) and bioinformatics analyses confirmed their probiotic potential, the absence of transferable antibiotic resistance genes, and the absence of virulence and pathogenicity factors. Bacteriocin mining identified a gene cluster encoding six plantaricins, suggesting their role in the antimicrobial activity exerted by these strains. In vitro cell-free protein synthesis (IV-CFPS) analyses was used to evaluate the expression of the plantaricin genes. The in vitro-synthesized class IIb (two-peptide bacteriocins) plantaricin E/F (PlnE/F) exerted antimicrobial activity against three indicator microorganisms, including the well-known ichthyopathogen Lactococcus garvieae. Furthermore, MALDI-TOF MS on colonies detected the presence of a major peptide that matches the dimeric form of plantaricins E (PlnE) and F (PlnF). This study emphasizes the importance of genome sequencing and bioinformatic analysis for evaluating aquaculture probiotic candidates. Moreover, it provides valuable insights into their genetic features and antimicrobial mechanisms, paving the way for their application as probiotics in larviculture, which is a major bottleneck in aquaculture.

Complete genome sequence of bacteriocin-producing Ligilactobacillus salivarius B4311 isolated from fecal samples of broiler chicken with anti-listeria activity.

Ligilactobacillus is a genus of Gram-positive lactobacilli commonly found in the intestinal tracts of vertebrates. It has been granted a Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA). One specific strain, Ligilactobacillus salivarius B4311, was isolated from fecal samples of broiler chickens from a farm associated with Chung-Ang University (Anseong, Korea). This strain was observed to have inhibitory effects against Listeria monocytogenes. In this paper, we present the complete genome sequence of Lig. salivarius B4311. The whole genome of strain B4311 comprises 2,071,255 bp assembled into 3 contigs representing a chromosome, repA-type megaplasmid, and small plasmid. The genome contains 1,963 protein-coding sequences, 22 rRNA genes, and 78 tRNA genes, with a guanine + cytosine (GC) content of 33.1%. The megaplasmid of strain B4311 was found to contain the bacteriocin gene cluster for salivaricin P, a two-peptide bacteriocin belonging to class IIb.

A Review on Enterocin DD14, the Leaderless Two-Peptide Bacteriocin with Multiple Biological Functions and Unusual Transport Pathway.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin (LLB) produced by Enterococcus faecalis 14, a human strain isolated from meconium. Studies performed on EntDD14 enabled it to show its activity against Gram-positive bacteria such as Listeria monocytogenes, Clostridium perfringens, Enterococcus faecalis, and Staphylococcus aureus. EntDD14 was also shown to potentiate the activity of different antibiotics such as erythromycin, kanamycin, and methicillin when assessed against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo in the NMRI-F holoxenic mouse model. Additionally, EntDD14 has an antiviral activity and decreased the secretion of pro-inflammatory IL-6 and IL-8 in inflamed human intestinal Caco-2 cells. The genome of E. faecalis 14 was sequenced and annotated. Molecular tools such as Bagel4 software enabled us to locate a 6.7kb-EntDD14 cluster. Transport of EntDD14 outside of the cytoplasm was shown to be performed synergistically by a channel composed of two pleckstrin-homology-domain-containing proteins, namely DdE/DdF and the ABC transporter DdGHIJ. This latter could also protect the bacteriocinogenic strain against extracellular EntDD14. Here, we focus on academic data and potential therapeutic issues of EntDD14, as a model of two-peptide LLB.

The development of a solid lipid nanoparticle (SLN)-based lacticin 3147 hydrogel for the treatment of wound infections

Chronic wounds affect millions of people globally. This number is set to rise with the increasing incidence of antimicrobial-resistant bacterial infections, such as methicillin-resistant Staphylococcus aureus (MRSA), which impair the healing of chronic wounds. Lacticin 3147 is a two-peptide chain bacteriocin produced by Lactococcus lactis that is active against S. aureus including MRSA strains. Previously, poor physicochemical properties of the peptides were overcome by the encapsulation of lacticin 3147 into solid lipid nanoparticles. Here, a lacticin 3147 solid lipid nanoparticle gel is proposed as a topical treatment for S. aureus and MRSA wound infections. Initially, lacticin 3147’s antimicrobial activity against S. aureus was determined before encapsulation into solid lipid nanoparticles. An optimised gel formulation with the desired physicochemical properties for topical application was developed, and the lacticin-loaded solid lipid nanoparticles and free lacticin 3147 aqueous solution were incorporated into separate gels. The release of lacticin 3147 from both the solid lipid nanoparticle and free lacticin gels was measured where the solid lipid nanoparticle gel exhibited increased activity for a longer period (11 days) compared to the free lacticin gel (9 days). Both gels displayed potent activity ex vivo against S. aureus-infected pig skin with significant bacterial eradication (> 75%) after 1 h. Thus, a long-acting potent lacticin 3147 solid lipid nanoparticle gel with the required physicochemical properties for topical delivery of lacticin 3147 to the skin for the potential treatment of S. aureus-infected chronic wounds was developed.Graphical abstract

Gallocin A, an Atypical Two-Peptide Bacteriocin with Intramolecular Disulfide Bonds Required for Activity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.

Advances in Characterizing the Transport Systems of and Resistance to EntDD14, A Leaderless Two-Peptide Bacteriocin with Potent Inhibitory Activity

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by the Enterococcus faecalis 14 strain previously isolated from meconium. This bacteriocin is mainly active against Gram-positive bacteria. Leaderless bacteriocins do not undergo post-translational modifications and are therefore immediately active after their synthesis. As a result, the cells that produce such bacteriocins have developed means of protection against them which often involve transport systems. In this and our previous work, we constructed different mutants deleted in the genes involved in the transport functions, thus covering all the supposed components of this transport system, using Listeria innocua ATCC 33090 as the indicator strain to assess the activity of externalized EntDD14. We also assessed the self-resistance of the WT and all its engineered derivative mutants against EntDD14, provided extracellularly, in order to evaluate their self-resistance. The results obtained highlight that the ABC transporter constituted by the DdG, H, I, and J proteins contributes to EntDD14 export as well as resistance to an external supply of EntDD14. Our results also have established the essential role of the DdE and DdF proteins as primary transporters dedicated to the externalization of EntDD14. Moreover, the in silico data showed that DdE and DdF appear to assemble in a formation that forms an essential channel for the exit of EntDD14. This channel DdEF may interact with the ABC transporter DdGHIJ in order to control the flow of bacteriocin across the membrane, although the nature of this interaction remains to be elucidated.

Identification and characterization of bacteriocin biosynthetic gene clusters found in multiple bacteriocins producing Lactiplantibacillus plantarum PUK6

The multiple bacteriocins produced by Lactiplantibacillus plantarum PUK6 isolated from misozuke-tofu (tofu pickled in miso) were identified as plantaricins A, EF, and NC8. The pln locus (21,847 bp) containing the three plantaricin structural genes and another newly found putative bacteriocin structural genes (orf1 and orf2) were determined, and a biosynthesis mechanism was proposed. Reverse transcription-PCR analysis revealed that orf1 and orf2, the putative two-peptide bacteriocin structural genes, were expressed after 8h (logarithmic growth phase) and 20h (stationary growth phase) of cultivation of the PUK6 strain. Additionally, the growth inhibition profile obtained using the chemically synthesized mature peptides of Orf1 and Orf2 (referred to as mOrf1 and mOrf2) showed that the equimolar mixture of mOrf1 and mOrf2 exhibited bactericidal effect against the indicator strain Latilactobacillus sakei subsp. sakei JCM 1157T. Furthermore, fluorescence microscopic analysis revealed disruption of the cell membranes. These findings indicate that orf1 and orf2 are structural genes encoding class IIb bacteriocins consisting of two peptides. Therefore, the novel bacteriocin encoded by plnPUK6α (orf1) and plnPUK6β (orf2) genes was named plantaricin PUK6. Since the PUK6 strain produces multiple bacteriocins, when used as a starter culture, it could contribute to improving the shelf life of fermented foods and preventing the appearance of bacteriocin-resistant bacteria.

Pharmaceutical design of a delivery system for the bacteriocin lacticin 3147

Lacticin 3147 is a dual-acting two-peptide bacteriocin which is generally active against Gram-positive bacteria, including Listeria monocytogenes and antimicrobial-resistant bacteria such as Closteroides difficile in the colon. L. monocytogenes infections can cause life-long effects in the elderly and vulnerable and can cause severe complications in pregnant women. C. difficile causes one of the most common healthcare-associated infections and can be fatal in vulnerable groups such as the elderly. Although lacticin 3147 is degraded by intestinal proteases and has poor aqueous solubility, encapsulation of the bacteriocin could enable its use as an antimicrobial for treating these bacterial infections locally in the gastrointestinal tract. Lacticin 3147 displayed activity in aqueous solutions at a range of pH values and in gastric and intestinal fluids. Exposure to trypsin and α-chymotrypsin resulted in complete inactivation, implying that lacticin 3147 should be protected from these enzymes to achieve successful local delivery to the gastrointestinal tract. The amount of lacticin 3147 dissolved, i.e. its solution concentration, in water or buffered solutions at pH 1.6 and 7.4 was low and varied with time but increased and was stabilized in gastrointestinal fluids by the phospholipid and bile salt components present. Thus, the feasibility of a solid lipid nanoparticle (SLN) delivery system for local administration of lacticin 3147 was investigated. Bacteriocin activity was observed after encapsulation and release from a lipid matrix. Moreover, activity was seen after exposure to degrading enzymes. Further optimization of SLN delivery systems could enable the successful pharmaceutical development of active lacticin 3147 as an alternative to traditional antibiotics.Graphical abstract

Novel Selective Inhibition of Lactobacillus iners by Lactobacillus-Derived Bacteriocins.

Lactobacillus iners is often associated with vaginal dysbiosis and bacterial vaginosis (BV), which are risk factors for adverse gynecological and obstetric outcomes. To discover natural inhibitors of L. iners, cell-free culture supernatants (CFSs) from 77 vaginal human Lactobacillus strains and 1 human intestinal strain were screened for inhibitory activity. Three active strains were identified, and Lactobacillus paragasseri K7 (K7), a human intestinal strain, produced the most potent L. iners-inhibitory activity. The active material was purified from the K7 CFS and yielded three active peptides, identified as components of two different class IIb, two-peptide bacteriocins, gassericin K7A (GasK7A) and gassericin K7B (GasK7B). The peptides corresponded to the GasK7A α peptide and the GasK7B α and β peptides. While all three peptides exhibited individual activity against L. iners, GasK7B α was the most potent, with an MIC of 23 ng/ml (4 nM). When combined in equal amounts, the GasK7B α and β peptides showed synergistic inhibition, with an MIC of 2 ng/ml (each peptide at 0.4 nM). Among the four major vaginal Lactobacillus species, the K7 bacteriocins selectively inhibited L. iners All 21 strains of L. iners tested (100%) were inhibited by the K7 bacteriocins, whereas <20% of the vaginal Lactobacillus crispatus, L. jensenii, and L. gasseri strains were inhibited. The combination of the BV treatment metronidazole and K7 bacteriocins completely killed both L. iners and Gardnerella vaginalis in a coculture experiment to mimic BV conditions. In contrast, this treatment did not inhibit L. crispatus cultures.IMPORTANCELactobacillus iners is a prevalent species of the vaginal microbiome, but unlike other major vaginal Lactobacillus species, it is not considered protective against BV and can coexist with BV-associated bacteria. L. iners is generally the first Lactobacillus species to emerge following the treatment of BV with metronidazole, and mounting evidence suggests that it may contribute to the onset and maintenance of vaginal dysbiosis. The discovery of highly potent bacteriocins that selectively kill L. iners while sparing protective vaginal lactobacilli may provide novel pharmacological tools to better understand the roles of this enigmatic bacterium in vaginal ecology and potentially lead to new and improved therapies for dysbiosis-related conditions such as BV.

Complete Genome Sequence of Enterococcus faecalis CAUM157 Isolated from Raw Cow’s Milk

Enterococcus faecalis CAUM157, isolated from raw cow’s milk, is a Gram-positive, faculta-tively anaerobic, and non-spore-forming bacterium capable of inhabiting a wide range of environmental niches. E. faecalis CAUM157 was observed to produce a two-peptide bacteriocin that had a wide range of activity against several pathogens, including Listeria monocytogenes, Staphylococcus aureus, and periodontitis-causing bacteria. The whole genome of E. faecalis CAUM157 was sequenced using the PacBio RS II platform, revealing a genome size of 2,972,812 bp with a G+C ratio of 37.44%, assembled into two contigs. Annotation analysis revealed 2,830 coding sequences, 12 rRNAs, and 61 tRNAs. Further, in silico analysis of the genome identified a single bacteriocin gene cluster.

A Leaderless Two-Peptide Bacteriocin, Enterocin DD14, Is Involved in Its Own Self-Immunity: Evidence and Insights.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by Enterococcus faecalis 14, a strain previously isolated from meconium. EntDD14 has a strong antibacterial activity against Gram-positive bacteria. Leaderless bacteriocins, unlike bacteriocins with leader peptides, are immediately active after their translation, and a producing strain has then to develop specific mechanisms to protect both intra and extracellular compartments. The in silico analysis of Ent. faecalis 14 genome allowed to locate downstream of structural ddAB genes, 8 other adjacent genes, designed ddCDEFGHIJ, which collectively may form three operons. To gain insights on immunity mechanisms of Ent. faecalis 14, mutant strains knocked out in ddAB genes encoding bacteriocin precursor peptides (Δbac) and/or ABC transporter (ΔddI) of EntDD14 were constructed and characterized. Importantly, Δbac mutant strains, from which structural ddAB genes were deleted, resulted unable to produce EntDD14 and sensitive to exogenous EntDD14 showing their involvement in the Ent. faecalis 14 immunity system. Moreover, the sensitivity of Δbac mutants appeared not to be associated with the down-regulation of ddCDEFGHIJ gene expression since they were similarly expressed in both Δbac and wild-type strains during the log phase while they were found significantly down-regulated in the Δbac mutant strain after 24 h of growth. Data gathered from this study suggest also the implication of the ABC transporter (ddHIJ) in the active export of EntDD14 but ruled-out its involvement in the primary self-immunity system. Interestingly, non-bacteriocin producing Ent. faecalis JH2-2 cells transformed with ddAB, or ddAB plus genes encoding the ABC transporter (ddAB-HIJ) did not produce EntDD14 and remained sensitive to its action. Of note, trans-complementation of the Δbac mutant strain with these constructions allowed to recover the WT phenotype. To the best of our knowledge, this is the first study delineating the role of the intracellular two-peptide leaderless bacteriocins in their self-immunity.

Biochemical, genetic and transcriptional characterization of multibacteriocin production by the anti-pneumococcal dairy strain Streptococcus infantariusLP90.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin.

Plantaricin NC8 \u03b1\u03b2 exerts potent antimicrobial activity against Staphylococcus spp. and enhances the effects of antibiotics

The use of conventional antibiotics has substantial clinical efficacy, however these vital antimicrobial agents are becoming less effective due to the dramatic increase in antibiotic-resistant bacteria. Novel approaches to combat bacterial infections are urgently needed and bacteriocins represent a promising alternative. In this study, the activities of the two-peptide bacteriocin PLNC8 αβ were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 α and β were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both L- and D-PLNC8 αβ caused rapid disruption of lipid membrane integrity and were effective against both susceptible and antibiotic resistant strains. The D-enantiomer was stable against proteolytic degradation by trypsin compared to the L-enantiomer. Of the truncated peptides, α1–22, β7–34 and β1–20 retained an inhibitory activity. The peptides diffused rapidly (2 min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 αβ substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 αβ is active against Staphylococcus spp. and may be developed as adjuvant in combination therapy to potentiate the effects of antibiotics and reduce their overall use.

The membrane topology of immunity proteins for the two-peptide bacteriocins carnobacteriocin XY, lactococcin G, and lactococcin MN shows structural diversity.

The two‐peptide bacteriocins produced by Gram‐positive bacteria require two different peptides, present in equimolar amounts, to elicit optimal antimicrobial activity. Producer organisms are protected from their bacteriocin by a dedicated immunity protein. The immunity proteins for two‐peptide bacteriocins contain putative transmembrane domains (TMDs) and might therefore be associated with the membrane. The immunity protein CbnZ for the two‐peptide bacteriocin carnobacteriocin XY (CbnXY) was identified by heterologously expressing the cbnZ gene in sensitive host strains. Using protein topology prediction methods and the dual pho‐lac reporter system, we mapped out the membrane topology of CbnZ, along with those of the immunity proteins LagC and LciM for the two‐peptide bacteriocins lactococcin G and lactococcin MN, respectively. Our results reveal wide structural variety between these immunity proteins that can contain as little as one TMD or as many as four TMDs.

Lactobacilli Isolated From Wild Boar (Sus scrofa) Antagonize Mycobacterium bovis Bacille Calmette-Guerin (BCG) in a Species-Dependent Manner.

Background: Wildlife poses a significant burden for the complete eradication of bovine tuberculosis (bTB). In particular, wild boar (Sus scrofa) is one of the most important reservoirs of Mycobacterium bovis, the causal agent of bTB. Wild boar can display from mild TB lesions, usually found in head lymph nodes, to generalized TB lesions distributed in different anatomical regions; but rarely clinical signs, which complicates the diagnosis of Mycobacterium bovis infection and bTB control. Among the possibilities for this variability in lesion distribution is the influence of the host-beneficial commensal-primed immune barrier. In this respect, beneficial microbes may delay bTB dissemination as a consequence of an antagonistic competition for nutrients and phagocytes. In order to explore this possibility, we have tested whether typical commensals such as lactobacilli have the capacity to reduce the survival rate of the surrogate M. bovis strain Bacillus Calmette-Guerin (BCG); and to modulate its phagocyte intake.Results: Three Lactobacillus species, L. casei, L. plantarum, and L. salivarius, isolated from wild boar feces displayed a pH-dependent inhibitory activity against BCG and influenced its intake by porcine blood phagocytes in a species-dependent manner. All lactobacilli showed a very significant bactericidal effect against BCG at low pH, but only isolates of L. plantarum and L. casei displayed such antimycobacterial activity at neutral pH. The genomes of these isolates revealed the presence of two-peptide bacteriocins whose precursor genes up-regulate in the presence of BCG cells. Furthermore, L. plantarum reduced significantly the BCG phagocytic intake, whereas L. casei had the opposite effect. L. salivarius had no significant influence on the phagocytic response to BCG.Conclusions: Our in vitro results show that lactobacilli isolated from wild boar antagonize BCG as a consequence of their antimycobacterial activity and a competitive phagocytic response. These findings suggest that commensal bacteria could play a beneficial role in influencing the outcome of bTB dissemination. Further work with lactobacilli as a potential competitive pressure to control bTB will need to take into account the complex nature of the commensal microbiome, the specific immunity of the wild boar and the in vivo infection context with pathogenic strains of M. bovis.

Biotechnological potential and in vitro safety assessment of Lactobacillus curvatus BCS35, a multibacteriocinogenic strain isolated from dry-salted cod (Gadus morhua)

The antimicrobial activity of Lactobacillus curvatus BCS35 is due to the production of, at least, two class IIa bacteriocins: sakacin P-H12Y (SakP-H12Y), which is a sakacin P (SakP) variant with tyrosine (Tyr) instead of histidine (His) at position 12, and sakacin X (SakX). Moreover, DNA sequence analysis revealed the presence of the genes encoding the two-peptide bacteriocin sakacin T. In order to compare the specific antimicrobial activities of SakP-H12Y and SakX with other class IIa bacteriocins (SakP from Lactobacillus sakei LTH673 and pediocin PA-1 (PedPA-1) from Pediococcus acidilactici 347), and to determine if the substitution His12/Tyr12 in SakP-H12Y/SakP exerts any effect on the bacteriocin potency and/or specificity, these four bacteriocins were purified and challenged against different indicator strains. Our results showed that (i) PedPA-1 was, in general, the most active peptide, and (ii) SakP-H12Y, which contains one less positively charged residue in the N-terminal half (His12/Tyr12), has a reduced antimicrobial potency against most of the tested indicators compared to SaKP, confirming the importance of the N-terminal cationic patch of SakP-H12Y/SakP in target-cell binding, potency and specificity. The in vitro safety of Lb. curvatus BCS35 was established by antibiotic susceptibility testing concluding that it may be considered as a safe strain.