Peptide Extracts from Native Lactic Acid Bacteria Generate Ghost Cells and Spheroplasts upon Interaction with Salmonella enterica, as Promising Food Antimicrobials

Abstract

Protecting foods from contamination applying peptides produced by lactic acid bacteria is a promising strategy to increase the food quality and safety. Interacting with the pathogen membranes might produce visible changes in shape or cell wall damage. Previously, we showed that the peptides produced by Lactobacillus plantarum UTNGt2, Lactobacillus plantarum UTNCys5-4, and Lactococcus lactis subsp. lactis UTNGt28 exhibit a broad spectrum of antibacterial activity against several foodborne pathogens in vitro. In this study, their possible mode of action against the commensal microorganism Salmonella enterica subsp. enterica ATCC51741 was investigated. The target membrane permeability was determined by detection of beta-galactosidase release from ONPG (o-nitro-phenyl-L-D-galactoside) substrate and changes in the whole protein profile indicating that the peptide extracts destroy the membrane integrity and may induce breaks in membrane proteins to some extent. The release of aromatic molecules such as DNA/RNA was detected after the interaction of Salmonella with the peptide extract. Transmission electronic microscopy (TEM) micrographs depicted at least four simultaneous secondary events after the peptide extract treatment underlying their antimicrobial actions, including morphological alterations of the membrane. Spheroplast and filament formation, vacuolation, and DNA relaxation were identified as the principal events from the Gt2 and Cys5-4 peptide extracts, while Gt28 induced the formation of ghost cells by release of cytoplasmic content, filaments, and separation of cell envelope layers. Gel retarding assays indicate that the Gt2 and Gt28 peptide extracts are clearly binding the Salmonella DNA, while Cys5-4 partially interacted with Salmonella genomic DNA. These results increased our knowledge about the inhibitory mechanism employed by several peptide extracts from native lactic acid bacteria against Salmonella. Further, we shall develop peptide-based formulation and evaluate their biocontrol effect in the food chains.

Next-Generation Sequencing for Whole-Genome Characterization of Weissella cibaria UTNGt21O Strain Originated From Wild Solanum quitoense Lam. Fruits: An Atlas of Metabolites With Biotechnological Significance

“…No difference in protein pattern was detected when different peptide concentrations were applied. These results were in concordance with our previous findings that some peptides might induce visible phenotypic changes in the whole protein of a target cell and might induce breaks to some extent in the membrane proteins ( Tenea, 2020c ), thus defining the possible bactericidal mode of action. These promising results will help in further developing antimicrobial formulations based on peptide extracts that might kill simultaneously both Gram-negative and Gram-positive microorganisms coexisting in the same food matrix, thus enhancing their safety.…”

Genome Characterization of Lactiplantibacillus plantarum Strain UTNGt2 Originated from Theobroma grandiflorum (White Cacao) of Ecuadorian Amazon: Antimicrobial Peptides from Safety to Potential Applications

“…BLAST against data core proteins indicated that the UTNGt2 genome sequence hit with rSAM-modified_RiPP_077 (Bit Score = 33.113), rSAM-modified_RiPP_075 (Bit Score = 31.187), bovicin_225_variant (Bit score = 32.7278) from Streptococcus bovis and Blp (Bit score = 30.0314) from Streptococcus pneumoniae TIGR4. These findings were in agreement with our previous results indicating the high inhibitory potential of this strain as more than one peptide act as an antimicrobial unit [ 21 ]. By treating Salmonella cells with UTNGt2 peptide extract at the final concentration of 1 X MIC, spheroplast formation was observed, the cells showed changed shape, the inner and outer membranes were intact, but they lost peptidoglycans layer, while by increasing the concentration of peptides (2 X MIC) along with the spheroplasts, some “ghost cells” were noted, indicating that target bacteria were devoid or near-devoid cytoplasm [ 21 ].…”

“…These findings were in agreement with our previous results indicating the high inhibitory potential of this strain as more than one peptide act as an antimicrobial unit [ 21 ]. By treating Salmonella cells with UTNGt2 peptide extract at the final concentration of 1 X MIC, spheroplast formation was observed, the cells showed changed shape, the inner and outer membranes were intact, but they lost peptidoglycans layer, while by increasing the concentration of peptides (2 X MIC) along with the spheroplasts, some “ghost cells” were noted, indicating that target bacteria were devoid or near-devoid cytoplasm [ 21 ]. An early study on conventional antibiotics, such as penicillin G, and beta-lactam, indicated that these convert bacteria into spheroplasts [ 37 , 38 ].…”

“…Moreover, coating the tomato fruits with peptide extracts-based suspension proves to be a promising approach to control spoilage growth, thus enhancing the safety of food products [ 16 ]. Additionally, the UTNGt2 strain induces spheroplast and filament formation along with cell vacuolation and DNA relaxation as a secondary killing event in Salmonella enterica [ 21 ].…”

Bioprospecting Antimicrobials from Lactiplantibacillus plantarum: Key Factors Underlying Its Probiotic Action

“…Being amphiphilic, plantaricins interact electrostatically with the negatively charged bacterial cell surface (mainly constituted by phospholipids and lipopolysaccharides) by their positively charged group (cationic plantaricins) [ 45 ], while their hydrophobic portion passes through the membrane lipid bilayer of target bacteria. After internalisation, the peptides aggregate to form polymers or complexes, which create holes in the cell wall and the membrane, causing a change in permeability with consequent leakage and depletion of intracellular compounds (e.g., genetic and proteinaceous material), eventually leading to cell membrane lysis [ 46 , 47 , 48 , 49 ].…”