Two New Secreted Proteases Generate a Casein-Derived Antimicrobial Peptide in Bacillus cereus Food Born Isolate Leading to Bacterial Competition in Milk

Abstract

Milk and dairy products harbor a wide variety of bacterial species that compete for both limited resources and space. Under these competitive conditions, bacteria develop specialized mechanisms to protect themselves during niche colonization and nutrient acquisition processes. The bacterial antagonism mechanisms include the production of antimicrobial agents or molecules that facilitate competitor dispersal. In the present work, a bacterial strain designated RC6 was isolated from Ricotta and identified as Bacillus cereus. It generates antimicrobial peptide (AMP) when grown in the presence of casein. The AMP was active against several species of Bacillus and Listeria monocytogenes. MALDI-TOF analysis of the RP-HPLC purified fractions and amino acid sequencing revealed a molecular mass of 751 Da comprised of a 6-residue sequence, YPVEPF. BLAST analysis showed that the AMP corresponds to the fractions 114–119 of bovine β-casein and represents the product of a specific proteolysis. Analysis of the purified proteolytic fractions from the B. cereus RC6 culture supernatant indicated that the presence of at least two different endoproteases is crucial for the generation of the AMP. Indeed, we were able to identify two new candidate endoproteases by means of genome sequencing and functional assignment using a 3D structural model and molecular docking of misannotated hypothetical proteins. In this light, the capacity of B. cereus RC6 to generate antimicrobial peptides from casein, through the production of extracellular enzymes, presents a new model of antagonistic competition leading to niche colonization. Hence, as a dairy product contaminant, this strategy may enable proteolytic B. cereus RC6 niche specialization in milk matrices.

Competitive and/or Cooperative Interactions of Listeria monocytogenes With Bacillus cereus in Dual-Species Biofilm Formation

“…Peptides and bacteriocins produced by this species may also inhibit L. monocytogenes, Bacillus subtilis, Escherichia coli, and Salmonella enteritidis, Salmonella typhi, and Staphylococcus aureus (Bizani et al, 2005;Yusra and Novelina, 2014). These observations are important on the development of solutions to eliminate biofilms in the food industry (Liu et al, 2016;Ouertani et al, 2018).…”

“…The behavior was similar for Experiments 1 and 2 (Figures 2A,B), indicating the antibiofilm activity of B. cereus against L. monocytogenes. The inhibition observed in Experiment 1 might be related to interference competition rather than exploitative competition, due to the production of secondary compounds or antimicrobial agents that facilitate competitor dispersal (Ouertani et al, 2018;Yuan et al, 2019). Antagonistic response depends on several factors, such as strain-specific response, substrates, and the interaction between species (Bartel et al, 2018).…”

“…There is a great interest in the food industry on the development of new strategies and antibiofilm agents (Møretrø et al, 2019;Ripolles-Avila et al, 2019). Bacteriophages, lysozymes, bacteriocins, antimicrobial peptides, enzymes, extracellular vesicles, lytic agents, essential oils, quorum sensing inhibitors, and surface modifications are promising alternatives (Ouertani et al, 2018;Friedlander et al, 2019;Yuan et al, 2019).…”

“…In a recent study, the use of B. cereus (AR156) in the presence of Ralstonia solanacearum bacteria and Meloidogyne incognita nematode was effective against plant diseases . Another study showed that B. cereus (RC6) was capable to secrete two enzymes that degrade casein, exhibiting antimicrobial activity to B. cereus ATCC 11778 (BC45) and L. monocytogenes DISTAM MACa1 (Ouertani et al, 2018).…”

A new class of antimicrobial molecules derived from kefir, effective against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA) strains

“…However, the possibility cannot be discounted that other microorganisms such as yeasts present in the microbiome of the grains could also be responsible for the capacity of kefir to kill bacteria. All Kefir serum samples were prepared with the same source of milk and in the exact same manner, so the antibacterial activity encountered cannot be associated with the direct action of the milk on the MRSA and P. www.nature.com/scientificreports/ aeruginosa strains, but rather by milk-derived components generated by the action of the enzymes produced by the microbiome organisms present in the grains during fermentation 28,29 . The MRSA and P. aeruginosa strains utilized in this study were acid resistant and could normally grow at pH 4.5, and the kefir serum sample had microbicidal effects against the MRSA strain even at pH 7.…”