Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria

Abstract

We present results of tests with recombinant Lactococcus lactis that produce and secrete heterologous antimicrobial peptides with activity against Gram-negative pathogenic Escherichia coli and Salmonella. In an initial screening, the activities of numerous candidate antimicrobial peptides, made by solid state synthesis, were assessed against several indicator pathogenic E. coli and Salmonella strains. Peptides A3APO and Alyteserin were selected as top performers based on high antimicrobial activity against the pathogens tested and on significantly lower antimicrobial activity against L. lactis. Expression cassettes containing the signal peptide of the protein Usp45 fused to the codon optimized sequence of mature A3APO and Alyteserin were cloned under the control of a nisin-inducible promoter nisA and transformed into L. lactis IL1403. The resulting recombinant strains were induced to express and secrete both peptides. A3APO- and Alyteserin-containing supernatants from these recombinant L. lactis inhibited the growth of pathogenic E. coli and Salmonella by up to 20-fold, while maintaining the host’s viability. This system may serve as a model for the production and delivery of antimicrobial peptides by lactic acid bacteria to target Gram-negative pathogenic bacteria populations.

Probiotic engineering: towards development of robust probiotic strains with enhanced functional properties and for targeted control of enteric pathogens

“…Antimicrobial compounds have a direct inhibition on several target pathogens [31]. The mechanisms used by probiotics to inhibit pathogenic bacteria are interconnected.…”

Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium

“…Cells were then grown at 37°C to an OD 600 of 0.4 to 0.5 at which point they were induced. L. lactis NZ9000/pNZCL was induced by adding NaCl to the medium to obtain final concentrations of 0.01, 0.05, 0.1, 0.3, and 0.5 M Cl Ϫ , and L. lactis NZ9000/pNZ8048L was induced by the addition of 5 ng/ml or 40 ng/ml nisin A as previously described (17). After induction, OD 600 readings and 1-ml samples were collected each hour.…”

pMPES: A Modular Peptide Expression System for the Delivery of Antimicrobial Peptides to the Site of Gastrointestinal Infections Using Probiotics

“…and probiotic Escherichia coli Nissle 1917 [1,14,15,16], have been engineered to produce specific AMPs. In our previous work, we demonstrated the expression of AMPs and their secretion in inhibitory amounts against various pathogens, including Enterococcus faecalis, Enterococcus faecium [1,14], Escherichia coli [15], and Salmonella enterica [16]. …”