Using Bacillus subtilis as a Host Cell to Express an Antimicrobial Peptide from the Marine Chordate Ciona intestinalis

Abstract

Ciona molecule against microbes-A24 (CiMAM) isolated from the marine chordate Ciona intestinalis is an antimicrobial peptide. To generate CiMAM-expressing transgenic Bacillus subtilis, we constructed a plasmid expressing recombinant CiMAM (rCiMAM) and introduced it into B. subtilis. Transgenic strains C117 and C166 were selected since they were able to highly and stably express rCiMAM. We studied the bactericidal activity of pepsin-digested extracts from rCiMAM-expressing strains against freshwater and euryhaline pathogens that commonly occur in aquaculture ponds and found no difference from that of lactoferricin-expressing strains. The bactericidal activity of 1-mL aliquot from a total 5.5 mL extracted from 5 mL of cultured C117 (1.45 × 108 CFU·mL−1) and C166 (2.17 × 108 CFU·mL−1) against halophilic bacteria was equivalent to the efficacy of 57.06 and 32.35 ng of Tetracycline against Vibrio natriegens, 47.07 and 25.2 ng against V. parahaemolyticus, and 58.17 and 36.55 ng against V. alginolyticus, respectively, indicating higher bactericidal activity of pepsin-extracts from rCiMAM-containing strains against halophilic bacteria compared to that from lactoferricin-containing strains. Since the antibacterial activity of rCiMAM-expressing B. subtilis strains shows higher competence against halophilic pathogens compared to that against freshwater and euryhaline pathogens, these strains are promising candidates to protect marine fish and shellfish from halophilic bacterial infection.

High antimicrobial activity of lactoferricin‐expressing Bacillus subtilis strains

“…Quite recently, Lee et al . (2021) reported that the antibacterial activity driven by the transgenic B. subtilis strains expressing recombinant CiMAM, which is an AMP originated from marine chordate, displays a higher competence against halophilic pathogens specifically. In this study, based on the results obtained from the bactericidal activity, the equivalent to the efficacious dose of Tetracycline against marine pathogen V. parahaemolyticus of CiMAM‐expressing C117 strain (Lee et al ., 2021) and lactoferricin‐expressing P263 strain (in this study) was 47 and 193 ng, respectively, suggesting that transgenic B. subtilis P263 strain exhibited a stronger bactericidal activity in seawater environment than that of CiMAM‐expressing C117 strain.…”

“…Quite recently, Lee et al (2021) reported that the antibacterial activity driven by the transgenic B. subtilis strains expressing recombinant CiMAM, which is an AMP originated from marine chordate, displays a higher competence against halophilic pathogens specifically. In this study, based on the results obtained from the bactericidal activity, the equivalent to the efficacious dose of Tetracycline against marine pathogen V. parahaemolyticus of CiMAM-expressing C117 strain (Lee et al, 2021) and lactoferricin-expressing P263 strain (in this study) was 47 and 193 ng, respectively, suggesting that transgenic B. subtilis P263 strain exhibited a stronger bactericidal activity in seawater environment than that of CiMAM-expressing C117 strain. Therefore, we conclude that the use of transgenic B. subtilis P245 and P263 strains able to produce large amounts of AMPs can both effectively kill pathogenic bacteria and mitigate the excessive use of the antibiotics, thereby reducing their harmful effects on the aquatic environment, including the elimination of antibiotic-resistant bacterial strains generated in fish and shellfish farm.…”

Nutraceuticals and Pharmaceuticals from Marine Fish and Invertebrates

“…Another new molecule, a Ciona molecule against microbes-A24 (CiMAM), was described by Lee et al [ 10 ] as an alternative to the antimicrobial peptide that was isolated from the marine chordate Ciona intestinalis , using a microorganism generally recognized as safe (GRAS), Bacillus subtilis , as a host cell. The CiMAM maintained its bactericidal activity, even in a very high salt and ion environment, making the application of recombinant CiMAM-expressing transgenic B. subtilis strains a promising candidate to protect marine fish and shellfish from halophilic bacterial infection.…”