ABSTRACT
Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers and children in resource-limited countries. ETEC colonization factors, fimbrial tip adhesins and enterotoxins are key virulence factors, and thus have been studied as vaccine candidates. Some prevalent colonization factors, including CFA/I and CS17, belong to the class 5 family. We previously found that passive oral administration of hyperimmune bovine colostral IgG (bIgG) raised against dscCfaE (donor strand complemented CFA/I tip adhesin) protected volunteers against CFA/I+ ETEC challenge, while anti-dscCsbD bIgG (CS17 tip adhesin) did not confer protection. These findings led us to develop and optimize a panel of alternative CsbD-based vaccine candidates based on allele matching and in silico protein engineering. Physicochemical characterizations revealed that an optimized vaccine candidate dscCsbDLSN139(P218A/G3) had the greatest thermal stability among the six tested dscCsbD adhesins, whereas the overall secondary structures and solubility of these adhesins had no obvious differences. Importantly, dscCsbDLSN139(P218A/G3) elicited significantly higher CS17+ ETEC hemagglutination inhibition titers in sera from mice intranasally immunized with the panel of dscCsbD adhesins, while no significant difference was observed among heterologous neutralizing titers. Our results strongly advocate for the incorporation of these modifications into a new generation of CsbD-based ETEC vaccine candidates.
ABSTRACT
Bacillus cereus, a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis, a deadly pathogen for humans, and Bacillus thuringenesis, an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli. Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus, B. anthracis, and other Bacillus species.
