ABSTRACT

Title of Dissertation: PATTERNS OF REACTIVITY OF LANTIBIOTICS SUBTILIN AND NISIN WITH MOLECULAR TARGETS IN Bacillus cereus AND Bacillus subtilis 168

Srilatha Kuntumalla, Doctor of Philosophy, 2005

Dissertation Directed By: Professor J. Norman HansenDepartment of Chemistry and Biochemistry

Subtilin and nisin belong to a unique class of antibiotics called lantibiotics that contain unusual dehydro and lanthionine amino acid residues. The gene-encoded antimicrobial peptides subtilin and nisin exhibit bactericidal effects against several Gram-positive bacteria and also inhibit bacterial spore outgrowth. 

Subtilin and nisin are structural analogs and possess similar mechanisms of antimicrobial action. Although nisin is very stable, subtilin previously isolated was highly unstable with loss of biological activity observed during storage. Subtilin isolated in this work using hydrophobic interaction chromatography was very stable, with biological activity retained for at least a few months after isolation.

The possibility that specificity of subtilin and nisin towards sensitive Gram-positive bacteria is due to interaction of these lantibiotics with specific target proteins in susceptible bacteria was explored in this work. Phage display experiments performed to detect peptides interacting with subtilin identified a 12-mer peptide with a KTTLL motif found in ATP binding proteins such as ABC transporters and protein synthesis initiation factor IF-2 (~78 kDa). Binding of subtilin to specific ABC transporters in bacterial cell membrane would contribute to its specificity. Binding of subtilin to IF-2 would result in inhibition of protein synthesis suggesting an alternative mechanism of action for subtilin.

Experiments performed to determine the nature of interaction of subtilin and nisin with bacterial cellular proteins detected both covalent and non-covalent interactions. The covalent interactions between bacterial proteins and subtilin or nisin were stable on boiling in SDS and analyzing by SDS-PAGE. These stable covalent adducts indicated that the electrophilic dehydro residues of subtilin and nisin were probably involved in covalent attachment with specific nucleophilic groups in bacterial protein targets. Covalent attachment of an antibiotic to its bacterial target has been previously observed with only a few antibiotics.

Sites of nisin attachment to bacterial spores as visualized by electron microscopy showed nisin binds to highly localized regions on spore surfaces. Attempts to identify bacterial protein targets of subtilin and nisin using monomeric avidin and anti-FITC columns, respectively, resulted in isolation of proteins in ~70-80 kDa range. Further characterization of these proteins should help in understanding the specificity and antimicrobial mechanism of action of nisin and subtilin.