Advisor

Coats, Karen

Committee Member

Smith, Leif James

Committee Member

Gordonm, Donna

Committee Member

Munn, Thibaudeau

Committee Member

Pharr, Todd Gregory

Other Advisors or Committee Members

Lawrence, Mark

Date of Degree

8-1-2011

Document Type

Dissertation - Open Access

Degree Name

Doctor of Philosophy

Abstract

Lantibiotics like mutacin 1140 are receiving a considerable amount of attention because of their broad spectrum of activity, high potency, low immunogenicity, and good structural stability. Mutacin 1140 is produced by Streptococcus mutans JH1140 and is a type A (I) lantibiotic. Lantibiotics are ribosomally synthesized bacteriocins that undergo post-translational modifications to form lanthionine or β-methyllanthionine rings as well as 2, 3-didehydroalanine (Dha), 2, 3-didehydrobutyrine (Dhb), and S-amino vinyl-D-cysteine (AviCys). Their mode of action is pore formation and/or abduction of lipid II from the site of new cell wall synthesis. In order to gain further knowledge of both the structural integrity and structureunction relationship of type A (I) lantibiotics, chemical modifications or site directed mutagenesis was utilized. In the first aim of this study, two type A (I) lantibiotics were used, nisin A produced by Lactococcus lactis and gallidermin produced by Staphylococcus gallinarium. They both share homology in rings A and B, the lipid II binding domain, with rings A and B of mutacin 1140. What was discovered was that oxidation of the lanthionine rings results in the complete loss of bioactivity due to the loss of affinity for lipid II. Interestingly, the lateral assembly ability of the oxidized variants is not affected. In the second aim, the dehydrated threonine residue (Dhb) at position 14 in gallidermin underwent chemical modification using several thiol-compounds. The results showed that this residue is amendable to modification through thiol chemistry with some loss of bioactivity. However, the MICs for the chemical variants were still in the nanomolar range. From this work the first ever in vivo images of gallidermin were produced. The last aim of the study utilized site directed mutagenesis of the mutA gene of mutacin 1140 to determine the role of various residues in ring A and the hinge region of the peptide. It was determined that neither Trp4, Dha5, nor Arg13 are important for bioactivity but a set distance between rings A and B is essential. The majority of the mutants constructed showed either similar or increased bioactivity.

URI

https://hdl.handle.net/11668/15342

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