Date of Award

Summer 2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Dayle A. Daines

Committee Member

Fred C. Dobbs

Committee Member

Christopher J. Osgood

Committee Member

Jing He

Abstract

Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminus (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). This dissertation presents three studies investigating type II TA modules in NTHi. The first study determined the crystal structure of the VapBC-1 complex. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of VapC-1 were constructed, and the effects of these mutations on pathogenesis were tested ex vivo. A novel model system was designed that consisted of an NTHi ΔvapBC-1 strain complemented in cis with the toxin mutants in tandem with the wild-type antitoxin controlled by the vapBC-1 native promoter in single copy. This determined that a single mutation to a conserved amino acid in the PIN domain significantly reduced the survival of NTHi. The second study measured the induction of a type II antitoxin in trans and the induction of a type II toxin in cis with or without an E. coli arabinose permease in cis in the background of NTHi. A bioassay was then designed to investigate the ability of NTHi to transport arabinose, the molecule necessary to induce the araBAD genes. Lastly, a bioinformatics study was performed on 12 recently-sequenced clinical isolates of NTHi to determine the conservation of four vap operons. Seven strains maintained all four operons, the other five strains maintained three and the operons all shared significant sequence homology. These three studies provide insight into the importance of the type II TA loci in NTHi and due to their conservation and contribution to pathogenesis, these modules prove to be promising candidates for the development of antimicrobial treatments.

DOI

10.25777/cmsb-qk23

ISBN

9781392648247

ORCID

0000-0002-6941-4450

Available for download on Tuesday, November 24, 2020

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