Tara Allison

Defense Date

4-22-2015

Graduation Date

Spring 1-1-2015

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Nancy Trun

Committee Member

Peter Castric

Committee Member

Jana Patton-Vogt

Committee Member

Joseph Horzempa

Keywords

glycan, glycosylation, group I, pilin, Pseudomonas aeruginosa, Type IVa pili

Abstract

Pseudomonas aeruginosa strains producing group I type IVa pili comprise over 50% of the clinical isolate population. The pilus lateral surface of these strains is characterized by the attachment of an O-antigen repeating unit to each pilin monomer. O-antigen (glycan) structural analysis provided information on two possible characteristics, charge and size. Solubility experiments of fully, partially, and non-glycosylated P. aeruginosa 1244 pili showed that a full length glycan was required for maximum pilus solubility. Reduced solubility suggested an increased pilus surface hydrophobicity which was supported by protein modeling. While having no effect on the wild type strain, ionic strengths found at many host infection sites inhibited surface and subsurface twitching motility of strain 1244G7, an isogenic mutant unable to glycosylate pilin. This effect was reversed by mutant complementation.

A PCR method for assigning R-type (glycan) was developed and conducted for 206 clinical isolates. It was expected that the R-types would be evenly distributed across all isolates, but there was a significant prevalence for the R5 type which encodes the O6 serotype. Verification of the R5 strains was conducted with O6 specific antibodies confirming the genetic classification of these isolates. Results suggest that the increased prevalence of the R5-type as the pilin glycan could potentially lead to its use in a multi-component vaccine or therapeutic for P. aeruginosa infections.

Clinical isolates were screened for group I pilin producing strains. Unexpectedly, strains producing non-glycosylated group I pilin were found, prompting the characterization of this altered phenotype. Sequencing and complementation results indicated that non-glycosylation was caused by defects in the main components of glycosylation: pilA, pilO, and the O-antigen biosynthesis pathway. Only one co-phenotype was found among these strains, reduced piliation. Additional characterization of the cause for reduced piliation showed that the presence of non-glycosylated pilin alone was enough to reduce surface pili in these mutant clinical isolates. A pilO knockout was engineered which also showed a loss of glycosylation and a marked reduction in piliation. Providing this mutant with a functional pilO complemented both phenotypes. Overall, these results show that the presence of glycan is important for pilus function, solubility and piliation.

Format

PDF

Language

English

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