Characterization of an Interaction Between the Pseudopilin XcpT and the Putative ATPase XcpR in the Type II Secretion System of Pseudomonas Aeruginosa

The secretion of alkaline phosphatase, phospholipase C, lipase, elastase, and exotoxin A are among several virulence factors that contribute to the opportunistic pathogenicity of Pseudomonas aeruginosa. The secretion of these exoproteins is achieved via the Type II secretion system. At least twelve genes named xcp have been demonstrated to encode the gene products responsible for this secretion. The roles for each individual Xcp protein or the protein-protein interactions that would comprise such an apparatus have yet to be determined (the only exception is PilD, the peptidase/methylase required for processing pseudopilins and pilin of both xcp secretion and Type IV pilus biogenesis, respectively). In this thesis, I have described the first genetic evidence of an interaction between two of the Xcp proteins, XcpT and XcpR. This was generated by isolating a temperature-sensitive mutation in the pseudopilin XcpT, which is the result of a serine to leucine mutation at residue 121 located in the carboxyl terminus of this subunit. Additional mutagenesis of a P. aeruginosa K strain containing the xcpTts allele allowed for the isolation of a suppressing mutation located in the amino terminus of the putative ATPase, XcpR. This is the result of a serine to phenylalanine change at position 84. Experiments investigating the nature of this potential interaction by site-directed mutagenesis of position 84 of XcpR and position 121 of XcpT are described. Additional secretion defective alleles in XcpT have been identified and suppression of these alleles by XcpR (S84F) and a subset of the larger, hydrophobic residues has been determined. The XcpR suppressors exhibit an altered mobility in SDS-PAGE. This shift appears to correlate with the suppression effect (ie., the greater the mobility shift, the more efficient the suppressor). Thus, the mobility shift has been examined for XcpR (S84F), the most efficient suppressor of XcpT (S121L), to determine the mode of suppression. Post-translational modification, conformational change, and hydrophobicity have been explored as possibilities for this shift. Additionally, biochemical studies to demonstrate an interaction between XcpT and XcpR and periplasmic accessibility of XcpR are discussed.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Ratliff, Melanie Christine

Contributors dc:contributor

• David N. Nunn

• Biology, Molecular

Language dc:language

eng

Identifier

(MiAaPQ)AAI9996675

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/86769