Presentation Title

Examination of Type III Toxin-Chaperone Interaction by Copurification

Advisor Information

Donald Rowen

Location

Milo Bail Student Center Gallery Room

Presentation Type

Oral Presentation

Start Date

8-3-2013 11:00 AM

End Date

8-3-2013 11:15 AM

Abstract

Type Three Secretion systems (TTSS) are an important virulence factor for many pathogenic bacteria that can infect humans including Pseudomonas aeruginosa. Type three secretion systems consist of a needle-like complex which can transport toxins directly from the bacterial cell cytoplasm to the cytoplasm of a cell of the infected host. The secretion of some toxins requires a chaperone protein to bind to the toxin, but the exact role of the chaperone is not completely understood. Previous results have suggested that the Type III secreted toxin ExoU of P. aeruginosa may be unusual in that residues near both the amino and carboxy-terminus are required for chaperone interaction. The C-terminus of ExoU contains a membrane localization domain (MLD), and chaperones have been hypothesized to act to mask MLDs. To confirm the importance of the C-terminal MLD of ExoU on SpcU interaction, I sought to test the ability of ExoU C-terminus truncation mutants to bind to a modified version of SpcU (His-tagged) that can be easily purified. For that experiment, I constructed plasmids that will express His tagged SpcU and either wild type or truncated forms of ExoU in E. coli cells. I have conducted trial experiment to optimize the conditions for the purification and to overcome some unexpected problems. Lack of purification of truncated forms of ExoU along with SpcU would support the hypothesis that SpcU binds to the C-terminus and N-terminus of ExoU.

This document is currently not available here.

COinS
 
Mar 8th, 11:00 AM Mar 8th, 11:15 AM

Examination of Type III Toxin-Chaperone Interaction by Copurification

Milo Bail Student Center Gallery Room

Type Three Secretion systems (TTSS) are an important virulence factor for many pathogenic bacteria that can infect humans including Pseudomonas aeruginosa. Type three secretion systems consist of a needle-like complex which can transport toxins directly from the bacterial cell cytoplasm to the cytoplasm of a cell of the infected host. The secretion of some toxins requires a chaperone protein to bind to the toxin, but the exact role of the chaperone is not completely understood. Previous results have suggested that the Type III secreted toxin ExoU of P. aeruginosa may be unusual in that residues near both the amino and carboxy-terminus are required for chaperone interaction. The C-terminus of ExoU contains a membrane localization domain (MLD), and chaperones have been hypothesized to act to mask MLDs. To confirm the importance of the C-terminal MLD of ExoU on SpcU interaction, I sought to test the ability of ExoU C-terminus truncation mutants to bind to a modified version of SpcU (His-tagged) that can be easily purified. For that experiment, I constructed plasmids that will express His tagged SpcU and either wild type or truncated forms of ExoU in E. coli cells. I have conducted trial experiment to optimize the conditions for the purification and to overcome some unexpected problems. Lack of purification of truncated forms of ExoU along with SpcU would support the hypothesis that SpcU binds to the C-terminus and N-terminus of ExoU.