Date of Award

Spring 2002

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Thomas G Pistole

Abstract

Salmonella enterica serovar Typhimurium expresses three major outer membrane proteins: OmpC, OmpF, and OmpD. Conflicting reports exist regarding the role of OmpC and OmpD in the pathogenesis of this organism. This study investigated the role of OmpC and OmpD in adherence to human U937 macrophages and T84 intestinal epithelial cells. OmpC and ompD mutant strains were created by transposon mutagenesis utilizing P22-mediated transduction of Tn10 and Tn 5 insertions into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with PKH26-labeled T84 cells at various bacteria-to-cell ratios for 1 h at 37 C. Single and dual fluorescence of samples was analyzed by flow cytometry to obtain mean fluorescence intensity (MFI). The MFI values of cells with adherent wild-type and mutant bacteria were compared to determine adherence levels. MFI values were used to estimate the number of bacteria bound per host cell. Dual-color fluorescence gave a more precise analysis of adherence. Analysis showed no significant difference in binding of ompC mutant and wild-type strains to the human cells but there was lower binding of the ompD mutant to these cells. Fluorescence microscopy was also used to measure adherence under the same conditions and data were obtained as number of bacteria bound per 100 cells. Results from this method correlated with those obtained from flow cytometry.

In conclusion, ompD but not ompC mutation yielded a significant difference in the binding of serovar Typhimurium to human U937 macrophages and T84 intestinal epithelial cells.

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