Date of Award

Winter 1992

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Frank G Rodgers

Abstract

The pneumonial agent, Legionella pneumophila, is the predominant bacterium responsible for Legionnaires' disease. Experimentally, these organisms have demonstrated the ability to adhere to host cells without the presence of a mucopolysaccharide layer.

A recombinant plasmid, pLP 116, resulting from the ligation of a Hae III digest from the L. pneumophila, Nottingham N$\sb7$ genome and Sma I-digested pUC 19 vector was shown to encode for a 25 kilodalton (kDa) major outer membrane protein (MOMP) of L. pneumophila by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS). This protein was detected on the surface of an E. coli clone (LP 116) by immunoassays. Virulence testing using the fertile chicken egg lethality assay determined that the clone experienced increased virulence over that of the parent strain. The E. coli parent strain was found to be non-adherent to U937 cells in culture while the clone LP 116 experienced a 40-55% increase in adherence. The L. pneumophila N$\sb7$ strain demonstrated 100% binding; however, L. pneumophila and clone LP 116 incubated with MOMP-specific monoclonal antibody experienced a complete loss of adherence to U937 cells. Organisms coated with the monoclonal antibody did not infect fertile chicken eggs at any dilution.

The outer membrane protein (OMP) profiles of the attenuated derivative of the L. pneumophila isolate used in this study showed a decrease in the 25 kDa protein and the presence of a 31 kDa protein not found in the OMP profile of the virulent strain. This laboratory strain experienced an increase in lethal dose (LD$\sb{50})$ values in the chicken embryonated egg lethality assay. When the recombinant plasmid pLP 116 was electroporated (electrically transformed) into the attenuated L. pneumophila derivative the 25 kDa protein was produced in greater amounts and the 31 kDa band was no longer present. The LD$\sb{50}$ values of the transformed attenuated L. pneumophila N$\sb7$ strain decreased to that of the original isolate.

This study has shown the first reported difference between what appears to be genotypically and phenotypically similar organisms. It also has demonstrated that the 25 kDa MOMP of L. pneumophila plays an important role in adherence of the organism and that the 25 kDa MOMP can be recognized as a virulence factor related to the ability of the organism to cause infection.

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