Student Theses and Dissertations

Debra Bessen

Date of Award

1987

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Enteroinvasive Escherichia coli (EIEC) preferentially invaded multinucleated ReLa cells which arose naturally in monolayer cultures at a low frequency. Data suggests that enhancement of the penetration step accounts partly for increased invasion of multinucleated cells. Treatment of ReLa cell monolayers with polyethylene glycol (PEG) led to cell fusion, and invasion of PEG-treated monolayers by EIEC was increased by an average of 7-fold compared to untreated ReLa cells. Treatment of ReLa cells with PEG had no significant effect on the low level of cell-association displayed by noninvasive organisms and thus, PEG-treated ReLa cells appeared to retain their selectivity for EIEC. The microfilament disrupting agent cytochalasin B (CB) reduced EIEC invasion by more than 50-fold. Furthermore, CB depressed the extent of EIEC invasion of PEG-treated and untreated ReLa cells to equivalent levels. The large, multinucleated ReLa cells generated by PEG treatment revealed a striking accumulation of·EIEC in cellular extensions. This study demonstrates that alteration of ReLa cell morphology results in increased invasion by EIEC. Colonial variants of Neisseria gonorrhoeae differ in their interactions with eucaryotic cells. Gonococci giving rise to the opaque colony type possess one or more proteins II in their outer membrane. When gonococci were cultivated with ReLa cell monolayers, the opaque phenotype became increasingly dominant in the subpopulation of organisms which adhered to the ReLa cells. Once bound, opaque organisms displayed very low levels of detachment. Adherent opaque gonococci exhibited generation times up to three-fold greater than cultures containing gonococci in the absence of HeLa cells. In addition, the progeny of adherent opaque organisms remained bound to the HeLa cell monolayer. Both piliated and transparent colony types attached to HeLa cells, but their progeny were retained less efficiently. Gonococci bound to HeLa cells were subjected to the bactericidal action of fresh rat serum and approximately 0.5% to 2.5% survived, irrespective of their opaque or piliation phenotype. Incubation with gentamicin resulted in a 10- to 50-fold further reduction in viablility. Pretreatment of HeLa cell monolayers with CB diminished gonococcal survival in either serum or gentamicin by up to 8-fold. In contrast, CB treatment did not decrease the survival of the commensal organism Neisseria sicca. The data suggests that very few gonococci are completely interiorized, and a small proportion of adherent gonococci are partially protected from the soluble-phase environment by HeLa cells. Binding of the opacity-associated Protein II (P.llop), P.lla, to eucaryotic macromolecules was studied. HeLa cell extracts were subjected to SDS-PAGE and transferred to nitrocellulose, and purified P.lla bound to approximately 30 to 50 distinct molecular species. The binding of P.lla to HeLa cell components was stable in high NaCI and nonionic detergent, and was not inhibited by monosaccharides. The binding behavior of P.lla was compared to that of two model carbohydrate-binding proteins, wheat germ agglutinin (WGA) and concanavalin A (ConA). Glycoproteins (ovomucoid, fetuin, mucin, ovalbumin) inhibited binding by P.lla, WGA, and ConA to variable degrees. HeLa cell glycopeptides, generated by pronase digestion of chloroform:methanol-extracted cells, were tested for their ability to inhibit binding by P.lla to Western blots of HeLa cell macromolecules. HeLa cell extracts inhibited P.lla binding prior to pronase treatment, but inhibitory activity was lost as a result of pronase digestion. Direct binding to defined glycosylated and nonglycosylated proteins revealed that ConA and WGA bound only glycoproteins, whereas P.lla bound to proteins lacking carbohydrate as well. The basic P.lla was compared to another P.llop whose overall net charge was significantly less (P.llc). Both P.lla and P.llc displayed similar binding specificities and affinities. The predominant binding interactions of P.lla and P.llc were with protein rather than eucaryotic carbohydrate, and the chemical nature of the interactions was more complex than the involvement of purely ionic or purely hydrophobic forces. Gonococci expressing P.lla, P.llc, neither or both were compared for colonial opacity, adherence to HeLa cells, and growth under stressful conditions. Both the quantity and type of P.II determined colonial opacity. city and Gonococcal adherence to HeLa cells correlated with both opa- P.II content. Environmental factors distinguished between gonococci expressing distinct P.llop molecules in a manner independent of opacity. The role of P.II as a determinant of tissue tropism is discussed.

Comments

A thesis submitted to the Faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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