Host-Parasite Interrelations: Bacterial Adherence to and Invasion of Epithelial Cells
Enteroinvasive Escherichia coli (EIEC) preferentially invaded multinucleated HeLa 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 HeLa 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 HeLa cells. Treatment of HeLa cells with PEG had no significant effect on the low level of cell-association displayed by noninvasive organisms and thus, PEG-treated HeLa 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 HeLa cells to equivalent levels. The large, multinucleated HeLa cells generated by PEG treatment revealed a striking accumulation of EIEC in cellular extensions. This study demonstrates that alteration of HeLa 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 HeLa cell monolayers, the opaque phenotype became increasingly dominant in the subpopulation of organisms which adhered to the HeLa 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.IIop), P.IIa, to eucaryotic macromolecules was studied. HeLa cell extracts were subjected to SDS-PAGE and transferred to nitrocellulose, and purified P.IIa bound to approximately 30 to 50 distinct molecular species. The binding of P.IIa to HeLa cell components was stable in high NaCI and nonionic detergent, and was not inhibited by monosaccharides. The binding behavior of P.IIa 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.IIa, 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.IIa to Western blots of HeLa cell macromolecules. HeLa cell extracts inhibited P.IIa 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.IIa bound to proteins lacking carbohydrate as well. The basic P.IIa was compared to another P.IIop whose overall net charge was significantly less (P.IIc). Both P.IIa and P.IIc displayed similar binding specificities and affinities. The predominant binding interactions of P.IIa and P.IIc 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.IIa, P.IIc, 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. Gonococcal adherence to HeLa cells correlated with both opacity and P.II content. Environmental factors distinguished between gonococci expressing distinct P.IIop molecules in a manner independent of opacity. The role of P.II as a determinant of tissue tropism is discussed.
Enteroinvasive Escherichia coli (EIEC), multinucleated HeLa cells, Protein II (P.II), gonococcal adherence, cytochalasin B, tissue tropism, Life Sciences
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