The Psa fimbriae of Yersinia pestis interact with phosphatidylcholine on alveolar epithelial cells and pulmonary surfactant

Abstract

The pH 6 antigen (Psa) of Yersinia pestis consists of fimbriae with adhesive properties of potential importance for the pathogenesis of plague, including pneumonic plague. The Psa fimbriae mediate bacterial binding to human alveolar epithelial cells. The Psa fimbriae bound mostly to one component present in the total lipid extract from type II alveolar epithelial cells of the cell line A549 separated by thin-layer chromatography (TLC). The Psa receptor was identified as phosphatidylcholine (PC) by TLC using alkali treatment, molybdenum blue staining, and Psa overlays. The Psa fimbriae bound to PC in a dose-dependent manner, and binding was inhibited by phosphorylcholine (ChoP) and choline. Binding inhibition was dose dependent, although only high concentrations of ChoP completely blocked Psa binding to PC. In contrast, less than 1 muM of a ChoP-polylysine polymer inhibited specifically the adhesion of Psa-fimbriated Escherichia coli to PC, and type I (WI-26 VA4) and type II alveolar epithelial cells. These results indicated that the homopolymeric Psa fimbriae are multimeric adhesins. Psa also bound to pulmonary surfactant, which covers the alveolar surface as a product of type II alveolar epithelial cells and includes PC as the major component. The observed dose-dependent interaction of Psa with pulmonary surfactant was blocked by ChoP. Interestingly, surfactant did not inhibit Psa-mediated bacterial binding to alveolar cells, suggesting that both surfactant and cell membrane PC retain Psa-fimbriated bacteria on the alveolar surface. Altogether, the results indicate that Psa uses the ChoP moiety of PC as a receptor to mediate bacterial binding to pulmonary surfactant and alveolar epithelial cells.

FIG. 1.

Psa binding to lipids from A549 cells on TLC plates. (A) Psa fimbriae were overlaid on TLC-separated lipids, and binding was detected with rabbit polyclonal anti-Psa antibody followed by incubation with HRP-conjugated anti-rabbit IgG. (B) Detection of phospholipids by molybdenum blue staining. (C) Detection of glycolipids with orcinol. Lane 1, A549 cell lipid extract corresponding to 300 μg of cellular protein; lane 2, A549 cell lipid extract after alkali methanolysis. Lane 3 contained commercial PE, PC, PS, PI, and SM (5.0 μg each) for panels A and B and neutral glycosphingolipid Qualimix (4.0 μg) containing GalCer, lactosylceramide (LacCer), globotriaosylceramide (Gb₃Cer), and globotetraosylceramide (Gb₄Cer) for panel C.

FIG. 2.

Psa binding to solid-phase-coated phospholipids. PC, SM, PS, PE, or PI was adsorbed onto polyvinyl chloride microtiter wells (1.0 μg per well) and incubated with increasing concentrations of Psa fimbria. Bound fimbria was detected with an antifimbrial antibody and an HRP-conjugated secondary antibody. The data are means ± standard error of three values and represent one of three reproducible experiments.

FIG. 3.

Inhibition of Psa binding to PC. PC was adsorbed onto polyvinyl chloride microtiter wells and incubated with Psa fimbriae (0.25 μg/well) and increasing concentrations of the following inhibitors: trypsinized polylysine-ChoP (empty circles), untreated polylysine-ChoP (filled circles), trypsinized polylysine-SerP (empty diamonds), or untreated polylysine-SerP (filled diamonds). PC adsorption onto the wells and Psa detection were as described in the legend to Fig. 2. The data are means ± standard error of three values and are representative of two reproducible experiments.

FIG. 4.

Psa binding to solid-phase-coated pulmonary surfactant. Polyvinyl chloride microtiter wells were used to adsorb rat surfactant (filled circles) or Survanta (empty squares). Binding of increasing concentrations of Psa fimbriae (A) and the inhibitory effect of ChoP on Psa binding (B) were determined as described in the legend to Fig. 2. The data are means ± standard error of three values and are representative of two reproducible experiments.