Roles of PilC and PilE proteins in pilus-mediated adherence of Neisseria gonorrhoeae and Neisseria meningitidis to human erythrocytes and endothelial and epithelial cells

Abstract

Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coli interacted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.

FIG. 1

Demonstration of PilC2_His6 proteins purified from N. gonorrhoeae and E. coli. The protein gel shows pure silver-stained PilC2_His6 proteins from recombinant E. coli H2627 (lane 1) and from N. gonorrhoeae (lane 2). Lane M, molecular weight markers (in thousands).

FIG. 2

Binding of purified PilC2_His6 proteins to different human epithelial cell types. (Left panels) Untreated epithelial cells. (Right panels) Epithelial cells after treatment with 600 ng of pure PilC2_His6 protein from N. gonorrhoeae ml⁻¹, except that panels c and d show cells not treated (c) and treated (d) with PilC2_His6 purified from E. coli. The bound proteins were stained with rabbit anti-PilC serum (AK217) and fluorescein isothiocyanate-labelled secondary antibody. Epithelial cells were ME-180 (a to d), Hec-1B (e and f), RT112 (g and h), and human primary cornea epithelial cells (i and k). Bars, 10 μm.

FIG. 3

(A) PilC purified from E. coli blocks the binding of N. gonorrhoeae to epithelial cells. (a and b) Binding patterns of piliated wild-type N. gonorrhoeae N138 without preincubation (a) and after preincubation (b) with 600 ng of PilC2_His6 protein purified from E. coli ml⁻¹ (b). (c to f) Competition of binding of N. gonorrhoeae N138 to human epithelial cells by PilC purified from N. gonorrhoeae. Shown are infected Hec-1B cells without pretreatment (c) and after pretreatment with 600 ng of PilC2_His6 protein ml⁻¹ (d) and infected RT112 cells without pretreatment (e) and after pretreatment with 600 ng of PilC2_His6 protein ml⁻¹ (f). (B) Competition of binding of N. meningitidis to human epithelial cells by PilC purified from N. gonorrhoeae. Target cells were ME-180 (a and b), Hec-1B (c and d), and RT112 (e and f) without pretreatment (a, c, and e) and after pretreatment with 600 ng of PilC2_His6 purified from gonococci ml⁻¹ (b, d, and f). (C) Competition of N. gonorrhoeae N138 binding to primary cornea epithelial cells by purified PilC2_His6 protein. Cornea cells were from human origin. Binding of N138 to target cells without pretreatment (a) and after pretreatment with 12 μg of PilC2_His6 purified from gonococci ml⁻¹ (b) is shown.

FIG. 4

Binding of N. gonorrhoeae and N. meningitidis to HUVEC (left panels) is inhibited by the addition of purified PilC2_His6 protein (right panels). (a to k) Bacterial strains tested were N. gonorrhoeae N138 (a and b), N. gonorrhoeae N137 (c and d), N. gonorrhoeae pilC double mutant N556 (e and f), N. meningitidis N862 (g and h), and Opa50-expressing N. gonorrhoeae N303 (i and k). (l and m) Binding of purified PilC2_His6 protein to HUVEC (m) and a control experiment without purified PilC2_His6 protein (l).

FIG. 5

Adherence of Covaspheres fluorescent particles to ME-180 cells as seen by fluorescent microscopy (left panels) and light microscopy (right panels). Particles were coated with fetuin (a and b), purified PilC2_His6 protein (c and d), pili purified from N. gonorrhoeae N137 (e and f), and pili from the pilC double mutant N556 (g and h). (i and k) Restoration of binding of Covasphere particles coated with pili from N556 after preincubation of the particles with 400 ng of purified PilC2_His6 protein ml⁻¹.