Pili prepared from Neisseria gonorrhoeae contain minor amounts of a 110 kd outer membrane protein denoted PilC. The corresponding gene exists in two copies, pilC1 and pilC2, in most strains of N.gonorrhoeae. In the piliated strain MS11(P+), only one of the genes, pilC2, was expressed. Inactivation of pilC2 by a mTnCm insertion resulted in a nonpiliated phenotype, while a mTnCm insertion in pilC1 had no effect on piliation. Expression of pilC was found to be controlled at the translational level by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Nonpilated (P‐), pilin expressing colony variants that did not express detectable levels of PilC were selected; all P+ backswitchers from these P‐, PilC‐ clones were found to be PilC+. The structural gene for pilin, pilE, was sequenced and found to be identical in one P‐, PilC‐ and P+, PilC+ pair. Most PilC‐ cells were completely bald whereas the PilC+ backswitcher had 10–40 pili per cell. Thus, a turn ON and turn OFF in the expression of PilC results in gonococcal pili phase variation. These results suggest that PilC is required for pilus assembly and/or translocation across the gonococcal outer membrane.

Different strains and isogenic variants of Neisseria gonorrhoeae were assayed for their ability to bind glycolipids extracted from various sources. Among a large number of reference glycolipids, binding was observed only to lactosylceramide [Gal( 1-4)Glc(.81-1)Cer], isoglobotriaosylceramide Gal(pl4)Glc(,l-l)Cer], gangliotriaosylceramide [GalNAc(fi14)Gal(pl4)Glc(f31-1)Cer], and gangliotetraosylceramide [Gal(,GalNAc(fBl4)Gal(fi1-4)Glc-(.B1-l)CerJ. The latter two glycolipids bound gonococci with the highest affinity. Lactosylceramide and gangliotriaosylceramide were found in glycolipid preparations from ME180 cells, an epithelial cell line derived from a human cervical carcinoma, and thus are possible receptors for gonococci. The gonococcal surface component that bound the above glycolipids is a protein distinct from pilin and protein II.The attachment of a pathogen to host tissues is an important step in the initiation of an infection. An increasing number of studies indicate the importance of animal cell surface carbohydrates as attachment sites for microbial ligands (1,2). Many of these are present as glycolipids. There is evidence that eukaryotic cell receptors for Neisseria gonorrhoeae (the gonococcus) contain sugar residues (1-5). However, no specific receptor structure has been identified, in part because of methodological difficulties (6). Piliated strains of gonococci attach to epithelial cells more readily than their nonpiliated counterparts, and binding of the gonococcus to human epithelial cells is thought to be mediated by pilin (7), the major subunit of the pilus fiber. In addition, other bacterial surface components are thought to be adhesins (6,8).We have used a recently developed binding assay to examine in detail gonococcal-host cell interactions. Glycosphingolipids were separated on thin-layer chromatograms (9, 10), and radiolabeled gonococci were incubated with the chromatograms to detect specific bacterial binding. Table 1) were isolated by DEAE-Sepharose chromatography (17), followed by chromatography on Iatrobead (6RS-8060, latron Laboratories, Tokyo) columns using chloroform/methanol/5 M NH3 in water (17) as eluant. Most of the individual nonacid glycolipids were prepared as acetylated derivatives (14) by Iatrobead column chromatography using chloroform/methanol (14) gradients and, after deacetylation (14), using chloroform/methanol/water gradients (14). Glycolipid 8 in Table 1 was obtained by degradation of 180 mg of glycolipid 18 in 30 ml of 0.1 M HCO at 1000C for 60 min, followed by Iatrobead column chromatography. Glycolipid 12 was obtainedfrom'Fuc(al-2)Gal(l31-4)GlcNAc(p81-3)Gal(f31-4)Glc-(,81-1)Cer (Cer = ceramide) of dog small intestine (18) by hydrolysis with bovine kidney a-fucosidase (Boehringer Mannheim) in which repeated additions of enzyme (1.5 units total) were made for 24 hr at 37°C to 4 ml of 0.1 M acetate buffer (pH 4.5) containing 10 mg of taurodeoxycholate. The structures of the isolated glycolipids were confirmed by mass spectrometry (sequence) and NMR spectrosc...

A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAca2-3GalP1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein proffle identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subfiava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GNO1. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was nonfimbrial in nature, with aggregates of the adhesin extended out from the cells in a patchy fashion.

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