Implications of phase variation of a gene (pgtA) encoding a pilin galactosyl transferase in gonococcal pathogenesis

Abstract

The pilin glycoprotein (PilE) is the main building block of the pilus of Neisseria gonorrhoeae (gonococcus [GC]). GC pilin is known to carry a disaccharide O-glycan, which has an alphaGal attached to the O-linked GlcNAc by a 1-3 glycosidic bond. In this report, we describe the cloning and characterization of the GC gene, pilus glycosyl transferase A (pgtA), which encodes the galactosyl transferase that catalyzes the synthesis of this Gal-GlcNAc bond of pilin glycan. A homopolymeric tract of Gs (poly-G) is present in the pgtA gene of many GC strains, and this pgtA with poly-G can undergo phase variation (Pv). However, in many other GC, pgtA lacks the poly-G and is expressed constitutively without Pv. Furthermore, by screening a large number of clinical isolates, a significant correlation was observed between the presence of poly-G in pgtA and the dissemination of GC infection. Poly-G was found in pgtA in all (24 out of 24) of the isolates from patients with disseminated gonococcal infection (DGI). In contrast, for the vast majority (20 out of 28) of GC isolated from uncomplicated gonorrhea (UG) patients, pgtA lacked the poly-G. These results indicate that Pv of pgtA is likely to be involved in the conversion of UG to DGI.

Figure 1.

Current model of gly-cosylation of pilin of N. gonorrhoeae. Vertical bar on the right, pilin polypeptide; Gal, galactose; GlcNAc, N-acetyl-glucosamine; Ser63, O-glycosylated serine at position 63 of GC pilin polypeptide.

Figure 2.

Molecular genetic characterization of the GC pgtA and its encoded activity. (A) Maps of the different GC DNA fragments that were cloned for analysis of the pgtA from various strains. Solid arrows, ORFs; vertical bars, poly-G tracts; open arrows, the location and direction of the oligonucleotide primers. (B) Alignment of PgtA homologues. Amino acid sequence of PgtA from GC strain FA1090 (GCPgtA) is compared with that of PglA from MC (MCPglA), RfpB of S. dysenteriae (ShigellaRfpB), and a functionally unknown ORF of P. aeruginosa (PsPgtAhlog). For this alignment, Jotun-Hein algorithm of the MegAlign program (DNASTAR) was used. The boxes represent sequence conservation. The tract of four consecutive glycines (residues 246–249) of PgtA, which correspond to the poly-G sequence, is marked by a thick solid line over it.

Figure 2.

Molecular genetic characterization of the GC pgtA and its encoded activity. (A) Maps of the different GC DNA fragments that were cloned for analysis of the pgtA from various strains. Solid arrows, ORFs; vertical bars, poly-G tracts; open arrows, the location and direction of the oligonucleotide primers. (B) Alignment of PgtA homologues. Amino acid sequence of PgtA from GC strain FA1090 (GCPgtA) is compared with that of PglA from MC (MCPglA), RfpB of S. dysenteriae (ShigellaRfpB), and a functionally unknown ORF of P. aeruginosa (PsPgtAhlog). For this alignment, Jotun-Hein algorithm of the MegAlign program (DNASTAR) was used. The boxes represent sequence conservation. The tract of four consecutive glycines (residues 246–249) of PgtA, which correspond to the poly-G sequence, is marked by a thick solid line over it.

Figure 3.

Galactosyl transferase activity in the membranes of E. coli XL1-Blue–expressing GC pgtA. The columns indicate the incorporation of radioactive galactosyl moiety from [¹⁴C]UDP Gal into the lipid-linked intermediates catalyzed by different E. coli membrane fractions expressing various plasmids. E. coli XL1-Blue (without any plasmid) and its derivative with cloning vector pBluescript II KS⁺ (pKS⁺) were used as controls for this enzymatic assay. The same host strain with plasmids ppgtA1 and ppgtA5 were assessed for their ability to transfer Gal under the same condition. The specificity of galactosyl transferase activity of PgtA from ppgtA5 was also tested by induction with IPTG (5 mM), as part of this pgtA transcript is likely transcribed from the Lac promoter of the cloning vector pKS⁺. The activity of ppgtA5-erm (pgtA knockout) was also evaluated. All assays were done in duplicate and standard deviations are shown by error bars (not visible on all columns).

Figure 4.

Western analysis of MS11A and MS11ApgtA pilins using antipilin mAb 1E8/G8, GSL1-B4 lectin, and polyclonal human anti–αGal Ab, respectively. Mouse laminin was used as a positive control for GSL1-B4 and anti-αGal reactions. Lanes marked MW contain SeeBlue prestained molecular weight marker (Novex).

Figure 5.

Matrix-assisted laser desorption/ionization time of flight MS study of pilins obtained from GC strain MS11A (top) and its isogenic mutant MS11pgtA (bottom). Bovine insulin peptide (molecular mass of 5,734.60 daltons) was added as the internal calibrant. Molecular masses of the major peaks are indicated.

Figure 6.

Monosaccharide composition analysis of pilins obtained from GC strain MS11A and its isogenic mutant, MS11ApgtA. The top chromatogram is of a standard mixture of monosaccharides containing 2 nmol of each component. The other chromatograms are hydrolysates of indicated GC pilins. Glc, glucose; Gal, galactose; Man, mannose; GalN, galactosamine; GlcN, glucosamine; C, coulomb.

Figure 7.

Presence and absence of the poly-G tract in the pgtA genes of several laboratory strains of GC. (A) Alignment of the pgtA DNA sequences from N. gonorrhoeae strains carrying (top four sequences) and not carrying (bottom six sequences) the phase-variable poly-G. The amino acid translation is given above or below the sequences. The BsrBI site, only present in the bottom sequences, is marked by an underline. Vertical bars, the identity of bases; dots, gaps in alignment; bold letters, the mismatches found within the poly-G tract region. (B) Southern analysis of BsrBI-digested GC genomic DNAs. On left, the picture of the DNA gel stained with ethidium bromide and on the right, the Southern autoradiograph are shown. A fluorescence-labeled 1-kb ladder (Amersham Biosciences) was used as the molecular weight marker. The source strain of each genomic DNA is indicated above the relevant lane.

Figure 7.

Presence and absence of the poly-G tract in the pgtA genes of several laboratory strains of GC. (A) Alignment of the pgtA DNA sequences from N. gonorrhoeae strains carrying (top four sequences) and not carrying (bottom six sequences) the phase-variable poly-G. The amino acid translation is given above or below the sequences. The BsrBI site, only present in the bottom sequences, is marked by an underline. Vertical bars, the identity of bases; dots, gaps in alignment; bold letters, the mismatches found within the poly-G tract region. (B) Southern analysis of BsrBI-digested GC genomic DNAs. On left, the picture of the DNA gel stained with ethidium bromide and on the right, the Southern autoradiograph are shown. A fluorescence-labeled 1-kb ladder (Amersham Biosciences) was used as the molecular weight marker. The source strain of each genomic DNA is indicated above the relevant lane.

Figure 8.

Poly-G–mediated Pv of the GC pgtA gene. (A) Western analysis of various PgtA–FLAG COOH-terminal fusion proteins expressed in E. coli XL-1 Blue host. On left, the Coomassie-stained gel shows the proteins present in the lysates of various E. coli hosts that carry different PgtA–FLAG fusions or controls. The corresponding Western blot, which was performed using anti-FLAG mAb M2, is shown on the right. The extreme lanes, marked MW, contain molecular weight marker. A firefly luciferase–FLAG fusion expressed from the plasmid, pTag4lux-FLAG (Stratagene), is the positive control for M2 reaction. The lysates from the cells carrying the vectors, pTag4b and pYUB631, are the two negative controls. ppgtA–F1a and ppgtA–F1b carry the in-frame and the out of frame fusions of FA1090 PgtA and FLAG, respectively. The in-frame and out of frame F62 PgtA-FLAG chimera are made by ppgtA–F2a and ppgtA–F2b, respectively. ppgtA–F1aFS1 is a putative phase variant (see below) of ppgtA–F1a resulting from a frameshift in pgtA poly-G. (B) The colony blot for the identification of potential phase variants of FA1090 pgtA cloned in E. coli XL-1 Blue host. The XL-1 Blue colonies carrying the positive control, pTag4lux-FLAG, are at the top spot. The negative control colonies, carrying pCMVTag4b vector, are at the bottom. The colonies of bacteria carrying the in-frame pgtA–FLAG (ppgtAF1a) and the out of frame pgtA–FLAG (ppgtAF1b) fusions are shown on the right and left, respectively. The potential phase-variant colonies are marked by arrows. (C) The DNA sequence of the poly-G tract of a potential phase variant (ppgtA–F1aFS1) obtained from the bacteria with the in-frame fusion (ppgtAF1a). The sequences from both DNA strands are shown with the leading strand data presented on the top. The direction of the transcription of pgtA for each sequence is indicated by an arrow. The G/Cs of the poly-G/C tract of pgtA are numbered in the direction of transcription.