In this study, we report the cloning and characterization of a GC pilin glycan biosynthetic gene that codes for a galactosyl transferase, which forms the α-glycosidic linkage between C3 of GalNAc and C1 of the αGal (). We named this gene pgtA
(pilus glycosyl transferase A) adhering to the convention of other GC glycosyl transferases (24
), particularly because we unambiguously demonstrated the enzyme encoded by it to be a galactosyl transferase. We first suspected this activity in this gene product because of its strong homology with the RfpB protein of Shigella
, which has been shown to be the transferase that catalyzes the formation of a Galα1–3GlcNAc bond in LPS O-antigen. The use of human anti–αGal antibodies and GSL1-B4 lectin, and Western analyses of the pilin proteins of GC strain MS11A and its isogenic pgtA
mutant, allowed a preliminary confirmation of our hypothesis. These two αGal-specific reagents reacted with wild-type MS11A pilin but not with the pilin of MS11ApgtA
. In addition, MS studies demonstrated a clear loss of a hexose due to the pgtA
mutation. HPAE-PAD–based monosaccharide composition analysis of pilin proteins provided the final confirmation of the proposed PgtA activity by demonstrating a quantitative loss of Gal in the pgtA
GC PgtA demonstrates very high homology (~95% identity) with MC PglA (31
), which has been proposed to synthesize the MC pilin Galα1–3DATDH bond, a linkage analogous to that of Galα1–3GlcNAc of GC pilin. However, the proposed substrate molecules for the two enzymes (GlcNAc for PgtA and DATDH for PglA) are quite different. Also, it should be noted that the α1,3 galactosyl transferase activity of PglA has yet to be shown unequivocally. Nevertheless, the clear characterization of pgtA
-encoded α1,3 galactosyl transferase supports the proposed activity of PglA (31
). Interestingly, the most remarkable difference between pglA
is that based on the presence of poly-G. The former always seems to have Pv as it is found with poly-G all the time, whereas the latter can either undergo Pv or constitutively express the glycosyl transferase activity depending on the presence or absence of the poly-G. Furthermore, although no pathogenic implication of PglA has been indicated in MC pathogenesis yet, here we report a clear role for PgtA in GC pathogenesis. Therefore, a reevaluation of pglA
might be needed to understand its role in MC pathogenesis. Lastly, it is also possible that PglA and PgtA may each act on both types of substrates (carrying DATDH or GlcNAc) and synthesize either type of pilin glycan depending on the specific background provided by different strains of MC and GC.
Notably, GC strains can be grouped into two categories based on the phenotype of phase variability of the pgtA
gene. Sequence analysis demonstrated that pgtA
s from certain GC strains and clinical isolates carried a poly-G tract, and that the number of Gs of this poly-G varied widely (between 9 and 20) from one isolate to another. In contrast, pgtA
from other GC strains and isolates does not carry the variable poly-G tract but a GGGAGCGGGG sequence instead, which differs from an analogous poly-G tract by only two bases. Still, the former allele of pgtA
, but not the latter, was expected to be phase variable because poly-G/C tracts with seven or more G/Cs can mediate Pv (22
). Using PgtA–FLAG fusions and colony blotting analyses, we demonstrated that the pgtA
carrying poly-G is phase variable, but the allele lacking poly-G is expressed constitutively without any variation. Thus, GC pgtA
is found in two mutually exclusive forms: one possessing and the other lacking Pv.
Our results indicate a possible significance for the presence or absence of the phase-variable poly-G tract in terms of GC pathogenesis. We observed that most GC associated with local infection only (uncomplicated inflammatory disease or PID in women) lack poly-G in pgtA
. PID isolates may have the poly-G–bearing pgtA
or the poly-G–lacking allele. However, all DGI-causing bacteria that we tested carried phase-variable pgtA
. Poly-G–mediated Pv of pgtA
may be advantageous for a GC isolate that disseminates for several reasons. One might be that anti-αGal IgGs are the most abundant Abs (~1% of total IgG population) in human sera (57
) and turning off pgtA
may help DGI isolates avoid these. In addition, Pv is known to increase the repertoire of neisserial antigens that often mimic human antigens and therefore may enhance tissue tropism of these bacteria (56
). Similarly, Pv of pilin glycan is likely to produce alternative glycoforms and therefore may lead to the expansion of potential host targets for GC that have disseminated. However, the nature of potential alternative pilin glycans having phase-variable pgtA
in GC strains has yet to be determined, because such pilin glycans have never been characterized. Future experiments will require analysis of the pilin glycans strains that have phase-variable pgtA
(such as GC strain FA1090).
Although previous studies have associated several phenotypes with DGI, the reason only a small percentage of untreated UG patients, and virtually no PID patients, develop DGI remains largely unknown (unpublished data). Clearly, serum resistance facilitates the systemic spread of GC by aiding survival in blood and can arise by several mechanisms (2
). In addition, the arginine-hypoxanthine-uracil auxotype, porin 1A expression, and a peptidoglycan hydrolase gene (atlA
) have been reported to correlate with DGI (59
). Although each of these factors is found in a much higher proportion of DGI isolates than in UG or PID isolates, none shows absolute correlation with DGI. Our analysis showed that all (24 out of 24) DGI isolates tested carried phase-variable pgtA
. DGI might be facilitated by multiple bacterial factors, none of which alone may be sufficient to cause dissemination.
Like phase-variable pgtA
lacking Pv may have its own advantages. This allele may help GC cause local infection because it is found in most UG (and PID) isolates and it may be relevant to examine whether a direct interconversion of these alleles is possible. Estimates from the 1970s suggest that ~1% of local gonorrhea proceeds to DGI (2
). Although hypothetical, a DGI organism with a poly-G tract in pgtA
could be directly selected from its counterpart that carries the corresponding nonhomopolymeric sequence, such a selection in vivo would require at least two point mutations when considering the normal frequency of mutational events. Nonetheless, the frequency of base changes (particularly for the second change, which would generate a full poly-G) in the GGGAGCGGGG sequence of the constitutive pgtA
allele can be significantly higher than the normal mutational rate of GC, due to this stretch's close resemblance to a poly-G tract.
A comparative analysis of neisserial genomes (strains MC58, Z2491, and FA1090) by Saunders et al. (22
) indicated the possible existence of a few genes that can be phase variable in one organism but not in another (62
). Similarly, our study has shown pgtA
to be a gene that exists in a phase-variable form in some strains of GC but not in others. To our knowledge, this is the first demonstration of a gene that can occur in both phase-variable and constitutive forms. This suggests the possibility that a gene might confer greater advantage to one strain of a pathogenic species as a “constitutive” gene, but to another strain of the same species as a “contingency” gene (63
). The presence of the contingency allele of pgtA
in DGI isolates would support the notion that these strains need greater adaptability in the more diverse and changing environment encountered at different systemic sites (e.g., blood, joints, skin, and occasionally the central nervous system and endocardium). In contrast, under perhaps the more “constant” environment of local genitourinary sites, the selection pressure for the contingency allele might be lessened. In this situation, a constitutive expression of the gene may be acceptable, or even preferred, if other benefits ensue.
Interestingly, from our search of existing databases, we observed poly-G tracts in numerous bacterial pathogens whose genomes have already been or are currently being sequenced (unpublished data). It is possible that poly-G–mediated Pv plays a role in the mechanism of pathogenesis in a number of bacteria. In addition, bacterial surface structures, particularly protein structures including the pili of other pathogenic bacteria, are increasingly being shown to be glycosylated (64
). In particular, the pgtA
homologue in P. aeruginosa
is especially significant because both pilus and flagella from Pseudomonas
are known to be glycosylated (64
). Future studies of bacterial surface glycans and their effects on Pv may yield important information concerning global pathogenic mechanisms.
Several types of glycosylation, present and absent, have been seen in different strains and variants of both GC and MC (17
). Organizational variation of the pgl
gene cluster among different MC strains (69
) also illustrates the degree of this diversity. Protein folding may influence glycosylation (71
) and even a single amino acid change in a polypeptide may alter the glycosylation pattern (72
). Diversification of glycosylation of neisserial pilus may stem from antigenic variation of pilin. Different pilin antigens may recruit separate sets of glycosyl transferases to produce distinct pilin glycosylation patterns. Therefore, synthesis of neisserial pilin glycan may involve yet unknown glycosylation enzymes. Notably, our HPAE-PAD analysis () detected novel sugars in MS11A pilin that were not described by our proposed model (unpublished data). A multiplicity of neisserial pilin glycoforms may arise because of involvement of phase-variable biosynthetic genes. As expected from this emerging diversity, the role of neisserial pilin glycans in pathogenesis of infection may take on an additional level of complexity.