Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili Finn Terje Hegge*†‡§, Paul G. Hitchen§¶, Finn Erik Aas*†‡, Heidi Kristiansen*‡, Cecilia Løvold*†‡, Wolfgang Egge-Jacobsen‡, Maria Panico¶, Weng Yee Leong¶, Victoria Bullʈ, Mumtaz Virjiʈ, Howard R. Morris¶**, Anne Dell¶, and Michael Koomey*†‡†† *Centre for Molecular Biology and Neuroscience, †Department of Molecular Biosciences, and ‡Biotechnology Centre of Oslo, University of Oslo, 0316 Oslo, Norway; ¶Department of Biological Sciences, Imperial College London, London SW7 2AY, United Kingdom; ʈDepartment of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, United Kingdom; and **M-Scan Mass Spectrometry Research and Training Centre, Silwood Park, Ascot SL5 7PZ, United Kingdom Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved May 11, 2004 (received for review April 5, 2004) Several major bacterial pathogens and related commensal species The importance of neisserial Tfp in host interaction and colonizing the human mucosa express phosphocholine (PC) at their disease pathogenesis is attested to by the notorious capacity of cell surfaces. PC appears to impact host–microbe biology by serving the PilE pilin subunit to undergo antigenic variation (8). Post- as a ligand for both C-reactive protein and the receptor for translational modifications provide additional sources for PilE platelet-activating factor. Type IV pili of Neisseria gonorrhoeae structural and functional diversity. In addition to proteolytic (Ng) and Neisseria meningitidis, filamentous protein structures processing and methylation of the N-terminal residue by PilD critical to the colonization of their human hosts, are known to react (9), three distinct posttranslational modifications have been variably with monoclonal antibodies recognizing a PC epitope. described. The first, reported for both Ng and Nm pilins, involves However, the structural basis for this reactivity has remained glycosylation. Characterization of the Ng strain MS11pilin crys- elusive. To address this matter, we exploited the finding that the tallographic structure localized a carbohydrate modification to PilE pilin subunit in Ng mutants lacking the PilV protein acquired serine 63 (S63), which was proposed to be Gal (␣1,3) GlcNAc the PC epitope independent of changes in pilin primary structure. (10). Concurrently, Stimson and colleagues (11) used MS to Specifically, we show by using mass spectrometry that PilE derived demonstrate that Nm strain C311 PilE was glycosylated with the from the pilV background is composed of a mixture of subunits trisaccharide Gal(␤1-4) Gal(␣1-3) 2,4-diacetamido-2,4,6- bearing O-linked forms of either phosphoethanolamine (PE) or PC trideoxyhexose (DATDH) at a serine or threonine between at the same residue, whereas the wild-type background carries residues 50 and 73. The site of this modification has yet to be only PE at that same site. Therefore, PilV can influence pilin identified, although most evidence implicates S63 (12). In con- structure and antigenicity by modulating the incorporation of trast, Marceau and colleagues (12) reported that some pilins these alternative modifications. The disaccharide covalently linked from Nm strain 8013SB were modified with Gal (␣1,3) GlcNAc, to Ng pilin was also characterized because it is present on the same the proposed Ng pilin glycan. Nm genes implicated in the pilin peptides bearing the PE and PC modifications and, contrary to gylcosylation pathway (termed pgl) have been identified by previous reports, was found to be linked by means of 2,4-diacet- virtue of mutations at these loci altering PilE biochemical amido-2,4,6-trideoxyhexose. Taken together, these findings pro- properties compatible with altered carbohydrate composition vide new insights into Ng type IV pilus structure and antigenicity (e.g., migration in SDS͞PAGE, reactivity with lectins and car- and resolve long-standing issues regarding the nature of both the bohydrate-specific antibodies, sugar composition profiles of PC epitope and the pilin glycan. purified pili, etc) (13–16). Many of these genes share strong homology with those implicated in the biosynthesis of the large number of Gram-negative pathogens initiating infec- proximal bacillosamine component of the N-linked glycans in Ation at mucosal surfaces use a unique family of protein- Campylobacter jejuni glycoproteins (17, 18). The mass data for aceous filaments termed Type IV pili (Tfp) in colonization. Nm pilin DATDH are consistent with bacillosamine, a DATDH These organelles have been extensively studied in the closely sugar, although its stereochemistry has not been resolved. Given related species Neisseria gonorrhoeae (Ng) and Neisseria menin- the distinctive glycan reported, it is surprising that Ng also gitides (Nm), the agents of gonorrhea and epidemic meningitis, contains a set of genes implicated in DATDH sugar synthesis respectively, where they mediate specific attachment to human (16). Moreover, with the exception of Nm galE mutants (11), the epithelial cells (1). A detailed understanding of the three- precise nature of the ensuing alterations in pilin glycosylation dimensional structure and chemistry of these Tfp and the PilE mutants have not been characterized at the molecular level. In pilin subunit is essential to the development of vaccines and the absence of supporting structural data, the functions associ- anti-infective agents designed to prevent and control human ated with the pgl gene products remain unsubstantiated and the disease. In both species, Tfp-associated adherence requires the pathway for pilin gylcosylation unresolved. A second pilin post- simultaneous expression of pili and the less abundant PilC translational modification was proposed from further analysis of adhesin protein, which copurifies with it (2–4). In Ng, the pilin-like PilV protein has also been shown to be required for This paper was submitted directly (Track II) to the PNAS office. efficient adherence (5). Explanations proposed to account for Abbreviations: CAD, collision-activated dissociation; DATDH, Gal(␤1-4) Gal(␣1-3) 2,4-diac- the pilV defect include the ineffective display of the PilC adhesin etamido-2,4,6-trideoxyhexose; Hex, hexose; PE, phosphoethanolamine; PC, phosphocho- and that PilV itself may have receptor-binding activity. In line; Tfp, type IV pili; Ng, Neisseria gonorrhoeae;Nm,Neisseria meningitidis;MS͞MS, addition, studies in both species have suggested that intrinsic tandem MS. properties of the PilE pilin subunit itself contribute to the §F.T.H. and P.G.H. contributed equally to this work. adherence process (4, 6, 7). Given these observations, the ††To whom correspondence should be addressed at: Department of Molecular Biosciences, molecular mechanisms by which neisserial Tfp promote human University of Oslo, 0316 Oslo, Norway. E-mail: [email protected]. cell adherence remain poorly understood. © 2004 by The National Academy of Sciences of the USA 10798–10803 ͉ PNAS ͉ July 20, 2004 ͉ vol. 101 ͉ no. 29 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402397101 Downloaded by guest on September 25, 2021 Ng pilin crystallographic data that revealed an electron density pilES68A (5Ј-CTTCTGCCGGCGTGGCAGCCCCCCCCA-3Ј) peak compatible with a phosphate group covalently linked to to introduce a thymine to guanosine substitution (bold italic) at serine 68 (S68) (19). A serine-linked phosphoglycerol has also codon 68 (bold). The PCR product was then digested by using been documented as being localized at residue 93 on Nm pilin the unique BglI and StuI sites and the resulting fragment ligated (20). The biological significance of these modifications remains into a derivative of p2͞16͞1 (34) containing the wild-type pilE largely unclear, given that dramatic phenotypic alterations have sequence, creating the plasmid pIga::pilES68A. This plasmid was not been correlated with their presence or absence. The Nm pilin used to introduce the pilES68A allele into the iga locus of strain glycan, which is exposed on native Tfp, may be a target for MW24 (pilEind) and strain GV12 (pilEind, pilVfs) by transforma- naturally occurring anti-Gal IgA antibodies capable of blocking tion and selection for the linked ermC marker, generating strain complement-mediated killing (21). GE108 (pilEind, iga::pilES68A) and GE108V (pilEind, iga::pilES68A, Evidence for a further pilin posttranslational modification pilVfs), respectively. Strain MW25V (pilEind, iga::pilE, pilVfs) was stems from the finding that some neisserial pilins react with the made by transforming strain MW25 (pilEind, iga::pilE) with DNA TEPC-15 monoclonal antibody recognizing a phosphocholine from a pilVfs (5) mutant strain harboring a chloramphenicol- (PC) epitope (22). The possibility that pilin may possess PC is resistant minitransposon downstream of pilV (unpublished particularly intriguing because this moiety is a surface constit- data). uent of many microbial pathogens. Activities attributed to the presence of the PC moiety in bacterial pathogens include Construction of Ng Strains Carrying Null Mutations in pgtA, pglC, pglD, promoting epithelial and endothelial cell adherence through and pglF. Plasmid ppgtA5-erm (35) was introduced into strain binding to the platelet-activating factor receptor (23–25) and N400 (wild type) and GV1 (pilVfs) by transformation and acting as an immune recognition target for C-reactive protein selection for the ermC marker, generating strain GGA and (25, 26) and PC-recognizing