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. 2004 Jul 20;101(29):10798-803.
doi: 10.1073/pnas.0402397101. Epub 2004 Jul 12.

Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili

Finn Terje Hegge  1 , Paul G Hitchen, Finn Erik Aas, Heidi Kristiansen, Cecilia Løvold, Wolfgang Egge-Jacobsen, Maria Panico, Weng Yee Leong, Victoria Bull, Mumtaz Virji, Howard R Morris, Anne Dell, Michael Koomey
Affiliations

Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili

Finn Terje Hegge et al. Proc Natl Acad Sci U S A. .

Abstract

Several major bacterial pathogens and related commensal species colonizing the human mucosa express phosphocholine (PC) at their cell surfaces. PC appears to impact host-microbe biology by serving as a ligand for both C-reactive protein and the receptor for platelet-activating factor. Type IV pili of Neisseria gonorrhoeae (Ng) and Neisseria meningitidis, filamentous protein structures critical to the colonization of their human hosts, are known to react variably with monoclonal antibodies recognizing a PC epitope. However, the structural basis for this reactivity has remained elusive. To address this matter, we exploited the finding that the PilE pilin subunit in Ng mutants lacking the PilV protein acquired the PC epitope independent of changes in pilin primary structure. Specifically, we show by using mass spectrometry that PilE derived from the pilV background is composed of a mixture of subunits bearing O-linked forms of either phosphoethanolamine (PE) or PC at the same residue, whereas the wild-type background carries only PE at that same site. Therefore, PilV can influence pilin structure and antigenicity by modulating the incorporation of these alternative modifications. The disaccharide covalently linked to Ng pilin was also characterized because it is present on the same peptides bearing the PE and PC modifications and, contrary to previous reports, was found to be linked by means of 2,4-diacetamido-2,4,6-trideoxyhexose. Taken together, these findings provide new insights into Ng type IV pilus structure and antigenicity and resolve long-standing issues regarding the nature of both the PC epitope and the pilin glycan.

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Figures

Fig. 1.
Fig. 1.
Influence of pilV on expression of the pilin PC epitope. Lanes: 1, N400 (wild type); 2, GV1 (pilVfs); 3, GV5 (pilVfs, iga::pilV); and 4, GV37 (pilVoe). (A) Coomassie-stained SDS/PAGE gel loaded with whole-cell lysates showing the relative migration of PilE. (B) Immunoblotting of whole-cell lysates by using monoclonal antibody TEPC-15 (1:1,000). (C) Coomassie-stained SDS/PAGE gel showing the relative amounts of PilE in purified pili. (D) Immunoblotting of purified pili by using monoclonal antibody TEPC-15. With regard to PilV expression, + denotes the wild-type allele, - denotes the PilVfs-null allele, -/+ denotes complementation of a pilV-null mutation by means of ectopic expression, and ++ denotes the pilVoe allele.
Fig. 2.
Fig. 2.
MS/MS of wild-type, modified PilE tryptic peptides. (A) Characterization of the species at [M + 2H]2+ (strains N400 and MW25). The peptide 57WPENNTSAGVASPPTDIK74 is modified with PE and the disaccharide HexDATDH. (B) Characterization of the modified PilE thermolysin/tryptic peptide at m/z 525 [M + 2H]2+ (strain N400). The peptide 66VASPPTDIK74 is modified with PE. Fragmentation patterns are shown in A Upper and B Upper.
Fig. 3.
Fig. 3.
MS/MS of modified PilE tryptic peptides in glycosylation mutants. (A) Characterization of the species at m/z 1,117.5 [M + 2H]2+ from a pgtA mutant (strain GGA). The peptide 57WPENNTSAGVASPPTDIK74 is modified with PE and DATDH. (B) Characterization of the species at m/z 1,003.5 [M + 2H]2+ from a pglC mutant (strain GGC). The peptide 57WPENNTSAGVASPPTDIK74 is modified with PE. Fragmentation patterns are shown in A Upper and B Upper. The same results were obtained for the corresponding tryptic peptides from the pglD and pglF mutants (Fig. 7, strains GGD and GGF).
Fig. 4.
Fig. 4.
MS/MS of modified PilE tryptic peptides in pilV mutants. (A) Characterization of the species at m/z 1,219.5 [M + 2H]2+ from a pilV background (strains GV1 and MW25V). The peptide 57WPENNTSAGVASPPTDIK74 is modified with PC and the disaccharide HexDATDH. (B) Characterization of the species at m/z 1,137 [M + 2H]2+ from a pilVoe background (strain GV37). The peptide 57WPENNTSAGVASPPTDIK74 is modified with the disaccharide HexDATDH. Fragmentation patterns are shown in A Upper and B Upper.
Fig. 5.
Fig. 5.
MS/MS of the modified tryptic peptide from the PilES68A mutant. Characterization of the species at m/z 1,129 [M + 2H]2+ (strain GE108). The peptide 57WPENNTSAGVAAPPTDIK74 is modified with the disaccharide Hex-DATDH. Fragmentation patterns are shown in Upper.
Fig. 6.
Fig. 6.
Effects of an alanine substitution at residue 68 on expression of the pilin PC epitope. Lanes: 1, MW25 (pilEind, iga::pilE); 2, MV25V (pilEind, iga::pilE, pilVfs); 3, GE108 (pilEind, iga::pilES68A); and 4, GE108V (pilEind, iga::pilES68A, pilVfs). (A) Coomassie-stained SDS/PAGE gel of whole-cell lysates showing the relative migration of PilE. (B) Immunoblotting of whole-cell lysates by using monoclonal antibody TEPC-15. (C) Coomassie-stained SDS/PAGE gel showing the relative amounts of PilE in purified pili. (D) Immunoblotting of purified pili by using monoclonal antibody TEPC-15. With regard to PilV expression, + denotes the wild-type allele and - denotes the pilVfs-null allele.
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