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. 2010 Jun;192(11):2816-29.
doi: 10.1128/JB.00101-10. Epub 2010 Apr 2.

Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species

Bente Børud  1 , Finn Erik Aas, Ashild Vik, Hanne C Winther-Larsen, Wolfgang Egge-Jacobsen, Michael Koomey
Affiliations

Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species

Bente Børud et al. J Bacteriol. 2010 Jun.

Abstract

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

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Figures

FIG. 1.
FIG. 1.
DATDH-based glycans are immunogenic and antigenic: immunoblotting of whole-cell lysates of N. gonorrhoeae pgl mutant strains and variants with (A) the rabbit polyclonal antibodies pAb1, pAb2, and pAb3 raised against purified PilE bearing the DATDH, Ac-Hex-DATDH and Ac-Hex-Hex-DATDH glycans, respectively, and (B) the rabbit monoclonal antibodies npg1, npg2 and npg3 raised against the corresponding glycans. The strains used all had a 4/3/1 background in which pilE was conditionally repressed and were KS105 (pglC), KS122 (pglA), KS101 (wt), KS314 (pglI), KS127 (pglEon), and KS315 (pglEon pglI). wt, wild type.
FIG. 2.
FIG. 2.
Neisserial glycoprotein profiles and glycan diversity. (Top panels) Immunoblotting of whole-cell lysates from strains of N. gonorrhoeae (Ngo) (N400 pglC, N400, and FA1090), N. meningitidis (Nme) (MC58, H44/76, Z2491, 8013, FAM18, BZ 10, and BZ 198), and N. lactamica (Nla) (ST-3787 and ST-640) with glycan-specific monoclonal antibodies. (Bottom panels) Immunoblotting using the SM1 MAb specifically recognizing class I pilin types and a polyclonal antiserum raised against the PilE-derived peptide 44KSAVTEYYLNHGKWPENNTSA64, which reacts with all pilin types. Thus, strains FAM18, BZ 10, ST-3787, and ST-640 all express class II pilins. Also, strains 8013, FAM18, BZ 10, and BZ 198 carry the pglB2 allele that is associated with synthesis of GATDH glycans, while all of the other strains have the pglB allele that is associated with synthesis of DATDH glycans.
FIG. 3.
FIG. 3.
Glycoprotein profiles and glycan antigenicity in strains N400, FA1090, and MC58. (A) Whole-cell lysates of N. gonorrhoeae N400 and FA1090 wild-type (wt) strains and pglA (monosaccharide-expressing) and pglC (glycosylation-null) mutants probed with MAb npg1. The strains used were KS100 (N400 wt), KS104 (N400 pglC), KS141 (N400 pglA), KS302 (FA1090 pglA), KS301 (FA1090 pglC), and KS300 (FA1090 wt). (B) Immunoblotting of whole-cell lysates of N. gonorrhoeae FA1090 and N. meningitidis MC58 wild-type strains and pglA (monosaccharide-expressing) and pglC (glycosylation-null) mutants probed with MAbs npg1, npg2, and npg3. The strains used were KS301 (FA1090 pglC), KS302 (FA1090 pglA), KS300 (FA1090 wt), KS317 (MC58 pglC), KS318 (MC58 pglA), and KS316 (MC58 wt).
FIG. 4.
FIG. 4.
Increased npg2 and npg3 immunoreactivity at high pH and in pglI mutants. Immunoblotting of whole-cell lysates of wild-type strains, pglI mutants, and high-pH-treated wild-type strains revealed increased reactivity with the npg2 and npg3 monoclonal antibodies due to the lack of an O-acetyl group in pglI mutants or with the high-pH treatment. The extraneous band that appeared during high-pH treatment is PilE (as determined by immunoblotting with SM1 [data not shown]). This band was not present with the 4/3/1 background, in which pilE was conditionally repressed. The strains used were KS100 (N400 wt), KS144 (N400 pglI), KS104 (N400 pglC), KS101 (4/3/1 wt), KS300 (FA1090 wt), KS303 (FA1090 pglI), and KS301 (FA1090 pglC). wt, wild type.
FIG. 5.
FIG. 5.
AniA and GNA1946 are glycosylated in MC58. (A) Equivalent whole-cell lysate samples of N. meningitidis MC58 were electrophoresed in adjacent lanes, and following transfer to a PVDF membrane, the membrane was divided in half lengthwise. The two pieces were then probed with MAb npg2 and antibodies to AniA. The results demonstrate that the same band corresponding to AniA was identified with both antibodies. (B) Identification of the glycopeptide 6DSAPAASASAAADNGAAK23 from lipoprotein GNA1946 derived by tryptic cleavage of a membrane protein-enriched fraction of MC58. High-energy collision dissociation (HCD) fragmentation of [M+2H]2+ at m/z 989.4505 revealed characteristic oxonium ions at m/z 433.182 and m/z 229.118 corresponding to O-Ac-Hex-DATDH and DATDH glycans, respectively. The signal at m/z 1545.716 is a signal from the peptide backbone after loss of the glycan moiety; the monoisotopic theoretical molecular mass is 1,545.714 Da.
FIG. 6.
FIG. 6.
MS analysis of intact PilE carrying DATDH- or GATDH-based glycan forms. ESI-MS analyses of intact PilE utilizing pili from strains carrying either pglBMC58 or pglB28013 in different pgl backgrounds were carried out to characterize the glycan structure. N400 pglBMC58 pglA produced two major signals that are enclosed in boxes and represent PilE carrying the DATDH monosaccharide with one (m/z 17530) or two (m/z 17653) PE modifications. The N400 pglB28013 pglA signals represent PilE carrying the GATDH monosaccharide with one (m/z 17576) or two (m/z 17699) PEs. For N400 pglBMC58 and N400 pglB28013, the major signals represent PilE with one or two PE modifications in conjunction with the Ac-Hex-DATDH and Ac-Hex-GATDH disaccharides, respectively (indicated by m/z values in boxes). For N400 pglBMC58 pglEon, major signals correspond to PilE carrying Hex-Hex-DATDH with one or two PE modifications (boxes). N400 pglB28013 pglEon major signals are enclosed in boxes and represent Hex-Hex-GATDH with one or two PEs. Table S1 in the supplemental material shows all of the ion species along with the m/z values and corresponding molecular weights. The strains used were KS306 (N400 pglBMC58 pglA), KS309 (N400 pglB28013 pglA), KS305 (N400 pglBMC58), KS308 (N400 pglB28013), KS307 (N400 pglBMC58 pglEon), and KS310 (N400 pglB28013 pglEon).
FIG. 7.
FIG. 7.
GATDH-based glycans are antigenically distinct from DATDH-based forms. Western blots of N400 (pglB), N400 pglBMC58, and N400 pglB28013 in different pgl backgrounds were incubated with the npg1, npg2, and npg3 monoclonal antibodies. The strains used were KS141 (N400 pglA), KS306 (N400 pglBMC58 pglA), KS309 (N400 pglB28013 pglA), KS100 (N400), KS305 (N400 pglBMC58), KS308 (N400 pglB28013), KS142 (N400 pglEon), KS307 (N400 pglBMC58 pglEon), and KS310 (N400 pglB28013 pglEon).
FIG. 8.
FIG. 8.
Protein-associated GATDH monosaccharide is both immunogenic and antigenic. An immunoblot of whole-cell lysates from strains with defined pgl backgrounds was first incubated with pGAb, a polyclonal antiserum raised against pili bearing the GATDH monosaccharide. The same filter was subsequently reprobed with the npg1, npg2, and npg3 MAbs with washing and developing steps between exposures. The strains used all had a 4/3/1 background, in which pilE was conditionally repressed, and were KS105 (4/3/1 pglC), KS122 (4/3/1 pglA), KS101 (4/3/1), KS127 (4/3/1 pglEon), KS312 (4/3/1 pglB28013 pglA), KS311 (4/3/1 pglB28013), and KS313 (4/3/1 pglB28013 pglEon).
FIG. 9.
FIG. 9.
Glycan MAbs react specifically with intact pili as determined by immunoelectron microscopy. Immunogold labeling and transmission electron microscopy using the monoclonal antibodies (npg1, npg2, and npg3) against different N400 pgl strains demonstrated their specificity by binding to pili of the corresponding strains. The strains used were KS100 (N400) (wild type [wt]), KS141 (N400 pglA), KS104 (N400 pglC), KS142 (N400 pglEon), KS144 (N400 pglI), and KS304 (N400 pglEon pglI). Note that for the npg2 and npg3 MAbs, the levels of immunoreactivity were dramatically enhanced in the pglI background, in which glycan O acetylation was not present.
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