Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue

Abstract

The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis, the etiological agent of cerebrospinal meningitis, crosses cellular barriers after adhering to host cells through type IV pili. Pilin glycosylation genes (pgl) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido-2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by "top-down" MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pili preparations originating from N. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacetamido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glyceramido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pglBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis.

Fig. 1.

FT-ICR analysis of PilE glycosylation. (A) Analysis of pili purified from the wild-type strain. An enlargement of the spectrum in the mass range of 17,480–17,520 Da showing a resolved isotopic envelope for the MH⁺ ion is depicted in Inset. (B) Analysis of the nonglycosylated forms of PilE. Pili from the pilE_S63A strain (red) and the pglD strain (blue) were compared with wild type (black). (C) PilE molecules subjected to ISD lose their sugar moiety, leading to the appearance of a secondary peak corresponding to the mass of the pilin without the sugar.

Fig. 2.

Determination of glycan structure by ISD and MS/MS. Crude pili preparations were analyzed with a Q-TOF Premier instrument. Samples were submitted to ISD to cleave the sugar from the rest of the protein and to form the corresponding oxonium ions. Ions were then analyzed by a quadrupole mass spectrometer, and chosen ions were further fragmented by collision and analyzed by TOF mass spectrometry. (A) Comparison of the ion profile in the presence (+ISD) and the absence (−ISD) of ISD. Ions of interest are indicated in bold. (B) Fragmentation of monosaccharides dissociated from PilE purified from the N. gonorrhoeae MS11 strain (Ng MS11, m/z 229.1 Da) and the N. meningitidis 8013 strain (Nm 8013, m/z 275.1 Da) by ISD. (C) Fragmentation of the 169.1-Da ion from both sugars.

Fig. 3.

Structures and fragmentation patterns of DATDH and GATDH. (A) Structures of DATDH and GATDH linked to serine-63 of PilE. (B) Proposed successive steps of fragmentation of DATDH and GATDH.

Fig. 4.

The pglB allele determines whether DATDH or GATDH is transferred to PilE serine-63. (A) Genetic organization of the pglB1 and pglB2 alleles from different Neisseria spp. strains. The gray shaded area indicates regions of homology between both strains. Arrows and the dotted line indicate the region amplified by PCR to insert the pglB2 allele into the pglB1-expressing MS11 strain. Tn indicates a transposon insertion site. (B) Q-TOF analysis of MS11 pilin glycosylation. Pili were purified from the MS11 strain (MS11), a pglA mutant strain (pglA), a pglD mutant strain (pglD), and the MS11 strain expressing the pglB2 allele (MS11pglB2).