Characterization of N-linked protein glycosylation in Helicobacter pullorum

Area of Science:

• Microbiology
• Glycobiology
• Molecular Biology

Background:

• The first bacterial N-linked glycosylation system identified was in Campylobacter jejuni, utilizing the oligosaccharyltransferase PglB.
• PglB orthologues are found in Deltaproteobacteria and Epsilonproteobacteria, including Helicobacter species.
• Unlike C. jejuni's single pglB within a gene cluster, Helicobacter species possess two distinct pglB genes (pglB1 and pglB2) outside glycosylation loci.

Purpose of the Study:

• To investigate the function of the two pglB genes in Helicobacter pullorum.
• To compare the N-linked glycosylation mechanism in H. pullorum with that of C. jejuni.
• To determine the essentiality of the pglB2 gene in H. pullorum.

Main Methods:

• Complementation experiments in Escherichia coli to assess PglB1 and PglB2 activity.
• In vitro oligosaccharyltransferase assays.
• Matrix-assisted laser desorption ionization (MALDI) mass spectrometry.
• Insertional knockout mutagenesis of the H. pullorum pglB2 gene.

Main Results:

• H. pullorum PglB1, but not PglB2, transferred the C. jejuni N-linked glycan onto an acceptor protein.
• PglB1-dependent N-glycosylation in H. pullorum utilized a linear pentasaccharide and required an acidic residue at the -2 position of the sequon.
• Insertional mutagenesis of pglB2 in H. pullorum was unsuccessful, indicating its essential role.

Conclusions:

• H. pullorum PglB1 functions as an oligosaccharyltransferase, mediating N-linked glycosylation similar to C. jejuni.
• The presence of two pglB genes and their distinct roles represent a fundamental difference from the C. jejuni system.
• The essentiality of pglB2 suggests a critical role in H. pullorum biology.