Evidence for the Sialylation of PilA, the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316

PLoS One. 2015 Sep 25;10(9):e0138103. doi: 10.1371/journal.pone.0138103. eCollection 2015.

Abstract

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adhesins, Bacterial / chemistry
  • Adhesins, Bacterial / genetics
  • Adhesins, Bacterial / metabolism*
  • Asparagine / chemistry
  • Asparagine / genetics
  • Asparagine / metabolism
  • Bacterial Adhesion / genetics
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Fimbriae Proteins / chemistry
  • Fimbriae Proteins / genetics
  • Fimbriae Proteins / metabolism*
  • Fimbriae, Bacterial / metabolism
  • Fimbriae, Bacterial / ultrastructure
  • Glucosyltransferases / metabolism
  • Models, Molecular
  • N-Acetylneuraminic Acid / metabolism*
  • Organisms, Genetically Modified
  • Plant Lectins / metabolism
  • Protein Binding
  • Protein Processing, Post-Translational*
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Ribosome Inactivating Proteins / metabolism
  • Streptococcus agalactiae* / genetics
  • Streptococcus agalactiae* / metabolism

Substances

  • Adhesins, Bacterial
  • Bacterial Proteins
  • Plant Lectins
  • Protein Subunits
  • Sambucus nigra lectins
  • Fimbriae Proteins
  • Asparagine
  • Glucosyltransferases
  • N-glycosyltransferase, Nocardia aerocolonigenes
  • Ribosome Inactivating Proteins
  • N-Acetylneuraminic Acid

Grants and funding

This study has received funding from the French National Research Agency (ANR-10-BLANC-1314 Glyco-Path) attributed to SD; and from the French Government's Investissement d'Avenir program, Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID) to PTC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.