title: Unconventional N-Linked Glycosylation Promotes Trimeric Autotransporter Function in Kingella kingae and Aggregatibacter aphrophilus.
abstract: Glycosylation is a widespread mechanism employed by both eukaryotes and bacteria to increase the functional diversity of their proteomes. The nontypeable Haemophilus influenzae glycosyltransferase HMW1C mediates unconventional N-linked glycosylation of the adhesive protein HMW1, which is encoded in a two-partner secretion system gene cluster that also encodes HMW1C. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. In the present study, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C that are not encoded near a gene encoding an obvious acceptor protein. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild-type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.
  
  
  OBJECTIVE
  This work emphasizes the importance of glycosylation for proper function of bacterial proteins. Here we show that the Kingella kingae Knh and the Aggregatibacter aphrophilus EmaA trimeric autotransporter proteins are N-glycosylated by novel homologues of the Haemophilus influenzae HMW1C glycosyltransferase, highlighting the first examples of trimeric autotransporters that are modified by HMW1C-like enzymes. In the absence of glycosylation, Knh and EmaA lack adhesive activity. This work has relevance to our understanding of bacterial pathogenicity and expression of potential vaccine antigens.
authors: Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
authors: Katherine A
authors: KA
authors: Rempe
authors: The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
authors: Lynn A
authors: LA
authors: Spruce
authors: The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
authors: Eric A
authors: EA
authors: Porsch
authors: The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
authors: Steven H
authors: SH
authors: Seeholzer
authors: Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark.
authors: Niels
authors: N
authors: Nørskov-Lauritsen
authors: The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA stgemeiiij@email.chop.edu.
authors: Joseph W
authors: JW
authors: St Geme
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064207:Aggregatibacter aphrophilus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000382:microbiology; D006020:Glycopeptides; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006193:Haemophilus influenzae / Q000235:genetics; D006801:Humans; D017882:Kingella kingae / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D053719:Tandem Mass Spectrometry; D000069320:Type V Secretion Systems
chemical_list: D018829:Adhesins, Bacterial; D006020:Glycopeptides; C117431:HMW1 protein, bacteria; D000069320:Type V Secretion Systems; D016695:Glycosyltransferases