Investigating the roles of O-linked Protein Glycosylation and Type Two Secretion in the Pathogenesis of Acinetobacter

• Author / Creator

  Kinsella, Rachel L.

• The Acinetobacter baumannii, A. nosocomialis and A. pittii are Gram-negative opportunistic human pathogens that are of importance to the healthcare system particularily because of their resistance to antibiotics. The thesis below focuses on investigating two virulence attributes of Acinetobacter that contribute to its pathogenesis, general O-linked protein glycosylation and Type II secretion. At least seven proteins are O-glycosylated in A. baumannii ATCC 17978 in an O-oligosaccharyltransferase dependent manner. Protein glycosylation is required for biofilm formation, and virulence in an amoeba, insect and murine models of infection. A. baumannii utilizes the same glycan to modify glycoproteins as is polymerized into capsular polysaccharide. Capsular polysaccharide is required for resistance to complement mediated killing and therefore also is necessary for colonization in mice. The importance and abundance of this carbohydrate in the pathogenesis of A. baumannii as well as the non-linear nature of glycan synthesis, lead us to investigate the diversity, composition and properties of the Acinetobacter glycoproteome using various mass spectrometry methods. Twenty-six glycoproteins were identified in the fifteen strains examined, revealing that similar proteins are targeted for glycosylation in Acinetobacter baumannii, A. calcoaceticus, A. pitti, A. baylyi, and A. nosocomialis. Glycosylation tends to occur at low complexity regions enriched in Proline, Alanine and Serine residues. There was extensive glycan variability between the strains and within the strains examined. All O-glycans identified have an N-acetyl hexosamine residue at the reducing end and tended to contain a negatively charged monosaccharide. Glycopeptides modified with more than one subunit of the O-glycan were identified in most strains, suggesting that sharing the glycan between protein glycosylation and capsule production is a common feature in Acinetobacter. Most glycoproteins identified required the sensitivity of Mass Spectrometry and Zwitter ionic hydrophilic interaction chromatography glycopeptide enrichment methods to be detected. Two glycoproteins, A1S3626 andA1S3744 were identified by two-dimensional differential gel electrophoresis separation of total membrane glycosylated and unglycosylated proteins, implying that these proteins are produced and modified in higher abundance. There is one 30 kDa periodic acid schiff stained glycoprotein band produced by A. baumannii ATCC 17978. We hypothesized that either A1S3626 or A1S3744 would be required for production of this 30 kDa glycoprotein. A1S3626 and A1S3744 are both not essential for production of the 30 kDa glycoprotein, suggesting there may be a yet unidentified glycoprotein. Both A1S3626 and A1S3744 are not required for biofilm formation in A. baumannii. Strains devoid of protein glycosylation display a virulence defect in Galleria mellonella. A1S_3744 is not required for virulence in G. mellonella. A. nosocomialis M2 is a medically relevant member of the Acinetobacter genus. Type II secretion is required for virulence and nutrient acquisition by several Gram-negative organisms. Here we show M2 has a functional Type II secretion system that is required for virulence in G. mellonella larvae and mice, and is responsible for the secretion of two lipases LipA and LipH as well as a protease, CpaA. LipA and CpaA required membrane anchored chaperones, LipB and CpaB respectively, for their secretion. Bioinformatic analysis revealed putative chaperones adjacent to several characterized Type II substrates, suggesting this may be a wide-spread phenomenon. CpaA is required for full virulence in the G. mellonella larvae and splenic colonization in a murine pulmonary infection model. We demonstrate the physical interaction between CpaA and its chaperone CpaB. The C-terminal periplasmic domain of CpaB is adequate for secretion of CpaA. Soluble, periplasmic CpaB is secreted with CpaA, suggesting the purpose of the membrane anchor may be to retain CpaB and only secrete free CpaA. One quarter of CpaA is sufficient to bind CpaB, indicating there are multiple points of interaction along the length of CpaA. We crystallized the CpaA-CpaB complex and have obtained diffraction data sets ranging from 3.5 to 7 angstrom resolution. There are no crystallized orthologs of CpaB, therefore we are pursuing the heavy-metal derivative in order to solve the crystal structure. The structural data may shed light on the mechanism of CpaB and provide the basis for design of specific CpaA inhibitors.

• Subjects / Keywords

  • Type II secretion
  • Chaperone
  • Protein glycosylation
  • Acinetobacter
  • Glycan diversity

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3GQ6RH4G

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.