Euan Thomson, Ph.D.

Plants are gaining recognition as useful infection models as the pathogenic and immunological parallels between plants and animals are delineated. The recent characterization of Common duckweed (Lemna minor), a small aquatic plant, as a model for Pseudomonas aeruginosa infection suggests its potential usefulness in studying the Burkholderia cepacia complex (Bcc), another group of important cystic fibrosis pathogens. The aims of this study are to investigate L. minor as a Bcc infection model and apply it to an array of purposes in Bcc pathogenesis research. The LD50 values of seventeen Bcc strains were determined in the duckweed model and, after plotting them against values previously obtained using the Greater wax moth larva model, it was observed that the relative virulence levels of the Bcc strains correlate strongly in the two infection models. Next, bacteriophage treatment of infected duckweed was attempted; plant rescue occurred only when phage were applied before 12 hours, suggesting bacterial invasion of tissues or cells. Finally, virulence genes were identified by challenging duckweed with 5,970 B. cenocepacia random plasposon mutants. Among the identified mutants were strains carrying insertions in known and uncharacterized virulence regulators, energy and amino acid synthesis clusters, and an antifungal toxin synthesis cluster.

Ritika Dwivedi, M.Sc.

The N-linked protein glycosylation pathway (Pgl) of Campylobacter jejuni is necessary for the biosynthesis of a unique heptasaccharide, GalNAc-GalNAc-(Glc)-GalNAc-GalNAc-GalNAc-diNAcBac (where diNAcBac is 2,4-di-acetamido-2,4,6-trideoxy-D-Glc) in the cytoplasm followed by the transfer onto the sequon D/E-X-N-X-S/T by the oligosaccharyltransferase, PglB. In addition, PglB exhibits hydrolase activity and releases the heptasaccharide in its free oligosaccharide (fOS) form into the periplasmic space. Recently, it was discovered that fOS levels are dependent on the osmotic conditions that C. jejuni encounters in its environment [1]. A related Campylobacter species, C. lari , is a halotolerant organism colonizing hosts found in high salinity environments (eg. marine animals and seagulls) and is able to tolerate 1.5% NaCl levels under laboratory conditions [2]. We are currently investigating how fOS contributes to the halotolerant phenotype of C. lari. Interestingly, MS and H1-NMR analyses indicate that C. lari fOS are hexasaccharides with a phosphate at the reducing end. In contrast, hexasaccharides are added directly to asparagine residues without further modification. As expected, MS analysis revealed that a C. lari pglB mutant does not produce fOS or N-linked glycans. In order to confirm that C. lari PglB in not able to cleave lipid-linked oligosaccharides in two positions, the C. lari PglB enzyme was expressed in a C. jejuni pglB mutant. In this experiment, only unphosphorylated fOS consistent with the C. jejuni heptasaccharide structure was detected, indicating that phosphorylation of fOS in C. lari is PglB independent. We are currently investigating into the mechanism that leads to phosphorylated fOS in C. lari. It is speculated that the anionic fOS modification in this species increases the periplasmic retention of the hexasaccharide. Future studies will focus on understanding the role of the fOS phosphate modification in the osmotolerance of C. lari. References: [1] Nothaft, H et al. (2009) Proc Natl Acad Sci U S A 106:15019-15024., [2] Miller, WG et al. (2008) Foodborne Pathog Dis, 5:371-386.

Sian Ford, 499

Emiliania huxleyi is a unicellular marine green algae that is notable in its ability to produce coccoliths using dissolved carbon dioxide as the carbon source. E. huxleyi is a major player in the marine carbon cycle, due to its ability to pull carbon into the deep ocean through the shedding of its coccoliths. The sequestration of carbon into the ocean allows carbon dioxide in the atmosphere to dissolve into the ocean, thus potentially influencing global climate change. E. huxleyi has a bacterial symbiont, Phaeobacter galleciencis, which undergoes a switch from beneficial to pathogenic in a manner postulated to form the basis of the cyclical nature of large scale algal blooms. P. galleciencis acts to promote algal growth until the bacterial cell density reaches a critical point, at which time it switches to a pathogenic role. P. galleciencis produces a roseobactercide which is a known algaecide, and upon its injection into E. huxleyi the algae undergoes cell death. It is believed that the roseobactercide induces E. huxleyi to undergo apoptosis, however the exact mechanism is yet unknown.

Karen Lithgow, 499

The Burkholderia cepacia complex (Bcc) is a group of gram-negative beta-proteobacteria with important roles in soils and the propensity to cause opportunistic infections in the lungs of immuno-compromised patients1. O-linked glycosylation is the enzymatic addition of glycan substrates onto hydroxylated amino acids (AAs) of acceptor proteins, purportedly contributing to immune evasion, stabilization from proteolysis, and adhesion2 in bacteria. During the en bloc transfer mechanism, sugars are sequentially built upon an inner membrane lipid carrier, which is translocated to the periplasm where an oligosaccharyltransferase (OTase) transfers the sugar to a target protein3. Although protein glycosylation is known to contribute to virulence in other beta-proteobacteria, it remains unexplored among Bcc bacteria. In this study we characterize the B. cenocepacia OTase (Bcal0960), demonstrating the presence of a general O-linked glycosylation system which functions by means of Bcal0960 in an en bloc transfer mechanism. Phenotypic work on an insertional mutant, bcal0960::Tet, showed important roles for this gene in virulence and motility. An enrichment for glycoproteins modified by Bcal0960 revealed fourteen glycoproteins in the WT that were absent from bcal0960::Tet; notably, the glycan-modified AA residue was identified from six of the fourteen glycoproteins. Further works aims to characterize Bcal0960 and the general O-linked glycosylation system. 1. Mahenthiralingam, E., et al. (2005) The multifarious…Burkholderia cepacia complex. Nat. Rev. Microb. 3:144-156. 2. Nothaft, H., et al. (2010). Protein glycosylation in bacteria: sweeter than ever. Nat. Rev. Microb. 8(11):765-778. 3. Faridmoayer, A., et al. (2007). Functional characterization of bacterial… J. Bact 189:8088-8098.

Robert Lees-Miller, M.Sc.

Acinetobacter baumannii is an emerging nosocomial pathogen of particular concern, mainly due to its extraordinary resistance towards antibiotics. Although several virulence factors have been identified, most are still poorly characterised. Previous work established that A. baumannii ATCC 17978 produces O-glycoproteins modified by a pentasaccharide with the presence of an uncommon tri-acetylated form of glucuronic acid. In silico analysis of the Ab genome revealed homologues for the genes required to synthesize this sugar, which are encoded within a locus that contains other genes presumably required for synthesis of the pentasaccharide. Furthermore, adjacently to this locus are genes encoding homologues of the required components for Wzy-dependent capsular polysaccharide. In this work, we show that PglC, the protein encoded by A1S_0061 is an initiating glycosyltransferase responsible for transferring the first monosaccharide of the glycan to the polyisoprenyl lipid carrier. Deletion of pglC in Ab resulted in the loss of both glycosylation and capsular polysaccharide (CPS). The CPS Structure was determined by NMR to be a polymer of the same oligosaccharide previously identified on O-glycoproteins. Ab ΔpglC produced unique, disordered biofilms, and exhibited loss of resistance to serum killing and attenuated virulence in animal models. These experiments suggest that PglC is a promising target for novel antibiotics against Ab.

Jessica Sacher, M.Sc.

Bacteriophages, or bacterial viruses, specifically target their bacterial hosts through receptor binding proteins (RBPs). The Campylobacter jejuni phage NCTC 12673 RBP has been used previously to demonstrate that phage RBPs can be effectively employed as bacterial diagnostics. However, in spite of the rapid progress in DNA sequencing technologies, phage genome sequencing remains a challenge. Furthermore, phage RBPs are difficult to identify through homology due to their wide ranges of host receptors. Thus, an alternative means of identifying phage RBPs is necessary in order to further exploit RBP-based technologies. We are developing a high-throughput method to screen for putative RBP genes in a phage genome. We expressed a C. jejuni phage RBP in Escherichia coli, immobilized cell lysates on a synthetic membrane, probed with intact C. jejuni cells and examined growth of bound colonies. Our preliminary results indicate that this method can be successfully employed to detect E. coli colonies expressing recombinant RBPs. We are now extending this methodology to examine total expression libraries of NCTC 12673 phage genomic DNA. Further progress in this direction would result in a rapid and inexpensive means of RBP identification, providing access to an untapped natural source of bacterial diagnostic tools.