Ittig, Simon Josef. The surface structures of "Capnocytophaga canimorsus" and their role in pathogenesis. 2012, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9936
The surface polysaccharides of commensal as well as pathogenic bacteria have to fulfil a multitude of functions to ensure viability. Besides phospholipids the outer membrane of Gram-negative bacteria consists of a unique carbohydrate component, the lipopolysaccharide (LPS). The LPS of gram-negative bacteria consists of three regions: the lipid A, the core-oligosaccharide, and the O-antigen. This work describes the structure of C. canimorsus lipid A, core-oligosaccharide and O-antigens.
The main features of the lipid A are that it is penta-acylated and composed of a ”hybrid backbone“ lacking the 4’ phosphate and having a 1-P-Etn at GlcN. C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human MD-2 was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2 or LBP/CD14 with the negative charge in the Kdo of the core might be needed to form the MD-2 – lipid A complex in case the 4’ phosphate is not present. Overall the properties of the lipid A-core may explain how this bacterium first escapes recognition by receptors of the innate immune system, but nevertheless is able to provoke a shock at the septic stage.
We further show that the C. canimorsus genome encodes in a single operon a lipid A 1 phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that LPS is modified after its synthesis by removal of the 1 phosphate and subsequent addition of a P-Etn group. In agreement with this prediction, deletion of lpxE or eptA led to increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP), where deletion of lpxE had a more severe effect. The endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant.
The structure of the complete LPS from C. canimorsus 5 (Cc5) was determined by chemical analysis, GLC-MS, ESI FT-ICR MS and NMR spectroscopy. Two different O-antigens (LPS I and LPS II) were found to be co-expressed. LPS I consists of repeating units of N-Acetylfucosamine (FucNAc), glucuronic acid (GlcA), N-Acetylquinovosamine (QuiNAc) and N-galacturonoyl-2-aminoglycerol (GalANgro) while LPS II O-antigen consists of five repeating units of N-Acetylglucosamine (GlcNAc) and L-Rhamnose (L-Rha). Several transposon mutants sensitive to complement killing isolated by a large screen turned out to be also sensitive to killing by Polymyxin B. All the mutations mapped in a 28-kb locus consisting of 29 genes involved in the biosynthesis and assembly of the sugars identified in LPS I and LPS II. All serum- and Polymyxin-sensitive mutants lacked LPS I but also a high molecular weight polysaccharide reacting with a specific anti LPS I antiserum. We inferred that this polysaccharide was a type 1 or 4 capsule consisting of the LPS I repeating units. The K-antigen, formed by LPS I and the related capsule, but not LPS II, were found to be assembled by a wzx/wzy dependent process. Deletion of wzz lead to deregulation of the length of LPS I, to the loss of the LPS I dependent capsule and to an altered surface as detected by TEM. Summarizing, we show that the C. canimorsus 5 K-antigen is responsible for the complement and Polymyxin B resistance.
|Advisors:||Cornelis, Guy R.|
|Committee Members:||Dehio, Christoph|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Molecular Microbiology (Cornelis)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||265 S.|
|Last Modified:||30 Jun 2016 10:49|
|Deposited On:||25 Jul 2012 12:19|
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