The surface structures of "Capnocytophaga canimorsus" and their role in pathogenesis

Ittig, Simon Josef. The surface structures of "Capnocytophaga canimorsus" and their role in pathogenesis. 2012, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_9936

Downloads: Statistics Overview


Since 1976 there have been numerous case reports about severe sepsis or meningitis in humans after dog bites or scratches. The bacterium causing these dramatic infections was identified as Capnocytophaga canimorsus. C. canimorsus belong to the family of Flavobacteriaceae in the phylum Bacteroidetes and are usual members of dog’s mouth flora. Human infections are rare and occur with an approximate frequency of one case per million inhabitants and year.
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)
UniBasel Contributors:Ittig, Simon Josef and Cornelis, Guy R. and Dehio, Christoph
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:9936
Thesis status:Complete
Number of Pages:265 S.
Identification Number:
edoc DOI:
Last Modified:22 Jan 2018 15:51
Deposited On:25 Jul 2012 12:19

Repository Staff Only: item control page