'Capnocytophaga canimorsus' : discovery of a deglycosylation mechanism that links metabolism to pathogenesis

We show that C. canimorsus (Cc) can serve as a recipient for RP4 mediated conjugation but there is neither replication of broad host range plasmid vectors nor expression of commonly used E. coli markers in C. canimorsus. We identified three selection markers, ermF, tetQ and cfxA leading to resistance against erythromycin, tetracycline and cefoxitin, respectively, that can be used in C. canimorsus. We engineered expression shuttle vectors using the replicon of a endogenous plasmid found in strain Cc7 and the promoter of one of the selection markers for gene expression. We developed a transposon mutagenesis strategy based on Tn4351 from Bacteroides fragilis and protocols for allelic exchange and electrotransformation. We carried out an extensive transposon mutagenesis and screened these mutants for different properties. We demonstrate that presence of mammalian cells, including phagocytes, favors growth of C. canimorsus 5 and this property was found to be dependent on direct cellular contacts. We isolated a Tn mutant unable to grow in presence of mammalian cells. The mutation occurred in a gene encoding a sialidase. The surface-exposed sialidase allows Cc5 to feed on internal aminosugars of glycan chains from host cell glycans. In addition, sialidase confers resistance to complement by promoting the binding of factor H. We developed an experimental mouse infection in which the read-out is bacterial persistence. In this infection model, Cc5, but not the sialidase deficient mutant, grew and persisted, showing the importance of this metabolic pathway in vivo. C. canimorsus by itself does not elicit the onset of an inflammatory response from macrophages. One strain, Cc5 turned out to have a mechanism that actively blocks the pro-inflammatory signaling of macrophages upon stimulation with endotoxic LPS. We screened the Tn mutant library for clones of Cc5 affected in this active mechanism. Isolated mutants have been mapped, characterized and complemented. The function of the mutated genes is presently under investigation as well as the mode of action of its gene product(s). The prevalence of C. canimorsus in dogs has not been clarified at present. We therefore sampled dog swabs to isolate C. canimorsus strains in Swiss dogs. We could identify 61 C. canimorsus isolates from 103 dogs, which represents 59.22% of the dogs tested. Besides this I also contributed to the analysis of LPS, to the study of resistance of Cc5 to complement mediated lysis, to sequencing of the genome, the assembly of the reads and the annotation.