"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.