Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus

Capnocytophaga canimorsus is a dog’s and cat’s oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus.


Sauder, Mohamed Chami and Guy R Cornelis
Supplementary data Data S1. The O-chain units are transported by Wzx As shown in Supplementary Fig. S2, deletion of the putative wzx (Ccan_23200) determined the disappearance of the CPS and of normal LOS (band C) and the appearance of lower molecular weight LOS bands.
However, trans complementation of wzx did not restore the wt LOS and CPS profiles but determined the formation of lower molecular weight LOS bands ( Supplementary Fig. S2) thus suggesting a polar effect of the deletion on the downstream genes. Indeed, the downstream gene Ccan_23210 encodes an A4GalT_like glycosyltransferase that could be responsible for the addition of galactose or glucose residues to the O-chain. We then co-expressed genes Ccan_23200 (wzx) and Ccan_23210 in trans in the wzx mutant strain and verified the LOS and CPS profiles. As shown in Supplementary Fig. S2, coexpression restored the wt phenotype thus confirming that deletion of wzx had a polar effect on Ccan_23210. Then, in order to determine the role of Ccan_23200, we expressed Ccan_23210 in trans in the wzx-deleted strain.
Expression of Ccan_23210 did not restore the wt phenotype ( Supplementary   Fig. S2) thus suggesting that Ccan_23200 could indeed encode for the Wzx O-chain transporter.

Data S2. The capsule is polymerized by Wzy
Trans complementation of the Ccan_23280 (wzy) mutant did not restore the wt LOS and CPS profiles thus suggesting a polar effect of the deletion on downstream genes ( Supplementary Fig. S2). Indeed, the wzy downstream gene codes for a glycosyltransferase of the family 1 that could be involved in the biosynthesis of the O-chain. We then co-expressed genes Ccan_23280 (wzy) and Ccan_23290 in trans in the wzy mutant strain and verified the LOS and CPS profiles. As shown in Supplementary Fig. S2, co-expression of Ccan_23280 and Ccan_23290 restored the CPS and LOS profiles thus confirming that deletion of wzy had a polar effect on Ccan_23290. Then, in order to determine the role of Ccan_23280, we expressed Ccan_23290 in trans in the wzy deletion strain. Expression of Ccan_23290 restored the wt LOS profile but not the CPS ( Supplementary Fig. S2.), suggesting that Ccan_23280 indeed encodes the Wzy O-antigen polymerase that is responsible for the assembly of the CPS but not of the LOS O-chain.

Data S3. The capsule assembly is controlled by Wzz
The Ccan_15550 (wza) deletion mutant lacked the CPS but surprisingly showed a ladder pattern of bands migrating higher than wt LOS (Fig. 4c), a phenotype resembling the one observed for the wzy (wzy + , 23290 + ) strain. We hypothesized that this ladder could be composed of LPS harboring O-chains of different lengths and that deletion of Ccan_15550 somehow affected the regulation of the LOS O-antigen length.
The length of the LPS O-chain is known to be regulated by Wzz (formerly Cld or Rol) 33,60 and the LPS profiles of several bacteria lacking this protein have been shown to be a ladder of different molecular weight bands. Ccan_15540, located immediately downstream of Ccan_15550 (wza) (Fig. 4c) is a wzz homolog and we thus hypothesized that the wza deletion could have a polar effect on wzz. To prove our hypothesis we generated a wzz (Ccan_15540) deletion strain and analyzed by western blot its polysaccharide structures.
The wzz mutant displayed the same phenotype as the wza one, i.e. a ladder pattern of bands and absence of the capsular polysaccharide ( Supplementary   Fig. S3). The lack of the CPS in the wzz mutant was somehow unexpected but it could be explained either by exhaustion of O-chain repeating units that are linked to lipid A generating a ladder pattern of LPS, or either by a direct involvement of Wzz in the CPS assembly. In order to understand the role of Wzz, we generated a double wzz and waaL mutant strain and analyzed its polysaccharide structures. As shown in Supplementary Fig. S3, the double mutant lacked both CPS and the laddering of LPS thus indicating that it is not the formation of an LPS ladder that prevents capsule formation but that, in C. canimorsus, Wzz could have a direct role in CPS assembly, i.e. controlling the CPS length. This is also supported by the absence of a Wzc homolog in Cc5 genome and by the genetic organization of the wza and wzz genes encoded in one operon (Fig. 4d). In addition, in the wzz mutant, the intensity of LOS bands C and C # was stronger than the one of the LPS ladder ( Supplementary   Fig. S3) suggesting that formation of normal LOS is independent from Wzz but could be controlled by another, yet unidentified, ruler-like protein.