Francesco Renzi

The genome and surface proteome of Capnocytophaga canimorsus reveal a key role of glycan foraging systems in host glycoproteins deglycosylation

Capnocytophaga canimorsus are commensal Gram‐negative bacteria from dogs mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria–Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus‐like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.

Oligomeric Coiled-Coil Adhesin YadA Is a Double-Edged Sword

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Suprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt) Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs) upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadAV98D,N99A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions.

New Iron Acquisition System in Bacteroidetes

ABSTRACT Capnocytophaga canimorsus, a dog mouth commensal and a member of the Bacteroidetes phylum, causes rare but often fatal septicemia in humans that have been in contact with a dog. Here, we show that C. canimorsus strains isolated from human infections grow readily in heat-inactivated human serum and that this property depends on a typical polysaccharide utilization locus (PUL), namely, PUL3 in strain Cc5. PUL are a hallmark of Bacteroidetes, and they encode various products, including surface protein complexes that capture and process polysaccharides or glycoproteins. The archetype system is the Bacteroides thetaiotaomicron Sus system, devoted to starch utilization. Unexpectedly, PUL3 conferred the capacity to acquire iron from serotransferrin (STF), and this capacity required each of the seven encoded proteins, indicating that a whole Sus-like machinery is acting as an iron capture system (ICS), a new and unexpected function for Sus-like machinery. No siderophore could be detected in the culture supernatant of C. canimorsus, suggesting that the Sus-like machinery captures iron directly from transferrin, but this could not be formally demonstrated. The seven genes of the ICS were found in the genomes of several opportunistic pathogens from the Capnocytophaga and Prevotella genera, in different isolates of the severe poultry pathogen Riemerella anatipestifer, and in strains of Bacteroides fragilis and Odoribacter splanchnicus isolated from human infections. Thus, this study describes a new type of ICS that evolved in Bacteroidetes from a polysaccharide utilization system and most likely represents an important virulence factor in this group.

Only a subset of C. canimorsus strains is dangerous for humans

Capnocytophaga canimorsus are gram-negative bacteria living as commensals in the mouth of dogs and cats. C. canimorsus cause rare but life-threatening generalized infections in humans that have been in contact with a dog or a cat. Over the last years we collected 105 C. canimorsus strains from different geographical origins and from severe human infections or healthy dogs. All these strains were analyzed by 16S rDNA sequencing and a phylogenetic tree revealed two main groups of bacteria instead of one with no relation to the geographical origin. This branching was confirmed by the whole-genome sequencing of 10 strains, supporting the evidence of a new Capnocytophaga species in dogs. Interestingly, 19 out of 19 C. canimorsus strains isolated from human infections belonged to the same species. Furthermore, most strains from this species could grow in heat-inactivated human serum (HIHS) (40/46 tested), deglycosylate IgM (48/66) and were cytochrome-oxidase positive (60/66) while most strains from the other species could not grow in HIHS (22/23 tested), could not deglycosylate IgM (33/34) and were cytochrome-oxidase negative (33/34). Here, we propose to call Capnocytophaga canis (Latin: dog) the novel, presumably less virulent dog-hosted Capnocytophaga species and to keep the name C. canimorsus for the species including human pathogens.Emerging Microbes & Infections (2015) 4, e48; doi:10.1038/emi.2015.48; published online 19 August 2015

Glycan-Foraging Systems Reveal the Adaptation of Capnocytophaga canimorsus to the Dog Mouth

ABSTRACT Capnocytophaga canimorsus is known to form two kinds of cells on blood agar plates (coccoid and bacillary), evoking phase variation. When grown in coculture with animal cells these bacteria appeared only as bacilli, but in the presence of vancomycin they were round, indicating that coccoid shapes likely result from weakening of the peptidoglycan layer. Polysaccharide utilization locus 5 (PUL5) and sialidase mutant bacteria, unable to retrieve glycans from glycoproteins, grew less than wild-type bacteria and also appeared polymorphic unless GlcNAc was added, suggesting that C. canimorsus is unable to synthesize GlcNAc, an essential component of peptidoglycan. Accordingly, a genome analysis was conducted and revealed that C. canimorsus strain 5 lacks the GlmM and GlmU enzymes, which convert glucosamine into GlcNAc. Expression of the Escherichia coli GlmM together with the acetyltransferase domain of GlmU allowed PUL5 mutant bacteria to grow normally, indicating that C. canimorsus is a natural auxotroph that relies on GlcNAc harvested from the host N-glycoproteins for peptidoglycan synthesis. Mucin, a heavily O-glycosylated protein abundant in saliva, also rescued growth and the shape of PUL5 mutant bacteria. Utilization of mucin was found to depend on Muc, a Sus-like system encoded by PUL9. Contrary to all known PUL-encoded systems, Muc cleaves peptide bonds of mucin rather than glycosidic linkages. Thus, C. canimorsus has adapted to build its peptidoglycan from the glycan-rich dogs mouth glycoproteins. IMPORTANCE Capnocytophaga canimorsus is a bacterium that lives as a commensal in the dog mouth and causes severe infections in humans. In vitro, it forms two kinds of cells (coccoid and bacillary), evoking phase variation. Here, we show that cell rounding likely results from weakening of the peptidoglycan layer due to a shortage of N-acetylglucosamine (GlcNAc). C. canimorsus cannot synthesize GlcNAc because of the lack of key enzymes. In its niche, the dog mouth, C. canimorsus retrieves GlcNAc by foraging glycans from salivary mucin and N-linked glycoproteins through two different apparatuses, Muc and Gpd, both of which are related to the Bacteroides starch utilization system. The Muc system is peculiar in the sense that the enzyme of the complex is a protease and not a glycosylhydrolase, as it cleaves peptide bonds in order to capture glycan chains. This study provides a molecular genetic demonstration for the complex adaptation of C. canimorsus to its ecological niche, the oral cavity of dogs. Capnocytophaga canimorsus is a bacterium that lives as a commensal in the dog mouth and causes severe infections in humans. In vitro, it forms two kinds of cells (coccoid and bacillary), evoking phase variation. Here, we show that cell rounding likely results from weakening of the peptidoglycan layer due to a shortage of N-acetylglucosamine (GlcNAc). C. canimorsus cannot synthesize GlcNAc because of the lack of key enzymes. In its niche, the dog mouth, C. canimorsus retrieves GlcNAc by foraging glycans from salivary mucin and N-linked glycoproteins through two different apparatuses, Muc and Gpd, both of which are related to the Bacteroides starch utilization system. The Muc system is peculiar in the sense that the enzyme of the complex is a protease and not a glycosylhydrolase, as it cleaves peptide bonds in order to capture glycan chains. This study provides a molecular genetic demonstration for the complex adaptation of C. canimorsus to its ecological niche, the oral cavity of dogs.

Modification of the 1-Phosphate Group during Biosynthesis of Capnocytophaga canimorsus Lipid A

ABSTRACT Capnocytophaga canimorsus, a commensal bacterium of dogs mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.

Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria

ABSTRACT Bacteria of the phylum Bacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread in Bacteroidetes. Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogen Capnocytophaga canimorsus corresponding to K-(D/E)2 or Q-A-(D/E)2. We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposed C. canimorsus lipoprotein. Furthermore, we identify a LES in Bacteroides fragilis and Flavobacterium johnsoniae surface lipoproteins that allow C. canimorsus surface protein exposure, thus suggesting that Bacteroidetes share a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane. IMPORTANCE Bacteria of the phylum Bacteroidetes are important human commensals and pathogens. Understanding their biology is therefore a key question for human health. A main feature of these bacteria is the presence of abundant lipoproteins at their surface that play a role in nutrient acquisition. To date, the underlying mechanism of lipoprotein transport is unknown. We show for the first time that Bacteroidetes surface lipoproteins share an N-terminal signal that drives surface localization. The localization and overall negative charge of the lipoprotein export signal (LES) are crucial for its role. Overall, our findings provide the first evidence that Bacteroidetes are endowed with a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane. Bacteria of the phylum Bacteroidetes are important human commensals and pathogens. Understanding their biology is therefore a key question for human health. A main feature of these bacteria is the presence of abundant lipoproteins at their surface that play a role in nutrient acquisition. To date, the underlying mechanism of lipoprotein transport is unknown. We show for the first time that Bacteroidetes surface lipoproteins share an N-terminal signal that drives surface localization. The localization and overall negative charge of the lipoprotein export signal (LES) are crucial for its role. Overall, our findings provide the first evidence that Bacteroidetes are endowed with a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane.

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.

Draft Genome Sequences of Three Capnocytophaga canimorsus Strains Isolated from Healthy Canine Oral Cavities

ABSTRACT Here, we present the draft genome sequences of three strains of Capnocytophaga canimorsus, each isolated from a different dogs mouth. Genome analysis provided evidence that these organisms may belong to a different nonpathogenic subtype of C. canimorsus.

Draft Genome Sequences of Three Capnocytophaga canimorsus Strains Isolated from Septic Patients.

ABSTRACT Capnocytophaga canimorsus is a bacterium from the normal oral flora of dogs and cats that causes rare generalized infections in humans. In an attempt to determine whether infections could be caused by a subset of strains and to identify pathogenicity factors, we sequenced the genomes of three strains isolated from human infections.