Abstract
The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b1–3) likely to control protein–protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC_P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b3 and the catalytic NLPC_P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.
Highlights
The cell wall (CW) of bacteria is an intricate mesh of macromolecules composed of peptidoglycan (PG), a complex polymer formed by linear glycan chains cross-linked by peptide stems
Three types of cell wall hydrolases (CWHs) exist, each displaying a specificity towards PG: (i) cell wall amidase (CWA) catalyzes the hydrolysis of the amide bond between N-acetylmuramic acid (NAM) and L-Ala at the N-terminal of the stem peptide; (ii) cell wall glycosidase (CWG) cleaves the glycosidic linkages; and (iii) cell wall peptidase (CWP) hydrolyses the amide bonds with the PG chains [8]
We show that CwpFM is an exported papain-like endopeptidase with, at the N-terminal end, three successive bacterial Src Homology 3 domains (SH3b1-3 ), and at the C-terminal end, a catalytic NLPC_P60 domain
Summary
The cell wall (CW) of bacteria is an intricate mesh of macromolecules composed of peptidoglycan (PG), a complex polymer formed by linear glycan chains cross-linked by peptide stems. The resulting cell wall fragments are recycled as signaling molecules to trigger bacterial communication, immune response or antibiotics resistance [5,7] They prime the insertion of supramolecular structures like secretion, flagella or pili systems [8]. Recent structural data evidence that CWHs show modularity, with a catalytic domain combined to one or several CW binding domains (CBDs) located at the N- or C-terminal ends This modular organization associated with CBD repeats is highly suspected to engage these hydrolases as a platform responsible for CW integrity. Due to their inherent flexibility and lack of structural characterization, the linker regions that connect the binding domains to the catalytic one have received little attention until now. Interest is increasing as they are shown to play a role in domains orientation or swapping, and dynamics that result in substrate specificity and affinity [1,2,3]
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