Abstract
Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.
Highlights
Clostridium difficile, a Gram-positive spore-forming anaerobe, is the leading cause of antibiotic-associated diarrhea in hospitalized patients [1]
Among 33 NAP1 isolates analyzed, we found a strain whose supernatant induced a cytopathic effect (CPE) different from the classic arborizing CPE observed for the other NAP1 strains
Since TcdBNAP1V clearly presents different phenotypic behavior than TcdBNAP1, we focused on differences at the sequence level
Summary
Clostridium difficile, a Gram-positive spore-forming anaerobe, is the leading cause of antibiotic-associated diarrhea in hospitalized patients [1]. In cell cultures treated with C. difficile toxins, monoglucosylation of RhoA, Rac, and Cdc disrupts the actin cytoskeleton and causes an arborizing cytopathic effect (CPE) [5]. C. difficile strains producing a variant TcdB have been previously reported, mainly in TcdA-negative strains [13,14,15] In cultured cells, these TcdB variants induce a CPE characterized by the. Collapse of the actin cytoskeleton with complete rounding of the cell body and detachment from the surface in contrast to the classic arborizing effect [13] This variant CPE is due to a different pattern of glucosylated GTPases since classic TcdB modifies RhoA, Rac, and Cdc whereas variant TcdB targets Rac, Cdc, Rap, Ral, and R-Ras [5, 13, 14, 16]. Variations based on the PaLoc sequence have classified these groups of strains in separate toxinotypes [17]
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