SummaryClostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infection (CDI). Here we carried out CRISPR/Cas9-mediated genome-wide screens and identified the members of the Wnt receptor Frizzled (FZDs) family as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity toward FZD1, 2, and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signaling. FZD1/2/7 triple-knockout (KO) cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7 KO mice, combined with knock-down of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7 KO mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

The large clostridial cytotoxins are a family of structurally and functionally related exotoxins from Clostridium difficile (toxins A and B), C. sordellii (lethal and hemorrhagic toxin) and C. novyi (alpha-toxin). The exotoxins are major pathogenicity factors which in addition to their in vivo effects are cytotoxic to cultured cell lines causing reorganization of the cytoskeleton accompanied by morphological changes. The exotoxins are single-chain protein toxins, which are constructed of three domains: receptor-binding, translocation and catalytic domain. These domains reflect the self-mediated cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and execution of their cytotoxic activity by an inherent enzyme activity. Enzymatically, the toxins catalyze the transfer of a glucosyl moiety from UDP-glucose to the intracellular target proteins which are the Rho and Ras GTPases. The covalent attachment of the glucose moiety to a conserved threonine within the effector region of the GTPases renders the Rho-GTPases functionally inactive. Whereas the molecular mode of cytotoxic effects is fully understood, the mechanisms leading to inflammatory processes in the context of disease (e.g., antibiotic-associated pseudomembranous colitis caused by Clostridium difficile) are less clear.

SummaryCell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac – although not essential – can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration.

Spinal muscular atrophy (SMA), a frequent neurodegenerative disease, is caused by reduced levels of functional survival of motoneuron (SMN) protein. SMN is involved in multiple pathways, including RNA metabolism and splicing as well as motoneuron development and function. Here we provide evidence for a major contribution of the Rho-kinase (ROCK) pathway in SMA pathogenesis. Using an in vivo protein interaction system based on SUMOylation of proteins, we found that SMN is directly interacting with profilin2a. Profilin2a binds to a stretch of proline residues in SMN, which is heavily impaired by a novel SMN2 missense mutation (S230L) derived from a SMA patient. In different SMA models, we identified differential phosphorylation of the ROCK-downstream targets cofilin, myosin-light chain phosphatase and profilin2a. We suggest that hyper-phosphorylation of profilin2a is the molecular link between SMN and the ROCK pathway repressing neurite outgrowth in neuronal cells. Finally, we found a neuron-specific increase in the F-/G-actin ratio that further support the role of actin dynamics in SMA pathogenesis.

The pathogenicity of Clostridium difficile is primarily linked to secretion of the intracellular acting toxins A (TcdA) and B (TcdB) which monoglucosylate and thereby inactivate Rho GTPases of host cells. Although the molecular mode of action of TcdA and TcdB is well understood, far less is known about toxin binding and uptake. It is acknowledged that the C-terminally combined repetitive oligopeptides (CROPs) of the toxins function as receptor binding domain. The current study evaluates the role of the CROP domain with respect to functionality of TcdA and TcdB. Therefore, we generated truncated TcdA devoid of the CROPs (TcdA1–1874) and found that this mutant was still cytopathic. However, TcdA1–1874 possesses about 5 to 10-fold less potency towards 3T3 and HT29 cells compared to the full length toxin. Interestingly, CHO-C6 cells even showed almost identical susceptibility towards truncated and full length TcdA concerning Rac1 glucosylation or cell rounding, respectively. FACS and Western blot analyses elucidated these differences and revealed a correlation between CROP-binding to the cell surface and toxin potency. These findings refute the accepted opinion of solely CROP- mediated toxin internalization. Competition experiments demonstrated that presence neither of TcdA CROPs nor of full length TcdA reduced binding of truncated TcdA1–1874 to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated endocytotic pathways. The second assumption was substantiated by TER measurements showing that basolaterally applied TcdA1–1874 exhibits considerably higher cytotoxic potency than apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.