Reactions of SleC, Its Structure and Inhibition in Mitigation of Spore Germination in Clostridioides difficile.

Abstract

Spore germination in Clostridioides difficile is initiated by a cascade of activities of several proteins that culminates in the activation of SleC, a cell-wall-processing enzyme. We report herein the details of the enzymatic activities of SleC by the use of synthetic peptidoglycan fragments and of spore sacculi. The reactions include the formation of 1,6-anhydromuramate─a hallmark of lytic transglycosylase activity─as well as a muramate hydrolytic product, both of which proceed through the same transient oxocarbenium species. Furthermore, we report the first X-ray structure of zymogenic prepro-SleC at 2.1 Å resolution. Additionally, the structure provides insights into the YabG and CspB cleavage sites necessary for the activation of the zymogen. The active site of SleC presents relevant differences in contrast to SpoIID, a homologous lytic transglycosylase involved in the sporulation Clostridioides species, explaining the ability of SleC to turn over the spore sacculus, a prerequisite for the germination event. A screening of an in-house library of compounds led to the discovery of an oxadiazole that binds to the mature (activated) form of SleC, whereby it shuts down the ability of spores to germinate in the presence of germinants. This is consistent with the SleC activity as an end-point for the germination cascade. The mechanistic knowledge and the inhibitor hold the promise in addressing an unmet medical need in intervention of recurrent infections by C. difficile.