The Caseinolytic Protease P System in Clostridium difficile

Abstract

Clostridium difficile is a Gram-positive, spore-forming, toxin producing pathogen that has rapidly gained resistance to several broad-spectrum antibiotics. C. difficile infection (CDI) is a leading cause of hospital-acquired illness and presents a unique challenge to therapeutic development, as it is both caused by and clinically managed with antibiotics that indiscriminately kill bacteria present in gut microbiota. As such, identifying new drug targets that affect novel pathways is urgently required in order to combat the rise of CDI. To address this, we focus on elucidating the caseinolytic protease P (ClpP) system in C. difficile, which has emerged as a promising new target for antibacterial development. Herein, we describe the biochemical characterization and phenotypic response to loss of ClpP1 and ClpP2 in vitro to establish: 1) proteolytic activity and susceptibility to chemo-activation differ between ClpP1 and ClpP2 and that these two isoforms are capable of functioning in an uncoupled fashion. 2) ClpP1 and ClpP2 together are required for sporulation and thus are viable drug targets. We conclude this work with our initial discovery of a novel ClpP activator, Sclerotiamde, and our work towards the optimization of another class of ClpP activators. The work herein describes the uniqueness of the ClpP system in C. difficile, opens new avenues of inquiry, and highlights added impact of additional detailed structural, genetic, and biological characterization of this system.