Identification of ClpP Dual Isoform Disruption as an Antisporulation Strategy for Clostridioides difficile

Abstract

The Gram-positive bacterium Clostridioides difficile is a primary cause of hospital-acquired diarrhea, threatening both immunocompromised and healthy individuals. An important aspect of defining mechanisms that drive C. difficile persistence and virulence relies on developing a more complete understanding of sporulation. C. difficile sporulation is the single determinant of transmission and complicates treatment and prevention due to the chemical and physical resilience of spores. By extension, the identification of druggable targets that significantly attenuate sporulation would have a significant impact on thwarting C. difficile infection. Using a new CRISPR-Cas9 nickase genome editing methodology, stop codons were inserted early in the coding sequence for clpP1 and clpP2 to generate C. difficile mutants that no longer produced the corresponding isoforms of caseinolytic protease P (ClpP). The data show that genetic ablation of ClpP isoforms leads to altered sporulation phenotypes with the clpP1/clpP2 double mutant exhibiting asporogenic behavior. A small screen of known ClpP inhibitors in a fluorescence-based biochemical assay identified bortezomib as an inhibitor of C. difficile ClpP that produces dose-dependent inhibition of purified ClpP. Incubation of C. difficile cultures in the presence of bortezomib reveals anti-sporulation effects approaching that observed in the clpP1/clpP2 double mutant. This work identifies ClpP as a key contributor to C. difficile sporulation and provides compelling support for the pursuit of small molecule ClpP inhibitors as C. difficile anti-sporulating agents. IMPORTANCE Due to diverse roles of ClpP and the reliance of pathogens upon this system for infection, it has emerged as a target for antimicrobial development. Biology regulated by ClpP is organism-dependent and has not been defined in C. difficile . This work identifies ClpP as a key contributor to C. difficile sporulation and provides compelling support for the pursuit of small molecule ClpP inhibitors as anti-sporulating agents. The identification of new approaches and/or drug targets that reduce C. difficile sporulation would be transformative and are expected to find high utility in prophylaxis, transmission attenuation, and relapse prevention. Discovery of the ClpP system as a major driver to sporulation also provides a new avenue of inquiry for advancing the understanding of sporulation.

Non-Toxin-Based Clostridioides difficile Vaccination Approaches

“…Another possible approach is to aim for proteins that are involved in the sporulation process. Bishop et al, showed that a genetic ablation in clpP1/clpP2 genes coding caseinolytic protease P (ClpP) impaired the sporulation process [ 24 ]. SpoA is a key regulator of sporulation as its mutation leads to impaired CD growth and reduced spore production [ 25 ].…”

Role and Regulation of Clp Proteases: A Target against Gram-Positive Bacteria

“…Another spore-forming pathogen is C. difficile, where it was shown that the 630 strain in sporulation process expressed only the ClpP1 peptidase and ClpX and ClpC chaperone. However, recent studies in the hypervirulent strain R20291 described that clpP1 and clpP2 participate directly in sporulation, considering that an absence of each ClpP isoform leads to altered sporulation phenotypes and the clpP1/clpP2 double mutant exhibiting asporogenic behavior [86] (Table 1).…”

Cellular functions of the ClpP protease impacting bacterial virulence

“…Moreover, while no regulatory substrates were identified, ClpP was found to be required for sporulation in C. difficile ( Bishop et al, 2022 ). The virulence and resilience of this pathogen to environmental stresses is critically dependent on spore formation ( Shen, 2020 ).…”

“…The virulence and resilience of this pathogen to environmental stresses is critically dependent on spore formation ( Shen, 2020 ). Spores formed from C. difficile clpP1 and clpP2 null mutants were found to be generally heat labile, morphologically abnormal, and they germinated significantly less than their wildtype counterpart ( Bishop et al, 2022 ). Therefore, ClpP appears to be involved in regulating the transition between different life stages of several pathogens to modulate their virulence.…”