Clostridium difficile ClpP Homologues are Capable of Uncoupled Activity and Exhibit Different Levels of Susceptibility to Acyldepsipeptide Modulation

Abstract

Caseinolytic protease P (ClpP) has emerged as a promising new target for antibacterial development. While ClpPs from single isoform expressing bacteria have been studied in detail, the function and regulation of systems with more than one ClpP homologue are still poorly understood. Herein, we present fundamental studies toward understanding the ClpP system in C. difficile, an anaerobic spore-forming pathogen that contains two chromosomally distant isoforms of ClpP. Examination of proteomic and genomic data suggest that ClpP1 is the primary isoform responsible for normal growth and virulence, but little is known about the function of ClpP2 or the context required for the formation of functional proteases. For the first time in a pathogenic bacterium, we demonstrate that both isoforms are capable of forming operative proteases. Interestingly, ClpP1 is the only homologue that possesses characteristic response to small molecule acyldepsipeptide activation. On the contrary, both ClpP1 and ClpP2 respond to cochaperone activation to degrade an ssrA-tagged substrate. These observations indicate that ClpP2 is less susceptible to acyldepsipeptide activation but retains the ability to interact with a known cochaperone. Homology models reveal no obvious characteristics that would allow one to predict less efficient acyldepsipeptide binding. The reported findings establish the uniqueness of the ClpP system in C. difficile, open new avenues of inquiry, and highlight the importance of more detailed structural, genetic, and biological characterization of the ClpP system in C. difficile.

Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX

“…11,32−34 In a recent study on Clostridium difficile , both ClpP isoforms are capable of forming functional peptidase independently. 42 Our results of ClpP isoform peptidase activity are in agreement with M. tuberculosis, where both isoforms are also inactive towards the di-peptide substrate. 36 It is also possible that Leptospira pure rClpP isoforms may show activity independently on other types of small peptides that are yet to be evaluated.…”

The functional ClpXP protease of Chlamydia trachomatis requires distinct clpP genes from separate genetic loci

“…Cross-contamination with ecClp proteins in particular may misguide experiments using ClpP proteins with significantly less activity than ecClpP, such as ctClpP1 or ctClpP2. As previously reported for the expression of C. difficile ClpP proteins using E. coli BL21(DE3), endogenous ecClpP was detected with a relative abundance of 10-30% of the total purified ClpP proteins 22 . Also in our hands, when we attempted to express ctClpP1 and ctClpP2 in E. coli BL21 (DE3), contamination with ecClp proteins was observed (data not shown).…”

“…On the genomic level, however, mtclpP genes differ from Chlamydia as they are organized in a single operon and are co-transcribed. In contrast, the genes encoding ctClpP1 and ctClpP2 are located on different operons at distant loci of the chromosome, similar to the clpP1 and clpP2 genes from Pseudomonas aeruginosa, C. difficile or L. monocytogenes [21][22][23] . In addition, ctClpP1 and ctClpP2 share rather low amino acid sequence identity, even when compared to homologous ClpP proteins from a variety of unrelated, multi-ClpP species.…”

“…In M. tuberculosis, mtClpP1 and mtClpP2, encoded by a single bicistronic operon, may either form inactive homo-tetradecamers or functionally active hetero-tetradecamers using the non-natural activator peptide Z-Leu-Leu 20 (regarded as active*). ClpP homologs from either C. difficile, L. monocytogenes, or P. aeruginosa, which are encoded in distinct operons, may form either functionally active homo-tetradecamers as well as hetero-tetradecamers [21][22][23] . Thus, in each of these situations, an active ClpP complex can result from the same operon or even a single ClpP homolog.…”

“…We here report on both the functioning of the Clp protease of Chlamydia trachomatis, an obligate intracellular pathogen and the causative agent of widespread sexually transmitted diseases and trachoma-associated infectious blindness in humans 18,19 , and the deregulation of the chlamydial ClpXP machinery by antibiotic action. Like other pathogenic bacteria, including listeria, clostridia, or pseudomonads 11,[20][21][22][23] , the genome of C. trachomatis encodes two clpP homologs (clpP1 and clpP2) 24 , that are organized in distinct operons. Also, C. trachomatis encodes the Clp-ATPases ClpX, ClpC, and the protein-disaggregating chaperone ClpB, the latter, however, is not commonly assumed to interact with ClpP 25 .…”