Letter pubs.acs.org/journal/aidcbc
Modulation of the Surface-Layer Protein of Clostridium dif f icile through Cwp84 Inhibition Major D. Gooyit and Kim D. Janda* Departments of Chemistry and Immunology and Microbial Science, The Skaggs Institute for Chemical Biology, and The Worm Institute of Research and Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States S Supporting Information *
ABSTRACT: Cysteine protease Cwp84 is responsible for surface-layer processing in Clostridium dif f icile and was also shown to cleave several human extracellular matrix components in vitro. To enable the facile identification and characterization of Cwp84 inhibitors, we developed a fluorogenic 10-mer peptide based on the enzyme’s natural substrate SlpA that is amenable for use in FRET-based highthroughput screening. The design of substrate-mimetic inhibitors led to epoxysuccinate 8c, which displayed an inactivation efficiency (kinact/KI) of (4.7 ± 0.3) × 104 M−1 min−1. Further evaluation of 8c demonstrated its ability to inhibit fibronectin cleavage and, more importantly, subvert surface-layer biogenesis in C. dif f icile. KEYWORDS: Clostridium dif f icile, S-layer, Cwp84, FRET assay, substrate mimetics
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disease. In a clindamycin-treated hamster model, Cwp84 immunization resulted in the abatement of intestinal colonization and also an increased overall survival rate relative to that of the unimmunized control group.13,14 However, the Cwp84-knockout strain was as competent as the wild-type strain at causing CDI in a hamster model.8 Indeed, the seeming discordance on the putative role of Cwp84 in C. dif f icile pathogenesis warrants further scrutiny. The absence of a screening assay for Cwp84 and the lack of inhibitors have, in part, precluded the investigation of the protease in CDI. Herein, we describe a robust FRET-based, high-throughput screening (HTS) assay for Cwp84 and its utility in the characterization of inhibitors. The administration of a potent Cwp84 inhibitor translated to the abrogation of S-layer processing in live C. dif f icile cells. In an effort to produce an ample amount of the enzyme for use in inhibitor screening, we attempted to purify truncated proform His6-Cwp8430−518 because this was previously reported to be enzymatically active under reducing conditions;15 however, we were unable to isolate His6-Cwp8430−518 from the soluble fraction. As a side note, all recombinant proteins described in this study were derived from strain 630, the most extensively studied strain of C. dif f icile. His6-Cwp8430−518 was mainly expressed as inclusion bodies in E. coli,;however, a major band (∼48 kDa) was evident in the soluble fraction that likely resulted after autocleavage of the His6-propeptide to generate
naerobic, Gram-positive, spore-forming bacterium Clostridium dif f icile was culpable for nearly half a million infections and approximately 29 000 deaths in the United States in 2011.1 C. dif f icile infections (CDI) stem from the disturbance of the normal microbial gut flora due to prolonged antibiotic therapy and the eventual proliferation of toxinproducing pathogenic species causing pseudomembranous colitis. The incidence and mortality rates of CDI have escalated following the emergence of hypervirulent NAP1 epidemic strains, which are characterized by increased toxin production, sporulation, and fluoroquinolone resistance.2,3 Clostridium dif f icile expresses a paracrystalline surface layer (S-layer) in its outer surface that serves to maintain its cell integrity and facilitates host-cell adherence and colonization.4 The S-layer of C. dif f icile is composed of two surface-layer proteins (Slps), the low-molecular-weight (LMW) Slp and high-molecular-weight (HMW) Slp, that originate from a single precursor SlpA.5 Upon expression, SlpA is presumably translocated through the membrane and undergoes posttranslational processing to generate the two fragments that then reassemble to form a stable noncovalently bound complex.6 Earlier studies have established cysteine protease Cwp84 as the enzyme responsible for SlpA cleavage and hence the maturation of the S-layer in C. dif f icile.7,8 The highly conserved Cwp849 has also been shown to cleave extracellular matrix components fibronectin, laminin, and vitronectin,10 implicating its relevance in host tissue degradation and bacterial colonization. Cwp84 was found to be highly immunogenic in patients with CDI11,12 and thus could present a potential vaccine candidate against the © XXXX American Chemical Society
Received: April 12, 2016
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DOI: 10.1021/acsinfecdis.6b00061 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
Letter
expression or after incubation under reducing conditions (Figure 1, lanes 3 and 4). Maturation of the C116A mutant to yield Cwp8492−517-C116A-His6 could, however, be effected in the presence of a catalytic amount of mCwp84 (via transmaturation) or trypsin (Figure 1, lanes 5 and 6, respectively).10,16 Cwp84 displays a high degree of conservation across C. dif f icile strains.9 To identify peptide substrates for evaluating mCwp84 activity, we surveyed the cleavage site sequences of the protease’s known substrate SlpA across clinical strains of C. dif f icile, as previously determined by N-terminal sequencing or homology.17 An analysis of SlpA sequences showed apparent conservation upstream from the cleavage sites with either lysine or tyrosine at the P2 position and serine/glycine (or arginine/ alanine in some strains) at the P1 site.17 We selected four of these strains (namely, 630, CDKK167, Y, and R13541) and synthesized the decapeptides spanning the HMW-LMW cleavage sites (Figure 1B in blue). The decapeptides were prepared using standard Fmoc-based solid-phase peptide synthesis and were then incubated with 30 nM mCwp84 and 2 mM DTT. An analysis of the reaction mixtures by LC/MS confirmed the cleavage sites of Cwp84 indicated in Figure 1B (data not shown). Of the four decapeptides, Ac-FSTYRATNYN-NH2 (based on the SlpA sequence from C. dif f icile strain CDKK167) proved to be the best synthetic substrate, which was completely cleaved after overnight incubation at 37 °C, as assessed by LC/MS (Figure S2). We consequently synthesized fluorogenic substrates by incorporation of the Abz/Tyr(NO2) FRET pair with varying lengths of the oligopeptide (Figure 1C). Abz-GFSTYRASTNY(NO2)-NH2 was most efficiently cleaved, with
