Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog Theodoros Goulas, Joan L. Arolas, and F. Xavier Gomis-Rüth1 Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas (CSIC); Barcelona Science Park, Helix Building, c/Baldiri Reixac 15-21, E-08028 Barcelona, Spain Edited* by Robert Huber, Max Planck Institute for Biochemistry, Planegg-Martinsried, Germany, and approved December 6, 2010 (received for review August 18, 2010) Enterotoxigenic Bacteroides fragilis is the most frequent disease- segments, and a ∼190-residue catalytic domain (CD). The latter causing anaerobe in the intestinal tract of humans and livestock encompasses two sequence elements that ascribe it to the metzin- and its specific virulence factor is fragilysin, also known as B. fragilis cin clan of MPs: (i) an extended zinc-binding consensus sequence toxin. This is a 21-kDa zinc-dependent metallopeptidase existing in (ZBCS), HEXXHXXG/NXXH/D, which comprises three histi- three closely related isoforms that hydrolyze E-cadherin and contri- dines that bind the catalytic zinc ion plus the general base/acid bute to secretory diarrhea, and possibly to inflammatory bowel glutamate involved in catalysis; and (ii) a conserved methionine disease and colorectal cancer. Here we studied the function and within a tight 1,4-β-turn, the Met-turn (5–8). However, upstream zymogenic structure of fragilysin-3 and found that its activity is of the ZBCS there is no significant sequence similarity to any repressed by a ∼170-residue prodomain, which is the largest hither- other metzincin, which suggests that fragilysin is a unique metzin- to structurally characterized for a metallopeptidase. This prodomain cin prototype (7). plays a role in both the latency and folding stability of the catalytic The enzyme exists in three closely related isoforms of identical domain and it has no significant sequence similarity to any known length: fragilysin-1, -2, and -3 alias BFT-1, -2, and -3, which dis- protein. The prodomain adopts a novel fold and inhibits the pro- play pairwise sequence identities of 93–96% (9). Analysis of clin- tease domain via an aspartate-switch mechanism. The catalytic ical isolates reveals that the three isoforms are generally present fragilysin-3 moiety is active against several protein substrates and simultaneously (2) and that fragilysin-1 is the most abundant (see its structure reveals a new family prototype within the metzincin table 9 in ref. 2). The three fragilysin isoforms are encoded by a clan of metallopeptidases. It shows high structural similarity despite chromosomal pathogenicity islet that is absent in nonenterotoxi- negligible sequence identity to adamalysins/ADAMs, which have genic strains. In addition to fragilysin, this island contains a sec- only been described in eukaryotes. Because no similar protein has ond gene, mpII, which is countertranscribed and encodes a been found outside enterotoxigenic B. fragilis, our findings sup- potential MP of similar size and moderate sequence identity port that fragilysins derived from a mammalian adamalysin/ADAM (28–30%) to the three fragilysin isoforms. However, its potential xenolog that was co-opted by B. fragilis through a rare case of role in ETBF pathogenesis remains to be established (2). horizontal gene transfer from a eukaryotic cell to a bacterial cell. The only proven substrate for fragilysin-1 in vivo is E-cadherin, Subsequently, this co-opted peptidase was provided with a unique an intercellular adhesion molecule. Shedding of E-cadherin by chaperone and latency maintainer in the time course of evolution to fragilysin-1 led to increased permeability of the epithelium render a robust and dedicated toxin to compromise the intestinal and, ultimately, cell proliferation, which supported a role for this epithelium of mammalian hosts. MP in colorectal carcinoma (10). In vitro, fragilysin-1 was shown to cleave type-IV collagen, gelatin, actin, fibrinogen, myosin, tro- bacterial endotoxin ∣ human pathogen ∣ zymogen activation pomyosin, human complement C3, and α1-proteinase inhibitor (5, 6). The protein is stable at room temperature and below, he gastrointestinal tract is that part of the interface between but it undergoes rapid autodigestion above 37 °C. Fragilysin-3, Tthe organism and its external environment where food diges- alias BFT-3 and BFT-Korea, was shown to cleave E-cadherin tion and nutrient uptake occur. The tract hosts bacteria that are in HT29/C1 cells similarly to isoforms 1 and 2 (9), but no further beneficial for the host by controlling invasion and proliferation of biochemical studies have been reported. pathogens, enhancing the immune system, processing indigestible Orally administered broad-spectrum antibiotics may remove food and providing essential nutrients. The most populated sec- enteropathogens from the gastrointestinal tract but they also af- tion of the tract is the large intestine, which is anaerobic and con- fect the beneficial and commensal flora. In the absence of this tains ten times more bacterial cells than the number of human flora, opportunistic microorganisms may colonize the intestine cells in the entire body (1). However, in certain circumstances, and lead to severe digestion alterations and gastrointestinal dis- this beneficial relationship can be disrupted and pathogenic eases. In addition, ETBF can be resistant to antibiotics such as bacteria can invade and proliferate, causing a number of distur- penicillin, ampicillin, clindamycin, tetracycline, and metronida- bances. Members of the genus Bacteroides comprise the majority zole (2). Accordingly, there is a substantiated need for better of intestinal obligate anaerobes, of which Bacteroides fragilis is most frequently associated with disease. Enterotoxigenic B. fragilis Author contributions: F.X.G.-R. designed research; T.G., J.L.A., and F.X.G.-R. performed (ETBF) strains colonize and affect humans and livestock, and research; F.X.G.-R. contributed new reagents/analytic tools; T.G. and F.X.G.-R. analyzed they have been linked to intraabdominal abscesses, diarrhea, data; and T.G., J.L.A., and F.X.G.-R. wrote the paper. inflammatory bowel disease, anaerobic bacteremia, and colon The authors declare no conflict of interest. cancer (1–3). In addition to the bacterial capsule, which induces *This Direct Submission article had a prearranged editor. abscess formation, the only identified virulence factor for ETBF is Data deposition: The atomic coordinates and structure factors have been deposited in the a 21-kDa zinc-dependent metallopeptidase (MP), termed fragily- Protein Data Bank, www.pdb.org (PDB ID code 3P24). sin alias B. fragilis toxin (BFT) (4–6). It is synthesized as a prepro- 1To whom correspondence should be addressed. E-mail: [email protected]. protein of 397 residues, with an 18-residue signal peptide for This article contains supporting information online at www.pnas.org/lookup/suppl/ secretion, a ∼170-residue prodomain (PD) flanked by flexible doi:10.1073/pnas.1012173108/-/DCSupplemental. 1856–1861 ∣ PNAS ∣ February 1, 2011 ∣ vol. 108 ∣ no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1012173108 Downloaded by guest on October 1, 2021 understanding ETBF targets such as fragilysin and for the design thermal denaturation of the latter (Fig. S2D). Taken together, of highly specific antimicrobials to tackle them, and detailed struc- all these results indicate that directly expressed fragilysin-3 is tural information greatly contributes to these aims. To explore unstable and strongly point to a role for the PD, in addition the mechanisms of fragilysin activation and activity, we examined to latency maintenance, as a chaperone that assists in the folding the proteolytic capacity of fragilysin-3 in vitro and the X-ray crystal and stabilization of CD, as previously reported for other MP structure of its zymogen, profragilysin-3, which provided a high- zymogens (14, 15). In the absence of this chaperone, fragily- resolution scaffold for the design of specific inhibitors. Taken sin-3 CD is not able to fold correctly in the environment provided together with the results of phylogenetic studies, these data by the bacterial expression host. enabled us to propose a mechanism for latency maintenance and activation of this enterotoxigenic MP, as well as a plausible Proteolytic Activity and its Inhibition. Mature wild-type fragilysin-3 hypothesis for its evolutionary origin based on xenology (from cleaved casein, fibrinogen, actin, and fibronectin (Fig. S1 D–G), ξεν´ oς, Greek for “stranger” or “alien”), which is gene homology as well as a casein fluorescein-conjugate, azocollagen, and azoca- that is the result of horizontal gene transfer (HGT) between spe- sein, but not azoalbumin in the conditions assayed. In addition, cies and not of Darwinian evolution. eight standard fluorogenic peptides were analyzed for cleavage, two of which were efficiently cleaved, including a matrix metal- Results and Discussion loproteinase (MMP) probe, NFF3 (16). The rest were cleaved Autolytic vs. Heterolytic Activation. Wild-type profragilysin-3 was moderately or not at all (Fig. S1I). Analysis of protein and pep- recombinantly overexpressed in Escherichia coli and purified to tide cleavage fragments revealed broad substrate specificity for homogeneity (Fig. S1A). Stepwise autolytic processing of profra- fragilysin-3 (see Table S1), and maximal activity was recorded gilysin-3 via various