Substrate cleavage profiling suggests a distinct function of Bacteroides fragilis metalloproteinases (fragilysin and metalloproteinase II) at the microbiome-inflammation-cancer interface

Abstract

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

Keywords: Bacterial Toxins; Bacteroides; Enzymes; Fragilysin; Metalloprotease; Microbial Pathogenesis; Toxins.

FIGURE 1.

Sequence alignment of the fragilysin isoforms (FRA1, FRA2, and FRA3) and MPII encoded by the B. fragilis pathogenicity island. Dots indicate identical residue positions. Asterisks indicate the conserved active site histidine residues.

FIGURE 2.

B. fragilis is predominantly associated with CRC tumors rather than normal tissue. The 501-bp fragment of the B. fragilis 16 S RNA gene was PCR-amplified in the CRC1-8 and XT1–22 tumor and matching normal tissue specimens. Numbers of B. fragilis-positive and -negative samples are shown in the bottom table. The arrows show the samples in which the amplified DNA bands were sequenced to determine their nucleotide sequence and to confirm their identity. The data are statistically significant (p = 0.035) as judged by two-sided Fisher's exact test.

FIGURE 3.

MPII and FRA3 constructs. A, recombinant MPII and FRA3. Left, B. fragilis wild type MPII and FRA3. The arrows indicate the cleavages at the prodomain excision sites in the proform. These cleavages release the prodomain and the mature protease. SP, PRO, and CAT, signal peptide, prodomain, and catalytic domain, respectively. Right, recombinant MPII and FRA3 tagged with the His₆ and FLAG tags. E352A and E349A indicate the catalytically inactive mutants of MPII and FRA3, respectively, in which the catalytically essential Glu residue of the active site was mutated into Ala. B, SDS-gel electrophoresis of the purified constructs. Note that the wild-type MPII is readily self-activated, whereas the wild-type FRA3 is significantly more stable in its proenzyme form during storage. CS, Coomassie staining; WB, Western blotting with an anti-FLAG tag antibody.

FIGURE 4.

Cleavage preferences of MPII and FRA3. A, representative MALDI-TOF MS spectra of the individual cleavage peptides. Top, GNKRRGGTAG was co-incubated with MPII. Middle and bottom, AGLRRAALGG and SGHMHAALTA, respectively, were co-incubated with FRA3. The spectra of the digest, the intact peptide, the enzyme alone, and the buffer alone are shown by red, black, green, and magenta lines, respectively. AU, arbitrary units. B, frequency plot of the cleavage sequences of MPII and FRA3 in an IceLogo format. The height of a character is proportional to the frequency of the amino acid residue at the individual position of the cleaved peptide and is normalized for the amino acid encoded in the entire human genome according to RefSeq. C, MPII cleaves anthrax PA83. MPII and furin were co-incubated with PA83 at the indicated enzyme-substrate molar ratio. The resulting PA63 and PA20 are shown by arrows. The digests were separated by SDS-gel electrophoresis. Where indicated, GM6001 was added to the reactions.

FIGURE 5.

Biochemical characteristics and inhibitors of MPII. The cleavage activity of MPII was measured using 5-FAM-SLGRKIQIQK(QXL520)-NH₂ as a fluorescence-quenched peptide substrate. A–D, the effect of buffer pH, glycerol concentration, salt concentration, and temperature on the MPII cleavage activity, respectively. E, selected inhibitors of MPII; representative dose-response curves. The EC₅₀ values of TIMP-2 and the hydroxamate inhibitors (GM6001 and AG3340) were measured using 5-FAM-SLGRKIQIQK(QXL520)-NH₂ as a fluorescence-quenched peptide substrate.

FIGURE 6.

Structural parameters of FRA1–3 and MPII. A, structural modeling of MPII and FRA3 with peptide substrates. Left, the structure of MPII with the PGRLR↓RSGAA modeled substrate. Right, the structure of FRA3 (PDB entry 3P24) with the PRPLR↓AWGAA modeled substrate. Substrates are shown as sticks. The molecular surface of MPII and FRA3 with the labeled substrate subsites is colored according to the electrostatic potential (red, blue, and white represent negative, positive, and neutral electrostatic potential values, respectively). The active site zinc ion is shown as a gray sphere. B, modeled structures of FRA1 and FRA2 versus FRA3 (PDB entry 3P24). The modeled PRPLR↓AWGAA substrate is shown as green sticks. The structures of the mature FRA1, FRA2, and FRA3 are shown as gray schematics in the background and as transparent electrostatic potential surfaces (red, blue, and white represent negative, positive, and neutral electrostatic potential values, respectively). The arrows point to the residue positions that may affect substrate binding and that are distinct in FRA1 and FRA2 versus FRA3.