The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

Abstract

To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.Enterotoxigenic Bacteroides fragilis (ETBF) is strongly linked epidemiologically to diarrheal disease in livestock, young children, and adults (22,23,27,28,30,35,40). The only recognized virulence factor of ETBF is a secreted 20-kDa zinc-dependent metalloprotease termed B. fragilis toxin (BFT) (19). BFT causes fluid accumulation in ligated intestinal loops of lambs, rats, rabbits, and calves (23,24,31). In vitro, BFT alters the morphology of certain human intestinal carcinoma cell lines, particularly cell line HT29/C1 (3,15,31,35). Subconfluent HT29/C1 cells treated with BFT develop striking changes in morphology, including loss of cell-to-cell attachments, rounding, swelling, and, in some cases, pyknosis. The mechanism of action and morphological changes stimulated by BFT are mediated, in part, by cleavage of the zonula adherens protein, E-cadherin (38). Recently, ETBF strains have also been associated with active inflammatory bowel disease and colorectal cancer (1,25,33). We and other workers (13,29,39) have shown that BFT stimulates interleukin-8 (IL-8) secretion by intestinal cells (HT29, T84, and Caco-2 cells) in vitro.Three highly related isotypes of BFT have been identified (termed BFT-1, BFT-2, and BFT-3) (4,8,12,37). All BFTs appear to be structurally similar. BFT is synthesized as a 44-kDa precursor (397 amino acid residues) containing the following three consecutive peptide domains: (i) a presignal sequence (18 amino acid residues), (ii) a propeptide (193 amino acid residues), and (iii) a mature protein (186 amino acid residues) (8, 14). The 44-kDa precursor protein is processed to a 20-kDa mature BFT that is secreted into the culture supernatant.Based on sequence analysis, BFT is predicted to be a member of the metzincin superfamily of zinc-dependent metalloprotease enzymes (19). Members of this superfamily contain an elongated zinc-binding metalloprotease motif (HEXXHX XGXXH) and present a perfectly superimposable methionine residue close to the zinc-b...

Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface

“…In turn, there is only a low, 25%, identity of the peptide sequence of MPII with that of FRAs (21,26,30). Whereas FRAs have been the focus of multiple studies by others (13,14,19,21,30,32,(63)(64)(65)(66)(67)(68)(69), the characteristics of MPII are completely unknown.…”

Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and γ-secretase-dependent E-cadherin cleavage

“…Further dissection of the initial steps in BFT interaction with the cell and the role(s) of the BFT protease domain and/or cellular proteases in the mechanism of action of BFT has been limited because we have not identified a protease inhibitor that specifically inhibits eukaryotic proteases, but not BFT. Similarly, mutational analyses of BFT suggest it is a highly conserved protein without, as yet, a defined binding domain distinct from its protease domain (Franco et al, 2005;Sears et al, 2006).…”

EnterotoxigenicBacteroides fragilis: a Rogue among Symbiotes

“…Although BFT has been reported to be autoproteolytic (61,116), BFT metalloprotease point mutations do not alter the intracellular processing and secretion of BFT by B. fragilis, suggesting that other intracellular B. fragilis proteases process the holotoxin to mature BFT (28). Additional mutational analysis of the C-terminal region of BFT indicated that this region is intolerant to modest amino acid deletions, suggesting that this region is also important for BFT activity (104). Truncation mutations removing only two C-terminal amino acids reduced BFT biologic activity, and removal of eight (or more) amino acids obliterated it.…”

“…Truncation mutations removing only two C-terminal amino acids reduced BFT biologic activity, and removal of eight (or more) amino acids obliterated it. BFT mutants lacking eight or more C-terminal amino acids were expressed similar to wild-type toxin, but the mutant BFTs were unstable (104).…”