Structural and functional diversity of metalloproteinases encoded by the Bacteroides fragilis pathogenicity island

Abstract

Summary Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal B. fragilis strains. The island encodes metalloproteinase II (MPII), a potential virulence protein, and one of three homologous fragilysin isozymes (FRA; also termed B. fragilis toxin or BFT). Here, we report biochemical data on the structural-functional characteristics of the B. fragilis pathogenicity island proteases by reporting the crystal structure of MPII at 2.13 Å resolution combined with detailed characterization of the cleavage preferences of MPII and FRA3 (as a representative of the FRA isoforms) identified using a high-throughput peptide cleavage assay with 18,583 substrate peptides. We suggest that the evolution of the MPII catalytic domain can be traced to human and archaebacterial proteinases, while the prodomain fold is a feature specific to MPII and FRA. We conclude that the catalytic domain of both MPII and FRA3 evolved differently relative to the prodomain, and that the prodomain evolved specifically to fit the B. fragilis pathogenicity. Overall, our data provide insights into the evolution of cleavage specificity and activation mechanisms in the virulent metalloproteinases.

High-Throughput Multiplexed Peptide-Centric Profiling Illustrates Both Substrate Cleavage Redundancy and Specificity in the MMP Family

“…This technology includes cleavage of a large number of peptide-cDNA fusions designed in silico and then synthesized in vitro . The technology has been tested and validated by determining cleavage preferences of several viral, bacterial and human proteinases (Kozlov, et al, 2012; Shiryaev, et al, 2014; Shiryaev, et al, 2013; Shiryaev, et al, 2013; Shiryaev, et al, 2012). …”

“…To determine cleavage preferences of MMPs, we employed a high throughput multiplexed peptide-centric profiling technology that we developed (Kozlov, et al, 2012; Shiryaev, et al, 2014; Shiryaev, et al, 2013; Shiryaev, et al, 2013). In this study, we treated a pool containing 18,583 peptide-cDNA fusions with 18 MMPs (one proteinase per reaction).…”

Randomized controlled trial on the impact of early-life intervention with bifidobacteria on the healthy infant fecal microbiota and metabolome

“…More specifically, we demonstrated that the supplementation of infant formula with bifidobacteria inversely correlated with B. fragilis colonization throughout the first year of life, showing similarities to the breastfed gut ecosystem, while placebo-fed infants were more often colonized by this species. B. fragilis contributes to the development and maturation of the infant immune system (47), but it also encodes virulent metalloproteases and other pathogenic features, making it one of the most popular anaerobic pathogens of the human gut (48). Moreover, one recent study showed that Bacteroides species are less abundant in Russian infants but are dominant in Finnish and Estonian infant cohorts.…”

Distinct Interactions with Cellular E-Cadherin of the Two Virulent Metalloproteinases Encoded by a Bacteroides fragilis Pathogenicity Island

“…FRAs exist in three homologous isoforms (FRA1, 2 and 3) with sequence identities of over 95%, while the sequence identity between FRAs and MPII is ∼25%. Both FRA and MPII are secretory metalloproteinases with a zinc-binding HEXXHXXGXXH motif and a characteristic Met-turn [9] – [11] in their catalytic domain that has a classic “metzincin” fold typical of eukaryotic matrix metalloproteases. The crystal structure of FRA3 was reported earlier [10] .…”