Jacobs, M. R. | bioRxiv Science

bioRxiv2024

Clonal Parabacteroides from Gut Microfistulous Tracts as Transmissible Cytotoxic Succinate-Commensal Model of Crohn's Disease Complications

Crohns disease (CD) has been traditionally viewed as a chronic inflammatory disease that cause gut wall thickening and complications, including fistulas, by mechanisms not understood. By focusing on Parabacteroides distasonis (presumed modern succinate-producing commensal probiotic), recovered from intestinal microfistulous tracts (cavernous fistulous micropathologies CavFT proposed as intermediate between mucosal fissures and fistulas) in two patients that required surgery to remove CD-damaged ilea, we demonstrate that such isolates exert pathogenic/pathobiont roles in mouse models of CD. Our isolates are clonally-related; potentially emerging as transmissible in the community and mice; proinflammatory and adapted to the ileum of germ-free mice prone to CD-like ileitis (SAMP1/YitFc) but not healthy mice (C57BL/6J), and cytotoxic/ATP-depleting to HoxB8-immortalized bone marrow derived myeloid cells from SAMP1/YitFc mice when concurrently exposed to succinate and extracts from CavFT-derived E. coli, but not to cells from healthy mice. With unique genomic features supporting recent genetic exchange with Bacteroides fragilis-BGF539, evidence of international presence in primarily human metagenome databases, these CavFT Pdis isolates could represent to a new opportunistic Parabacteroides species, or subspecies ( cavitamuralis) adapted to microfistulous niches in CD.

genomics↗

bioRxiv2023

Uridine Bisphosphonates Differentiate Phosphoglycosyl Transferase Superfamilies

Complex bacterial glycoconjugates are essential for bacterial survival, and drive interactions between pathogens and symbionts, and their human hosts. Glycoconjugate biosynthesis is initiated at the membrane interface by phosphoglycosyl transferases (PGTs), which catalyze the transfer of a phosphosugar from a soluble uridine diphospho-sugar (UDP-sugar) substrate to a membrane-bound polyprenol-phosphate (Pren-P). Two distinct superfamilies of PGT enzymes, denoted as polytopic and monotopic, carry out this reaction but show striking differences in structure and mechanism. With the goal of creating non-hydrolyzable mimics (UBP-sugars) of the UDP-sugar substrates as chemical probes to interrogate critical aspects of these essential enzymes, we designed and synthesized a series of uridine bisphosphonates (UBPs), wherein the diphosphate bridging oxygen of the UDP and UDP-sugar is replaced by a substituted methylene group (CXY; X/Y = F/F, Cl/Cl, (S)-H/F, (R)-H/F, H/H, CH3/CH3). These compounds, which incorporated as the conjugating sugar an N-acetylglucosamine (GlcNAc) substituent at the {beta}-phosphonate, were evaluated as inhibitors of a representative polytopic PGT (WecA from Thermotoga maritima) and a monotopic PGT (PglC from Campylobacter jejuni). Although CHF-BP most closely mimics pyrophosphate with respect to its acid/base properties, the less basic CF2-BP conjugate most strongly inhibited PglC, whereas the more basic CH2-BP analogue was the strongest inhibitor of WecA. These surprising differences indicate different modes of ligand binding for the different PGT superfamilies implicating a modified P-O- interaction with the structural Mg2+, consistent with their catalytic divergence. Furthermore, at least for the monoPGT superfamily example, this was not the sole determinant of ligand binding: the two diastereomeric CHF-BP conjugates, which feature a chiral center at the P-CHF-P{beta} carbon, exhibited strikingly different binding affinities and the inclusion of GlcNAc with the native -anomer configuration significantly improved binding affinity. UBP-sugars are a valuable tool for elucidating the structures and mechanisms of the distinct PGT superfamilies and offer a promising scaffold to develop novel antibiotic agents for the exclusively prokaryotic monoPGT superfamily. TABLE OF CONTENTS GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/558431v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@3479edorg.highwire.dtl.DTLVardef@130f028org.highwire.dtl.DTLVardef@1e6d4c1org.highwire.dtl.DTLVardef@199f3e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗