Heptylmannose-functionalized cellulose for the binding and specific detection of pathogenic E. coli

International audience; We developed a chemical method to covalently functionalize cellulose nanofibers and cellulose paper with mannoside ligands displaying a strong affinity for the FimH adhesin from pathogenic E. coli strains. Mannose-grafted cellulose proved efficient to selectively bind FimH lectin and discriminate pathogenic E. coli strains from non-pathogenic ones. These modified papers are valuable tools for diagnosing infections promoted by E.coli, such as cystitis or inflammatory bowel diseases, and the concept may be applicable to other life-threatening pathogens. The worldwide spread of antibiotic resistances raises serious health problems, and has driven the identification of new virulence factors and development of alternative antibacterial therapeutics. Mannose-binding FimH adhesin, expressed by Escherichia coli strains has been extensively studied as a target for disrupting bacterial attachment to the host cells. 1 Impressive results were obtained in the context of urinary tract infections (UTI), a prevalent infection type generally mediated by the attachment of uropathogenic E. coli strains (UPEC) to the highly mannosylated uroplakin transmembrane protein of urothelial lining cells. FimH antagonists orally administered to in vivo UTI mouse models, were shown to decrease the E.coli load in the bladder by several orders of magnitude, 2-4 making them competitive with conventional antibiotic treatment. 5,6 This concept was more recently extended to Crohn's disease (CD), an inflammatory disorder of the intestine where an altered gut microbiota, particularly the presence of adherent-invasive E. coli strains (AIEC), is suspected to play a key role in the pathogenesis. 7 Synthetic derivatives of heptylmannoside (HMan), a nanomolar FimH antagonist, 8 were shown to lower the AIEC bacterial level, signs of colitis and gut inflammation when administered per os (10 mg/kg) in CD mouse models. 9,10 Sensitive and rapid diagnostic systems are essential to evaluate the presence of E. coli expressing FimH adhesin in gut microenvironments in order to properly stratify patients before treatment. While the high prevalence of UPEC in the normally sterile urinary tract environment facilitates diagnosis, the AIEC niche is more complex, located at the ileal mucosa in 21 to 63% of CD patients, 11,12 within an ecological community of hundreds of symbiotic microorganisms. Furthermore, no specific biomarkers are currently effective at distinguishing AIEC from other commensal E. coli of the gut microbiota. Previously, it has been shown that AIEC pathobiont possesses specific allelic variants in the fimH gene, conferring them a high ability to adhere in vitro and to colonize the gut of CEABAC10 mice. 13 Establishing an approach to discriminate the strong mannose-binders from other bacteria would therefore be of tremendous importance for efficient diagnosis. In this work, we developed a heterogeneous support to specifically trap and accumulate pathogenic E.coli from biological fluids. Heptyl-α-D-mannoside (HMan) was grafted by click chemistry techniques onto cellulose nanofibers (CN) and cellulose paper (CP). HMan was previously identified as a potent binder of the isolated FimH lectin domain. 8 It should be noted that the lectin domain represents the high-affinity state of FimH under mechanical force and that full-length FimH display a lower affinity for mannosides. 14,15 Covalently functionalized CN or CP were characterized by Fourier transform spectroscopy (FTIR), elemental analysis, X-ray photo-electron spectrometry (XPS), and scanning electron microscopy (SEM). HMan-CN was first compared in vitro against CN grafted with lower FimH affinity ligands i.e. Man-CN lacking the hydrophobic heptyl chain, and HGlc-CN an analog with a glucose sugar that is not recognized by FimH lectin (Scheme 1). In addition, we switched the anomeric oxygen atom to a sulfur and synthesized HSMan-CN to prevent potential sugar hydrolysis from the surface by mannosidases. The modified CN were first assessed for their faculty to bind FimH and to prevent AIEC adhesion to intestinal cells. HMan-CN was then orally administered to the CEABAC10 mouse model of CD to assess its capacity to decrease AIEC in the feces of AIEC-infected mice and to act as a potential CD therapeutic. HM was next coated on CP and the capacity of the HMan-CP to selectively catch AIEC in solution was analyzed. Scheme 1. Chemical synthesis of the sugar-coated cellulose fibers.