Biofilms are communities of self-enmeshed bacteria in a matrix of exopolysaccharides. The widely distributed human pathogen and commensal Escherichia coli produces a biofilm matrix composed of phosphoethanolamine (pEtN)-modified cellulose and amyloid protein fibers, termed curli. The addition of pEtN to the cellulose exopolysaccharide is accomplished by the action of the pEtN transferase, BcsG, and is essential for the overall integrity of the biofilm. Here, using the synthetic co-substrates p-nitrophenyl phosphoethanolamine and β-D-cellopentaose, we demonstrate using an in vitro pEtN transferase assay that full activity of the pEtN transferase domain of BcsG from E. coli (EcBcsG ΔN ) requires Zn 2+ binding, a catalytic nucleophile/acid-base arrangement (Ser 278 /Cys 243 /His 396 ), disulfide bond formation, and other newly uncovered essential residues. We further confirm that EcBcsG ΔN catalysis proceeds by a ping-pong bisubstrate−biproduct reaction mechanism and displays inefficient kinetic behavior (k cat /K M = 1.81 × 10 −4 ± 2.81 × 10 −5 M −1 s −1 ), which is typical of exopolysaccharide-modifying enzymes in bacteria. Thus, the results presented, especially with respect to donor binding (as reflected by K M ), have importantly broadened our understanding of the substrate profile and catalytic mechanism of this class of enzymes, which may aid in the development of inhibitors targeting BcsG or other characterized members of the pEtN transferase family, including the intrinsic and mobile colistin resistance factors.

The genital epithelial barrier is a crucial rst line of defence against HIV, and epithelial disruption may enhance HIV susceptibility. Assessment of genital epithelial integrity require biopsies, but their collection is not practical in many research settings. A validated biomarker of genital epithelial barrier integrity would therefore be useful. E-cadherin, an adhesion component of the cell-cell junction in epithelial tissues, may be released as soluble E-cadherin (sE-cad) upon epithelial trauma, and so we evaluated sEcad as a marker of genital epithelial disruption. First, using an in vitro monolayer of immortalised endocervical epithelial cells, we demonstrate that sE-cad, IL-1β, and IL-1α levels are immediately increased after physical disruption, followed by a delayed increase in IL-6 levels compared to undisrupted controls. In vivo studies con rmed that sE-cad levels in cervicovaginal secretions were signi cantly elevated 6 hours after endocervical cytobrush sampling. Furthermore, the level of sE-cad in coronal sulcus swabs from Ugandan men was inversely correlated with the amount of membrane-bound Ecadherin within the overlying foreskin tissues. Our results validate the use of soluble E-cadherin as a marker of epithelial disruption and demonstrate that the processes of physical disruption and in ammation in the genital tract are strongly intertwined.

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