RESUMO
E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1â CFU mL-1 of E. coli in water within 6â hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV ) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Polímeros/metabolismo , Tiofenos/metabolismo , Sequência de Aminoácidos , Ânions , Proteínas da Membrana Bacteriana Externa/química , Contagem de Colônia Microbiana , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Peptídeo Hidrolases/química , Espectrometria de Fluorescência , Especificidade por Substrato , Microbiologia da ÁguaRESUMO
Cylindrical poly(styrene sulfonate) brushes were combined with a tetravalent double-DABCO based counterion. With increasing addition of counterion, hydrodynamic radius and radius of gyration decrease and AFMreveals a conformation change from wormlike to curled structures. Rg/Rh changes from 1.3 to 0.9. Above charge stoichiometry, brushes become interconnected into finite size assemblies, which are stable in aqueous solution for charge ratios (ratio of the molar concentration of charged counterion groups to the molar concentration of charged monomer units of the polymer brush) 1 < l < 1.2. They are compact and consist of the brushes in curled (bent) conformation, which is in difference to network-like aggregates from cylindrical poly(styrene sulfonate) brushes with tetravalent porphyrin counterions (Gröhn et al. Macromolecules 2009) and to a wormlike - bent conformation - helix transition found for another brush counterion system (Müller et al. Soft Matter 2009).