Enterohemorrhagic Escherichia coli (EHEC) is a food-borne cause of bloody diarrhea and the hemolyticuremic syndrome (HUS) in humans. Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions. A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide. However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island. Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS. Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions. Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an ϳ90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine. In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21. This chromosomal region comprises four open reading frames, lpfA to lfpD, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933. In a survey of EHEC of other serotypes, homologues of lpfA O113 were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains. Deletion of the putative major fimbrial subunit gene, lpfA, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that lpf O113 may function as an adhesin in LEE-negative isolates of EHEC.Shiga toxin-producing strains of enterohemorrhagic Escherichia coli (EHEC) are a prominent cause of acute gastroenteritis, hemorrhagic colitis, and the hemolytic-uremic syndrome (HUS) in humans (17,23). HUS is characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, and those most at risk of developing renal disease are children under 10 years and the elderly. In approximately 3 to 5% of affected children, HUS is fatal, while 12 to 30% suffer serious impairment of renal function or neurologic damage (17, 23).Although the carriage of bacteriophage-encoded Shiga toxin genes (stx) is a defining feature of virulence, strains of EHEC also harbor a large ϳ90-kb plasmid, pEHEC, and/or a large chromosomal pathogenicity island, termed the locus for enterocyte effacement (LEE) (6, 14). Strains of EHEC carrying LEE belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-effacing (A/E) lesions. This group of organisms also includes the human pathogen enteropathogenic E. coli (EPEC) and the animal pathogens rabbit-specific enteropathogenic E. coli (REPEC) and Citrobacter rodentium. A/E lesions are characterized by localized destruction (effacement) of intestinal brush border microvilli, intimate attachment of the bacteria to the host cell ...

Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. In particular the use of fluorescent ligands as chemical tools to study proteins such as G protein-coupled receptors (GPCRs) has received ongoing interest. Methods to improve the efficient chemical synthesis of fluorescent ligands remain of paramount importance to ensure this area of bioanalysis continues to advance. Here we report conversion of the non-selective GPCR adenosine receptor antagonist Xanthine Amine Congener into higher affinity and more receptor subtype-selective fluorescent antagonists. This was achieved through insertion and optimisation of a dipeptide linker between the adenosine receptor pharmacophore and the fluorophore. Fluorescent probe 27 containing BODIPY 630/650 (pK(D) = 9.12 ± 0.05 [hA3AR]), and BODIPY FL-containing 28 (pK(D) = 7.96 ± 0.09 [hA3AR]) demonstrated clear, displaceable membrane binding using fluorescent confocal microscopy. From in silico analysis of the docked ligand-receptor complexes of 27, we suggest regions of molecular interaction that could account for the observed selectivity of these peptide-linker based fluorescent conjugates. This general approach of converting a non-selective ligand to a selective biological tool could be applied to other ligands of interest.

A series of indolequinones including derivatives of EO9 bearing various functional groups and related indole-2-carboxamides have been studied with a view to identifying molecular features which confer substrate specificity for purified human NAD(P)H:quinone oxidoreductase (DT-diaphorase), bioreductive activation to DNA-damaging species, and selectivity for DT-diaphorase-rich cells in vitro. A broad spectrum of substrate specificity exists, but minor changes to the indolequinone nucleus have a significant effect upon substrate specificity. Modifications at the 2-position are favorable in terms of substrate specificity as these positions are located at the binding site entrance as determined by molecular modeling studies. In contrast, substitutions at the (indol-3-yl)methyl position with bulky leaving groups or a group containing a chlorine atom result in compounds which are poor substrates, some of which inactivate DT-diaphorase. Modeling studies demonstrate that these groups sit close to the mechanistically important amino acids Tyr 156 and His 162 possibly resulting in either alkylation within the active site or disruption of charge-relay mechanisms. An aziridinyl group at the 5-position is essential for potency and selectivity to DT-diaphorase-rich cells under aerobic conditions. The most efficient substrates induced qualitatively greater single-strand DNA breaks in cell-free assays via a redox mechanism involving the production of hydrogen peroxide (catalase inhibitable). This damage is unlikely to form a major part of their mechanism of action in cells since potency does not correlate with extent of DNA damage. In terms of hypoxia selectivity, modifications at the 3-position generate compounds which are poor substrates for DT-diaphorase but have high hypoxic cytotoxicity ratios.