The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. In this study, we identified several peptides of AIDA-I modified by the addition of heptoses by use of mass spectrometry and N-terminal sequencing of proteolytic fragments of AIDA-I. One threonine and 15 serine residues were identified as bearing heptoses, thus demonstrating for the first time that AIDA-I is O-glycosylated. We observed that unglycosylated AIDA-I is expressed in smaller amounts than its glycosylated counterpart and shows extensive signs of degradation upon heat extraction. We also observed that unglycosylated AIDA-I is more sensitive to proteases and induces important extracytoplasmic stress. Lastly, as was previously shown, we noted that glycosylation is required for AIDA-I to mediate adhesion to cultured epithelial cells, but purified mature AIDA-I fused to GST was found to bind in vitro to cells whether or not it was glycosylated. Taken together, our results suggest that glycosylation is required to ensure a normal conformation of AIDA-I and may be only indirectly necessary for its cell-binding function.
A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10 4 S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.
To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells 1 . These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology' 2 . Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories 3,4 . These assays are now practiced by most laboratories working on bacterial pathogenesis.Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 2787 5 , a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787ΔaidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before 6 . Protocol Preliminary: Bacterial strains and epithelial cells.Manipulations of cells and bacteria are performed aseptically, under a laminar flow hood. , and C600 from glycerol stocks on Lysogeny Broth (LB) agar plates (1% tryptone, 0.5% sodium chloride, 0.5% yeast extract, 1.5% agar) and grow at 37°C. To minimize variability in the assay, it is advised to always use freshly plated strains and to keep the strains at 4°C on sealed Petri plates only for a maximum of a couple of weeks. 2. Culture HEp-2 cells (ATCC CCL-23) in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated bovine serum (heat inactivation is performed at 56°C for 30 min). During routine culture we also add 10 U/ml penicillin and 10 μg/ml streptomycin. 3. Grow Hep-2 cells at 37°C in a cell incubator with an atmosphere containing 5% CO2. Use standard cell culture procedures to maintain the cells, which are grown in 75 cm 2 flasks and subcultured every time they reach confluence. We use our cultured cells until they reach 30 to 40 passages and then discard them. 4. Prepare an assay when the HEp-2 cells in a 75 cm 2 flask are nearly reaching confluence and therefore are ready for an adherence assay. DAY 1: Preparing the inoculum and the epithelial cells DAY 2: Infection of cells1. Wash the HEp-2 cells in the flask once with warm Dulbecco's phosphate buffered saline (DPBS: 8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 0.21 g/L Na2HPO4:7H2O) 2. The cells are incubated with 0.05% trypsin -EDTA for 5 min before adding fresh warm complete medium. After fresh medium i...
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