Journal: PLoS ONE

Article Title: H-NST Induces LEE Expression and the Formation of Attaching and Effacing Lesions in Enterohemorrhagic Escherichia coli

doi: 10.1371/journal.pone.0086618

Figure Lengend Snippet: The effect of wild type H-NST and mutant derivatives on LEE -encoded protein levels was determined by western analysis as described in Materials and Methods . EHEC strain TUV93-0 containing the empty vector pQE80 (lane 1), TUV93-0 containing constructs producing wild type EPEC H-NST (lane 2) or the H-NST mutant derivatives H-NST A16V (lane 3), H-NST A16L (lane 4), H-NST R60Q (lane 5) and HNST R60Q/R63Q (lane 6) were grown in LB to a density of OD 600 ∼0.5 and hnsT expression was induced by 0.5 mM IPTG for 60 min. Levels of EspA, EspB and GroEL were detected in total protein by western analysis using polyclonal antisera against the respective proteins as indicated. GroEL served a loading control for total protein. The relative levels of EspA and EspB normalized to that of GroEL are indicated by numbers below the protein bands. Data shown are representative of four independent experiments.

Article Snippet: Oligonucleotide sequences used for plasmid constructions are listed in . pQEH-NST: A 262 bp DNA fragment encoding hnsT was PCR amplified from EPEC E2348/69 gDNA using the primer set QEH-NST F/QEH-NSTF R, digested with Bam HI and Hind III and cloned into the corresponding sites in pQE80 (QIAGEN).

Techniques: Mutagenesis, Western Blot, Plasmid Preparation, Construct, Expressing, Control

Journal: PLoS ONE

Article Title: H-NST Induces LEE Expression and the Formation of Attaching and Effacing Lesions in Enterohemorrhagic Escherichia coli

doi: 10.1371/journal.pone.0086618

Figure Lengend Snippet: FAS assays were used to determine the effect of H-NST on A/E lesion formation of EHEC as described in Materials and Methods . HeLa cell monolayers were co-cultured for four hours with EHEC strain TUV93-0 containing the empty vector pQE80 (A), constructs producing wild type EPEC H-NST (B) or the H-NST mutant derivatives H-NST A16V (C), H-NST A16L (D), H-NST R60Q (E) and HNST R60Q/R63Q (F). The images of FITC phalloidin-stained actin of infected HeLa cells are representative of three independent experiments. Arrows indicate examples of A/E lesions.

Article Snippet: Oligonucleotide sequences used for plasmid constructions are listed in . pQEH-NST: A 262 bp DNA fragment encoding hnsT was PCR amplified from EPEC E2348/69 gDNA using the primer set QEH-NST F/QEH-NSTF R, digested with Bam HI and Hind III and cloned into the corresponding sites in pQE80 (QIAGEN).

Techniques: Cell Culture, Plasmid Preparation, Construct, Mutagenesis, Staining, Infection