Journal: Cellular Microbiology
Article Title: The E Block motif is associated with Legionella pneumophila translocated substrates
Figure Lengend Snippet: SidC fusion assay allows identification of carboxyl terminal sequences from L. pneumophila proteins that promote protein translocation into macrophages. A. Flowchart of procedure that allowed identification of carboxyl terminal translocation signals. B. Construction and screening of gene bank. The plasmid pZL204 ( Experimental procedures ) encodes the sidC gene under Ptac control, has a multi-cloning site and a premature truncation removing 100 codons from the 3′ end of the gene, allowing expression of a protein that is translocation defective. Rescue of the translocation defect was performed by introducing a bank of 600 nucleotide-long fragments amplified from the 3′ ends of 442 genes encoding candidate translocated substrates ( sidC- X). The resulting gene fusion constructions were introduced into L. pneumophila strain Lp02 ΔsidC and used to challenge bone marrow-derived macrophages for 1 h before fixation and staining with anti- L. pneumophila and anti-SidC antisera in 96-well microtiter plates. The stained samples were subjected to image analysis by automated microscopy, and the efficiency of translocation was assayed quantitatively ( Experimental procedures ). C. Examples of images captured during high throughput screening. Macrophage infections are displayed after challenge with three different strains. SidC + : L. pneumophila encoding intact sidC gene under ptac control. SidCΔ100: L. pneumophila harbouring pZL204 plasmid. SidC-1798: L. pneumophila harbouring a plasmid encoding sidCΔ100 fused to the 3′ end sequence of Lpg1798 . Cells were immunostained using: α- L.p. , mouse monoclonal antibody directed against L. pneumophila ; α-SidC, rabbit antisera directed against the SidC protein. Rightmost panels are merged images of the two antibody probings, coloured as noted, with yellow spots denoting bacteria translocating SidC, and red indicating bacteria defective for SidC translocation.
Article Snippet: Four images from each microtiter well were captured with a Nikon 20× plan-Apo lens using the Texas Red and FITC filter sets to identify bacteria and exported SidC respectively.
Techniques: Single Vesicle Fusion Assay, Translocation Assay, Plasmid Preparation, Clone Assay, Expressing, Amplification, Derivative Assay, Staining, Microscopy, High Throughput Screening Assay, Sequencing