Analysis of the novel c-terminal secretion signal of the Escherichia coli haemolysin protein.
thesisposted on 19.11.2015, 08:53 by Brendan. Kenny
The release of a haemolytic toxin from some urinopathogenic strains of E. coli is unique in that it is the only reported polypeptide to be truly secreted from this Gram-negative organism. This secretion system comprises four genes on a contiguous, approximately 7.5Kb DNA fragment, encoding the toxin itself (HlyA, 107KD), two membrane localised export proteins (HlyB,D) and the cytoplasmic HlyC protein, whose only apparent function is to post-translationally activate the toxin. Recently, another unlinked gene, tolC, encoding a minor outer membrane protein has also been reported to be required for the export process together with HlyB,D. A novel feature of this system is the presence of a C-terminal targeting signal to direct HlyA from the cell. In this study I show that the HlyA C-terminal targeting signal can be harnessed to secrete the majority of both the mammalian prochymosin and the E. coli cytoplasmic, LacZ, proteins in an HlyB,D dependent manner. I have also shown that the efficiency of secretion dramatically decreases when either the C-terminal domain is reduced in size, from 23KD to 4KD, or as the "passenger" domain increases in size. These results suggest that the HlyA signal domain is composed of sequences required for both efficiency of secretion and targeting and that the secretion process is inhibited by heterologous passenger domains, the effect increasing with size presumably due to the adoption of more stable folded conformations. This study has also been concerned with the investigation into the nature of the novel hlyA targeting signal by deploying a series of in vitro mutagenesis methods (hydroxylamine, site directed and "saturation"), to introduce point mutations. This work has generated a bank of mutants, the analysis of which, has highlighted several residues essential for efficient secretion and also indicated a minimal region containing the signal motif. However, this information together with comparisons with other molecules carrying similar targeting signals have not yet identified a common signal motif.