AbstractThe Type VII secretion system (T7SS), is a protein secretion pathway found in Gram positive bacteria. In S. aureus the T7SS, termed Ess, is dispensable for laboratory growth but is essential for virulence. The Ess system is encoded by the ess locus, the first six genes of which esxA, esaA, esaB, essA, essB and essC code for the core components of the secretion apparatus.
The genetic organisation of the ess locus was investigated in five closely related S. aureus strains. It was shown that esxA is a monocistronic gene in strains RN6390 and Newman, but is part of a transcriptional unit with esaA in strains COL, USA300 and SA113. The eleven genes downstream of esxA were shown to be co-expressed in all strains tested. A transcriptional start site for esaA was identified in the esxA-esaA intergenic region in strain RN6390. It was shown that esxA is expressed in large excess over esaA regardless of these differences in transcriptional organisation.
Analysis of EsaD, encoded at the ess locus, showed that it was a large secreted substrate of the T7SS. Bioinformatic predictions suggested that EsaD has a nuclease domain at its C-terminus and in support of this EsaD was shown to lead to DNA damage when produced in E. coli. in vitro nuclease assays using the purified EsaD C-terminal domain confirmed that it has Mg2+-dependent DNase activity. EsaG, encoded directly downstream of esaD at the ess locus, is a cytoplasmic protein that possesses the ability to interact with the nuclease domain of EsaD and block its activity. EsaE, a third ess-encoded protein was also shown to interact with EsaD, but with the N-terminal region rather than the nuclease domain. Co-purification experiments show that the three proteins form a ternary complex. EsaE and EsaD were seen to be co-secreted whereas EsaG remained in the cytoplasm. Fractionation and crosslinking experiments showed that a proportion of EsaE localised to the cell membrane and interacted with the integral membrane T7SS core component EssC, suggesting it may target the complex for secretion. Secreted EsaD was shown to inhibit the growth of closely related strains of S. aureus indicating that it plays a role in intra-species competition.
|Date of Award||2017|
|Sponsors||China Scholarship Council|
|Supervisor||Tracy Palmer (Supervisor)|