Aurodox, a specialised metabolite from the soil bacterium Streptomyces goldiniensis was discovered in 1973 and was originally investigated for its antibacterial properties. However, aurodox has been recently identified from large-scale compound screens as an inhibitor of the Enteropathogenic Escherichia coli (EPEC) Type III Secretion System (T3SS).Therefore, to gain an understanding of its mechanism of action and to assess the suitability of this molecule for repurposing as an anti-virulence compound, a multidisciplinary approach to understanding aurodox was used. The biosynthesis of aurodox by S. goldiniensis was investigated through sequencing the whole genome of S. goldiniensis to enable the identification of the putative aurodox biosynthetic gene cluster (BGC).This BGC was then cloned and expressed in multiple heterologous hosts including Streptomyces coelicolor M1152, confirming that this BGC is responsible for aurodox production. In-depth analysis of the BGC supports a model of a polyketide synthase pathway involving a combination of both cis and transacyltransferases which synthesise the aurodox polyketide backbone before tailoring enzymes are recruited to form the final aurodox precursor, kirromycin, which is also produced by S. collinus.These studies suggest that the SAM-dependent Omethyltransferase AurM* is responsible for catalysing the conversion of kirromycin to aurodox in S. goldiniensis through the methylation of the pyridone moiety. Furthermore, multiple aurodox resistance genes at distinct loci have been identified and their role in aurodox resistance has been explored. The mechanism of action of aurodox has also been investigated.Whole transcriptome analysis, cell infection and GFP-reporter assays were used to demonstrate that aurodox transcriptionally downregulates the expression of the Locus of Enterocyte Effacement (LEE) pathogenicity island- which encodes for the T3SS, acting via its master regulator, Ler. We have also observed these effects across other enteric pathogens carrying a homologous T3SS such as Enterohemorrhagic Escherichia coli (EHEC). Significantly, unlike traditional antibiotics, aurodox does not induce the production of shiga toxin.
|Date of Award||11 Aug 2020|
- University Of Strathclyde
|Sponsors||University of Strathclyde|
|Supervisor||Paul Hoskisson (Supervisor) & Robin Plevin (Supervisor)|