| Summary: | Staphylococcus aureus is a high priority pathogen which has generated resistance to multiple antibiotics meaning it is difficult to treat. Due to this, novel treatments for S. aureus infections are being investigated including the development of virulence factor inhibitors. Inhibiting the virulence of the bacterium will assist immune clearance, reducing the need for exposure to bactericidal antibiotics which provide pressure for resistance to emerge. In S. aureus virulence is largely controlled by the agr quorum sensing (QS) system. This is a means of bacterial communication via the release and detection of a signal molecule autoinducing peptide (AIP).
AIPs are produced by post-translational processing of precursor peptide AgrD by cytoplasmic endopeptidase AgrB. AgrD is directed to the cell membrane via the N-terminal amphipathic leader. Here, AgrB recognises AgrD and facilitates cleavage of the C-terminus and formation of a thiolactone ring which is essential for AIP function. Subsequent removal of the AgrD N-terminus releases the mature AIP. Once released externally, AIP binds to histidine kinase AgrC on neighbouring cells activating a signal transduction pathway via AgrA, upregulating AIP biosynthesis and virulence gene expression. The exact mechanism of AgrD processing, however, is poorly understood. Research into this pathway will aid the development of inhibitors which target AIP biosynthesis which can be used as alternative therapies to prevent S. aureus infections.
The present study sought to develop bioreporters for agr QS activation and inhibition. Novel luciferases Gaussia luciferase and NanoLuc were shown to be effective reporters for agr expression in S. aureus through evaluation of chemical agent-mediated activation and inhibition of agr. These reporters enabled quantitative analysis of agr inhibition and can be used to screen for agr QS inhibitors.
This study also sought to explore the mechanism of biosynthesis of AIP, the pheromone of the agr QS system. This is produced by post-translational processing of precursor peptide AgrD by integral membrane protease AgrB. AgrB dimerization was posited to be essential for AgrD processing from analysis of computational modelling of AgrB and AgrD interactions in a lipid membrane. Oligomerization of AgrB was also demonstrated in living cells through the use of novel NanoBRET and NanoBiT assays developed to show protein proximity in live cells. This is the first confirmation of AgrB oligomerisation in living cells which it is thought to be essential for AgrD processing.
Additionally, an agrBD expression construct was developed to increase understanding of AIP maturation. An AgrBD enzyme bound intermediate was discovered to form in E. coli membranes which was detectable by Western blot against AgrB. This intermediate was found to be essential in the processing of AgrB to form AIP. This complex was analysed with a range of inactivating mutations in AgrB identifying residues in AgrB which are essential for recognition of AgrD. It was also analysed in the presence of protease inhibitors including the natural product ambuic acid and several ambuic acid analogues and enabled insights into the mechanism of action of these compounds.
To investigate AIP processing further, AgrB was purified and was shown to be able to perform C-terminal but not N-terminal cleavage of AgrD. MroQ, another protease which has been reported recently to have a potential role in AgrD processing was also purified and, in the presence of purified AgrB and MroQ, mature AIP was formed. This provided evidence for the function of MroQ in the N-terminal cleavage of AgrD, releasing mature AIP.
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