Antimicrobials and Antimicrobial Resistance (AAMR)

The antibiotic resistance crisis is threatening the future of modern healthcare and the discovery of solutions will involve collaboration and investigation into multiple avenues of research. This work involved two projects, the first investigating plasmid transmission and the spread of antibiotic resistance genes (ARGs). The second focussed on developing tools to monitor Staphylococcus aureus biofilm growth. ARGs can be spread between bacteria of the same or different species by horizontal gene transfer on plasmids. Transmission of plasmids can be specific to each host- plasmid interaction therefore it is important to monitor plasmid transmission in clinically relevant hosts. This project involved developing a fluorescence-based system used for monitoring the spread of ß-lactamase carrying plasmids. The system has been established for monitoring transmission of pCT and pKpQIL in single-species environments, the aim was to establish this with pNDM-HK, a globally disseminated AMR plasmid. The second focus was to develop the system with pCT and pKpQIL to enable monitoring of plasmid movement in multi-species environments. The assay can be used to screen for inhibitors of plasmid conjugation to prevent the spread of antibiotic resistance plasmids. The second project involved developing tools for evaluation of S. aureus biofilm growth. Growth in biofilms protects bacteria from antibiotics and the host immune response making infections hard to clear. Phenotypic analysis of S. aureus biofilm growth was performed on ceramic beads as tooth models and cytodex beads which are used as supports in virulence models in vivo. Biofilms were visualised using confocal microscopy. Biofilms were grown in artificial saliva media to mimic biofilm growth in the mouth which is a reservoir for staphylococci that can disseminate around the body. A strain with a knockout of the agr system was analysed as this has a role in control of biofilm growth. Confocal microscopy was also used to determine the spatial heterogeneity of S. aureus biofilms to give insights for drug target development

Gaussia luciferase as a reporter for quorum sensing in staphylococcus aureus

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Gaussia luciferase (GLuc) is a secreted protein with significant potential for use as a reporter of gene expression in bacterial pathogenicity studies. To date there are relatively few examples of its use in bacteriology. In this study we show that GLuc can be functionally expressed in the human pathogen Staphylococcus aureus and furthermore show that it can be used as a biosensor for the agr quorum sensing (QS) system which employs autoinducing peptides to control virulence. GLuc was linked to the P3 promoter of the S. aureus agr operon. Biosensor strains were validated by evaluation of chemical agent-mediated activation and inhibition of agr. Use of GLuc enabled quantitative assessment of agr activity. This demonstrates the utility of Gaussia luciferase for in vitro monitoring of agr activation and inhibition

Gaussia Luciferase as a Reporter for Quorum Sensing in Staphylococcus aureus

Gaussia luciferase (GLuc) is a secreted protein with significant potential for use as a reporter of gene expression in bacterial pathogenicity studies. To date there are relatively few examples of its use in bacteriology. In this study we show that GLuc can be functionally expressed in the human pathogen Staphylococcus aureus and furthermore show that it can be used as a biosensor for the agr quorum sensing (QS) system which employs autoinducing peptides to control virulence. GLuc was linked to the P3 promoter of the S. aureusagr operon. Biosensor strains were validated by evaluation of chemical agent-mediated activation and inhibition of agr. Use of GLuc enabled quantitative assessment of agr activity. This demonstrates the utility of Gaussia luciferase for in vitro monitoring of agr activation and inhibition

Antimicrobials and Antimicrobial Resistance (AAMR)

The antibiotic resistance crisis is threatening the future of modern healthcare and the discovery of solutions will involve collaboration and investigation into multiple avenues of research. This work involved two projects, the first investigating plasmid transmission and the spread of antibiotic resistance genes (ARGs). The second focussed on developing tools to monitor Staphylococcus aureus biofilm growth. ARGs can be spread between bacteria of the same or different species by horizontal gene transfer on plasmids. Transmission of plasmids can be specific to each host- plasmid interaction therefore it is important to monitor plasmid transmission in clinically relevant hosts. This project involved developing a fluorescence-based system used for monitoring the spread of ß-lactamase carrying plasmids. The system has been established for monitoring transmission of pCT and pKpQIL in single-species environments, the aim was to establish this with pNDM-HK, a globally disseminated AMR plasmid. The second focus was to develop the system with pCT and pKpQIL to enable monitoring of plasmid movement in multi-species environments. The assay can be used to screen for inhibitors of plasmid conjugation to prevent the spread of antibiotic resistance plasmids. The second project involved developing tools for evaluation of S. aureus biofilm growth. Growth in biofilms protects bacteria from antibiotics and the host immune response making infections hard to clear. Phenotypic analysis of S. aureus biofilm growth was performed on ceramic beads as tooth models and cytodex beads which are used as supports in virulence models in vivo. Biofilms were visualised using confocal microscopy. Biofilms were grown in artificial saliva media to mimic biofilm growth in the mouth which is a reservoir for staphylococci that can disseminate around the body. A strain with a knockout of the agr system was analysed as this has a role in control of biofilm growth. Confocal microscopy was also used to determine the spatial heterogeneity of S. aureus biofilms to give insights for drug target development

Investigating agr Quorum Sensing and Autoinducing Peptide Biosynthesis in Staphylococcus aureus

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

Investigating agr Quorum Sensing and Autoinducing Peptide Biosynthesis in Staphylococcus aureus

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

Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo