Engineering drug sensitivity to screen for new classes of antibiotics for the emerging, drug resistant fungal pathogen Candida auris.
Lead Research Organisation:
King's College London
Department Name: Pharmaceutical Sciences
Abstract
This Basic Bioscience project addresses BBSRC priority 'Combatting Antimicrobial Resistance'
Objectives:
i) To develop a drug sensitive strain of Candida auris that will enable discovery of new classes of antibiotics for this drug resistant fungal pathogen
ii) To use fungal molecular genetics to determine the mechanism that underpins the toxicity of bolalipids against fungi.
The frequency of nosocomial fungal infections has risen over the past few decades. The most significant fungal pathogens are the Candida sp., including the emerging pathogen C.auris which has an alarming multi-drug resistance profile, with isolates resistant to all four class of antifungals used in the clinic. There is a pressing need for new broad antifungals as well as antifungals targeted to emerging pathogens like C.auris.
Understanding of the mechanisms of drug resistance in C.auris is limited, but it is evident that effectiveness of screening for novel antimicrobials is restricted by drug resistance mechanisms, including activity of multi-drug resistance pumps. Improving the understanding of how these pumps impact on current drugs would be valuable in predicting resistance in clinical isolates. They also impact on screening for new drugs, because many compounds which may have a modest biocidal effect, but could be useful as lead compounds in structure activity relationship studies, slip through the net as they are pumped out of the cells before they can exert an effect. S.cerevisiae strains harbouring mutations in the genes encoding the Pdr1 and Pdr3 pleiotropic drug-resistance transcription factors and a gene involved in the biosynthesis of the sterol component of yeast membranes, feature a combination of increased membrane fluidity and compromised drug efflux, significantly increasing intracellular concentration of drugs, enhancing their activity. We aim to generate the same genetic background in C.auris. Development of the C.auris molecular genetic toolkit that enables manipulation of its genome is in its infancy. However, the genome sequence for this fungal pathogen is available as is a marker gene that permits selection of transformants. We will characterise the resistance phenotype, more specifically our manipulation of the ergosterol pathway should affect membrane fluidity; we will assess this by fluorescence polarization and HRMAS NMR metabolomics. At the same time we will use this drug sensitive C.auris strain to screen chemical libraries. Subsequently, the Galleria mellonella invertebrate model of systemic candidiasis will be employed to explore the impact of drugs that emerge from such screens.
Dequalinium is a quaternary ammonium cation and bola-amphiphile commonly used as an antiseptic. Several derivatives of this compound display improved antibacterial activity (Mason, unpublished). However, the most potent antimicrobial activity was that displayed against C.albicans by two of these bolalipids. The same two compounds are also effective against C.auris, We aim to use fungal molecular genetics to determine the mechanism that underpins this toxicity. Initially we will use the extensive molecular-genetic toolkit that has been developed in the baker's yeast S. cerevisiae, initially using a simplified screen comprising a subset of yeast gene deletion signature strains to probe bolalipid mode of action. The idea behind such profiling is that instead of assays involving isolated proteins against test compounds, collections of yeast gene deletion strains can be used to measure the sensitivity of whole cells, a strategy which is less laborious, and relatively inexpensive. This will reveal facets of cell biology that are involved in bolalipid toxicity. Data can be validated by modulation of the relevant biological pathways (via gene deletion or over-expression) followed by assessing how this affects bolalipid toxicity.
Objectives:
i) To develop a drug sensitive strain of Candida auris that will enable discovery of new classes of antibiotics for this drug resistant fungal pathogen
ii) To use fungal molecular genetics to determine the mechanism that underpins the toxicity of bolalipids against fungi.
The frequency of nosocomial fungal infections has risen over the past few decades. The most significant fungal pathogens are the Candida sp., including the emerging pathogen C.auris which has an alarming multi-drug resistance profile, with isolates resistant to all four class of antifungals used in the clinic. There is a pressing need for new broad antifungals as well as antifungals targeted to emerging pathogens like C.auris.
Understanding of the mechanisms of drug resistance in C.auris is limited, but it is evident that effectiveness of screening for novel antimicrobials is restricted by drug resistance mechanisms, including activity of multi-drug resistance pumps. Improving the understanding of how these pumps impact on current drugs would be valuable in predicting resistance in clinical isolates. They also impact on screening for new drugs, because many compounds which may have a modest biocidal effect, but could be useful as lead compounds in structure activity relationship studies, slip through the net as they are pumped out of the cells before they can exert an effect. S.cerevisiae strains harbouring mutations in the genes encoding the Pdr1 and Pdr3 pleiotropic drug-resistance transcription factors and a gene involved in the biosynthesis of the sterol component of yeast membranes, feature a combination of increased membrane fluidity and compromised drug efflux, significantly increasing intracellular concentration of drugs, enhancing their activity. We aim to generate the same genetic background in C.auris. Development of the C.auris molecular genetic toolkit that enables manipulation of its genome is in its infancy. However, the genome sequence for this fungal pathogen is available as is a marker gene that permits selection of transformants. We will characterise the resistance phenotype, more specifically our manipulation of the ergosterol pathway should affect membrane fluidity; we will assess this by fluorescence polarization and HRMAS NMR metabolomics. At the same time we will use this drug sensitive C.auris strain to screen chemical libraries. Subsequently, the Galleria mellonella invertebrate model of systemic candidiasis will be employed to explore the impact of drugs that emerge from such screens.
Dequalinium is a quaternary ammonium cation and bola-amphiphile commonly used as an antiseptic. Several derivatives of this compound display improved antibacterial activity (Mason, unpublished). However, the most potent antimicrobial activity was that displayed against C.albicans by two of these bolalipids. The same two compounds are also effective against C.auris, We aim to use fungal molecular genetics to determine the mechanism that underpins this toxicity. Initially we will use the extensive molecular-genetic toolkit that has been developed in the baker's yeast S. cerevisiae, initially using a simplified screen comprising a subset of yeast gene deletion signature strains to probe bolalipid mode of action. The idea behind such profiling is that instead of assays involving isolated proteins against test compounds, collections of yeast gene deletion strains can be used to measure the sensitivity of whole cells, a strategy which is less laborious, and relatively inexpensive. This will reveal facets of cell biology that are involved in bolalipid toxicity. Data can be validated by modulation of the relevant biological pathways (via gene deletion or over-expression) followed by assessing how this affects bolalipid toxicity.
