Periodic Reporting for period 4 - VisTrans (Visualising transport dynamics of transmembrane pumps)
Periodo di rendicontazione: 2022-03-01 al 2023-08-31
The resistance of bacterial infections to antibiotic treatment is a growing problem internationally. The origins of drug resistance in bacteria are multifaceted and complex, with numerous processes contributing to the
capacity of pathogenic species to evade antibiotic action during infection. One important mechanism by which drug resistance is conferred is through the intrinsic and inducible activities of multi-drug efflux
pumps. In Gram-negative bacteria, these pumps are nano-machines that span the cell envelope and use energy of electrochemical gradients or ATP hydrolysis to expel antibiotics and other toxic compounds from
the cell, and in this way contribute to resistance phenotypes. The machines are formed of three types of components: an inner membrane protein, outer membrane component that transduces energy, and a protein
residing in the periplasm that physically links the membrane proteins. An analogous machine that exports virulence factors uses paralogous components and has a similar tripartite architecture. Structural data are
available of representative efflux pumps from the applicant’s laboratory and other research groups that use proton electrochemical gradients (AcrA-AcrB-TolC) and ATP (MacA-MacB-TolC) (Du et al., 2014; Wang et
al., 2017; Fitzpatrick et al., 2018). Targeting these machines to impede their activities in pathogens such as Pseudomonas aeruginosa may be a useful strategy to treat resistant and threatening infections. Fungal species use ATP-dependent transporters that
confer drug resistance and pathogenicity, and our structural data have illuminated the mechanism of these machines.
The problem is important for society due to the growing number of cases of life threatening bacterial infections that are resistant to known antibiotics. Finding compounds to inhibit the pumps in Pseudomonas and other
pathogens is challenging due to the permeability barrier presented by the formidable cell envelope, but the structural information provided from these and related studies are helping to identify alternative ways of accessing sites to inhibit the pumps.
The overall objectives of the project is to understand how the machines work in sufficient detail that it might be possible to understand how to inactivate them and enable treatments with antibiotics.
capacity of pathogenic species to evade antibiotic action during infection. One important mechanism by which drug resistance is conferred is through the intrinsic and inducible activities of multi-drug efflux
pumps. In Gram-negative bacteria, these pumps are nano-machines that span the cell envelope and use energy of electrochemical gradients or ATP hydrolysis to expel antibiotics and other toxic compounds from
the cell, and in this way contribute to resistance phenotypes. The machines are formed of three types of components: an inner membrane protein, outer membrane component that transduces energy, and a protein
residing in the periplasm that physically links the membrane proteins. An analogous machine that exports virulence factors uses paralogous components and has a similar tripartite architecture. Structural data are
available of representative efflux pumps from the applicant’s laboratory and other research groups that use proton electrochemical gradients (AcrA-AcrB-TolC) and ATP (MacA-MacB-TolC) (Du et al., 2014; Wang et
al., 2017; Fitzpatrick et al., 2018). Targeting these machines to impede their activities in pathogens such as Pseudomonas aeruginosa may be a useful strategy to treat resistant and threatening infections. Fungal species use ATP-dependent transporters that
confer drug resistance and pathogenicity, and our structural data have illuminated the mechanism of these machines.
The problem is important for society due to the growing number of cases of life threatening bacterial infections that are resistant to known antibiotics. Finding compounds to inhibit the pumps in Pseudomonas and other
pathogens is challenging due to the permeability barrier presented by the formidable cell envelope, but the structural information provided from these and related studies are helping to identify alternative ways of accessing sites to inhibit the pumps.
The overall objectives of the project is to understand how the machines work in sufficient detail that it might be possible to understand how to inactivate them and enable treatments with antibiotics.
We have developed methods to prepare the pumps in an environment that mimics the natural membrane in which the proteins are embedded. This has enabled us to obtain high resolution images of the molecules and understand how the proteins can bind and push drugs through the membrane in an energy dependent process. This work has now been published (Du, D., Neuberger, A., Newman, C., Wu Orr, M., Hsu, P.-C. Samsudin, F., Ramos, L.M. Debela, M., Khalid, S., Storz, G., Luisi, B.F. (2020) Allosteric modulation of an RND transporter by a transmembrane small protein and lipid. Structure. S0969-2126(20)30095-2. doi: 10.1016/j.str.2020.03.013. PMC7267776). We have been developing methods to reconstitute the entire tripartite efflux pumps in membrane-mimicking conditions for high resolution reconstructions by electron microscopy.
We have also been developing an approach to enable the visualisation of tripartite efflux pumps in situ for tomographic reconstruction, and this has been published (Shi, X., Chen, M., Yu, Z., Bell, J.M. Wang, H., Forrester, I., Villarreal, H., Jakana, J., Du, D., Luisi, B.F. Ludtke, S.J. and Wang, Z. (2019) In situ structure and assembly of the multidrug efflux pump AcrAB-TolC. Nature Communications, 10:2635. PMC6570770). The resolution the reconstructions has now been extended, enabling visualisation of conformational changes in the pump in situ with inhibitor binding (Wang, Z., Chen, M., Shi, X., Yu, Z., Fa, G., Serysheva, I.I. Baker, M., Luisi, B. and Ludtke, S.J. (2020) In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution. biorxivs, doi: https://doi.org/10.1101/2020.06.10.144618). We are extending this work for tripartite assemblies based on ABC transporters. We have solved the structure of a multidrug transporter from the ABC family and identified a conformational cycle for the efflux mechanism (manuscript in preparation).
We have prepared a general review of multi-drug efflux mechanisms (Du, D, Wang-Kan, X., Neuberger, A., van Veen HW, Pos, K.M. Piddock. LJV, and Luisi, B.F. (2018) Multidrug efflux pumps: structure, function and regulation. Nat Rev Microbiol. doi: 10.1038/s41579-018-0048-6).
We have solved the structure of a fungal ATP-binding cassette transporter in complex with transport substrates that illuminates the transport mechanismHarris, A., Wagner, M., Du, D., Raschka, S., Gohlke, H., Smits, S.H.J. Luisi, B.F. Schmitt, L. (2021) Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5. Nature Communications, 12:5254 doi.org/10.1038/s41467-021-25574-8. PMC8421411).
We have also been developing an approach to enable the visualisation of tripartite efflux pumps in situ for tomographic reconstruction, and this has been published (Shi, X., Chen, M., Yu, Z., Bell, J.M. Wang, H., Forrester, I., Villarreal, H., Jakana, J., Du, D., Luisi, B.F. Ludtke, S.J. and Wang, Z. (2019) In situ structure and assembly of the multidrug efflux pump AcrAB-TolC. Nature Communications, 10:2635. PMC6570770). The resolution the reconstructions has now been extended, enabling visualisation of conformational changes in the pump in situ with inhibitor binding (Wang, Z., Chen, M., Shi, X., Yu, Z., Fa, G., Serysheva, I.I. Baker, M., Luisi, B. and Ludtke, S.J. (2020) In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution. biorxivs, doi: https://doi.org/10.1101/2020.06.10.144618). We are extending this work for tripartite assemblies based on ABC transporters. We have solved the structure of a multidrug transporter from the ABC family and identified a conformational cycle for the efflux mechanism (manuscript in preparation).
We have prepared a general review of multi-drug efflux mechanisms (Du, D, Wang-Kan, X., Neuberger, A., van Veen HW, Pos, K.M. Piddock. LJV, and Luisi, B.F. (2018) Multidrug efflux pumps: structure, function and regulation. Nat Rev Microbiol. doi: 10.1038/s41579-018-0048-6).
We have solved the structure of a fungal ATP-binding cassette transporter in complex with transport substrates that illuminates the transport mechanismHarris, A., Wagner, M., Du, D., Raschka, S., Gohlke, H., Smits, S.H.J. Luisi, B.F. Schmitt, L. (2021) Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5. Nature Communications, 12:5254 doi.org/10.1038/s41467-021-25574-8. PMC8421411).
We hope to gain detailed structural and mechanistic understanding of how efflux pumps and related toxin export machinery in bacteria are assembled and operate in the complex cellular environment.