Defining the interactions between components of RND efflux complexes in Salmonella typhimurium

Alav, Ilyas ORCID: 0000-0002-7212-3371 (2022). Defining the interactions between components of RND efflux complexes in Salmonella typhimurium. University of Birmingham. Ph.D.

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Resistance-nodulation-division (RND) efflux pumps contribute to the multidrug resistance of Gram-negative bacteria by exporting clinically relevant antibiotics and preventing them from accumulating at sufficient concentrations. RND pumps, including the principal multidrug efflux pump AcrAB-TolC in Salmonella enterica, are tripartite systems with an inner membrane RND-transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). The role of the PAPs in the assembly of tripartite efflux complexes and the specific residues involved in the interaction between the PAP and RND-transporter is poorly understood. Previous work in this laboratory predicted four 3D sites involved in the interaction between the PAP AcrA and the AcrB-transporter that corresponded to nine discrete binding sequences referred to as binding boxes within the β-barrel domain and membrane-proximal domain (MPD) of AcrA. These binding boxes are highly conserved within the phylogenetically and structurally related PAPs AcrA and AcrE, which have been shown to be interchangeable.

In this study, the functional role of the residues within these predicted binding boxes was validated by site-directed mutagenesis. Mutations mapping to binding boxes 1, 4, 5, 6 and 9 of AcrA had a significant impact on antimicrobial susceptibility, efflux function, and virulence, indicating their importance in AcrB-binding. Furthermore, a mutation mapping to the MPD of AcrA was found to impact the access of substrates to the entry channels of AcrB, suggesting a potential role for the PAP MPD in modulating substrate channel access. Using the newly defined RND-binding boxes, the PAPs AcrA and AcrE were shown to bind to the AcrB-transporter in the same way by using equivalent residues. Furthermore, the AcrD-transporter was also found to function with AcrA and AcrE to form an efflux competent pump by using the same residues identified based upon AcrA-AcrB interaction.

Overall, this study demonstrates that AcrA and AcrE use the same residues to interact with their RND-transporter partners to form functional tripartite efflux pumps. Targeting the PAP AcrA has been shown to be a good strategy to inhibit the activity of the AcrAB-TolC pump. However, since AcrA and AcrE are interoperable, any potential PAP-inhibitor would need to inhibit the function of both proteins. The sequence and function of the RND-binding boxes defined in this study are conserved between AcrA and AcrE and so could be attractive drug target sites for the development of efflux inhibitors capable of inhibiting both PAPs.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Medical & Dental Sciences
School or Department: Institute of Microbiology and Infection
Funders: Biotechnology and Biological Sciences Research Council
Subjects: Q Science > QR Microbiology


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