Fig 1.
A. A general view of the PAP-RND assembly as exemplified by the E. coli AcrAB sub-complex seen in the asymmetric cryo-EM structure (5o66.pdb) [24]. B. AcrB trimer organisation illustrated by side view of the AcrB trimer from the same assembly as in 1A. Protomers coloured with different colours and principal domains indicated. C. Domain organisation of a typical PAP based on the experimental structure of E.coli AcrA (protomer G from 5o66.pdb above). The chain is coloured in rainbow from N-terminus (blue) to C-terminus (red).
Fig 2.
A. Positions of PAP1 (violet) and PAP2 (red) protomers relative to RND-protomer within the assembly exemplified by the AcrAB sub-complex discussed in Fig 1 above. Binding is discussed relative to the green protomer of AcrB. B. Binding regions of PAP1 relative to the RND protomer (using AcrA chain G in 5o66.pdb for illustration). The region of PAP1-RND interaction is magnified and annotated highlighting the 4 principal binding sites and structural features of the surface. Further details provided in the text. C. PAP2 –RND binding regions on the example of AcrA (chain H) in 5o66.pdb.
Fig 3.
The 3D-binding sites of PAPs relative to the RND-transporter can be reduced to discrete linear sequence “binding boxes”.
A. A multiple sequence alignment of the 4 Salmonella PAPs combined with the structural alignment of the experimental E. coli AcrA structure (based on 5o66.pdb chainG) (top) reveals clear domain boundaries and correspondingly high likelihood of secondary structure conservation. Identical residues are coloured red. The proposed “binding boxes” are annotated from 1 to 9 and delineated using rectangles. Figure produced using Espript. B. Mapping of the binding boxes (in blue) onto the 3D structure of the PAPs (AcrA) shows that they are restricted solely to β-barrel and MPD domains and map predominantly to one face of the PAP protomer, which faces the RND transporter. C. A Consurf sequence conservation map based on the 150 unique PAP sequences (sequence identity from 95% to 45%) projected onto the AcrA structure. Highly conserved residues are indicated with deep magenta, hypervariable regions in cyan. D. A composite image combining the Consurf map from 3C with the space-fill representation of the residues comprising the binding boxes, demonstrating the strong conservation of the sequence elements within them.
Fig 4.
The effect of loss of combinations of PAPs on the phenotype of Salmonella.
A. Efflux of ethidium bromide in strains lacking single or combinations of PAPs. Bacteria were treated with ethidium bromide and CCCP for 60 min and then re-energized with glucose. Data presented is the time taken for the fluorescence to decrease by 25% +/- SE. (Data for 10% and 50% drop can be seen in S1 Table) B. Survival of Galleria mellonella wax moth larvae infection model. C. The frequency of resistance to ciprofloxacin. Data is displayed as the mean of at least 14 biological replicates +/- SE. Data analysed by one-way ANOVA and strains whose frequency of resistance was significantly different (p<0.05) from SL1344 are indicated by *.
Table 1.
Antimicrobial susceptibility in strains lacking all combinations of one, two, three or four PAPs.
Table 2.
Antimicrobial susceptibility following complementation of the Δ4PAP strain with each individual PAP.
Fig 5.
Bacteria were treated with ethidium bromide and CCCP for 60 min and then re-energized with glucose. Data presented is the time taken for the fluorescence to decrease by 25% +/- SE. (Data for 10% and 50% drop can be seen in S1 Table). A. Shows data for the Δ4PAP strain with and without complementation of single PAPs B. Shows data for Δ4PAP that also lacks AcrB or AcrF with and without complementation with acrA or acrE.
Table 3.
Antimicrobial susceptibility in Δ4PAP strain also lacking major pumps AcrB or AcrF and complemented with acrA, acrE, mdtA or mdsA.
Fig 6.
Efflux activity in M15 is restored due to increased expression of acrEF.
A. Ethidium bromide efflux. B. Hoechst accumulation. C. Schematic of RamR with duplication marked by red box d. Real-time reverse transcriptase PCR. Data from A and B are the mean of three biological replicates +/- SE mean.
Table 4.
Antimicrobial susceptibility of ciprofloxacin selected mutants in a ΔacrA background and corresponding gyrA genotype.
Table 5.
Antimicrobial susceptibility of ciprofloxacin selected strain M15 and derivatives.
Fig 7.
A. Efflux of ethidium bromide by strains complemented with mutated versions of AcrA. Mutants labelled above bar according to which binding box is mutated. Bacteria were treated with ethidium bromide and CCCP for 60 min and then re-energized with glucose. Data presented are the mean of three independent biological replicates and are shown as the time taken for the fluorescence to decrease by 50% +/- SE. Data were analysed by one way ANOVA. B. Mapping of PAP box mutations to the structure of the assembled complex based on the cryo-EM structure of E. coli AcrAB-TolC. The PAP 1 protomer bound to the green RND protomer is colored blue; PAP 2 protomer is colored red. For clarity the hairpins of both PAP 1 and PAP 2 protomers are removed. Mutations of residues with prominent phenotypic effect on efflux are colored magenta and the residues responsible are presented in spacefill. Mutations colored orange were responsible for a measurable (although not statistically significant effect) while mutations in green had no measurable effect. The mapping reveals that the majority of the mutations with a statistically significant effect are mapping to the beta-barrel domain of the PAP. In particular, it is notable that the efflux-sensitive mutations cluster around the two beta-hairpins at the crown of the porter domains of the RND-transporter and that the same residues appear to be grasping the hairpin 1 and hairpin 2 respectively in PAP 1 and PAP 2 in a pincer-like fashion. This finding strongly supports the primary role of the beta-hairpins in the PAP assembly. C. Western Blot analysis of the expression and stability of selected mutated AcrA constructs with pronounced phenotypic effects. C-terminal His-tagged versions of the proteins were expressed from pET20b in Salmonella Δ4PAP background and protein expression visualised using a monoclonal anti-His AP-conjugated antibody.