Mutations in the Pseudomonas aeruginosa needle protein gene pscF confer resistance to phenoxyacetamide inhibitors of the type III secretion system

Abstract

The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics in P. aeruginosa infections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition of P. aeruginosa T3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS gene pscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles of pscF, together with its chaperone and cochaperone genes pscE and pscG, to a ΔpscF P. aeruginosa strain demonstrated that each of the single-codon mutations in pscF is necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.

FIG 1

Structures of small-molecule inhibitors of P. aeruginosa T3SS used in this study. (A) Five analogs of the phenoxyacetamide series, including MBX 2359, which was used for selection of resistant mutants. (B) Representatives of three published nonphenoxyacetamide scaffolds.

FIG 2

Stereoselective inhibition of T3SS-mediated secretion by phenoxyacetamide inhibitors. Inhibition of T3SS-mediated secretion of ExoS-BLA from P. aeruginosa strain MDM973 is shown. Inhibitors include the original screening hit, racemate MBX 1641 (Δ), its (R)-isomer, MBX 1684 (O), its (S)-isomer, MBX 1686 (□), and an analog optimized for potency, the racemate MBX 2359 (▲), together with its (R)-isomer MBX 2401 (●) and (S)-isomer MBX 2402 (■). The fraction of secretion of ExoS-BLA into culture medium with respect to the control (uninhibited secretion), as measured by the change in absorbance at 490 nm per min resulting from the cleavage of nitrocefin (ΔA490/min), is plotted versus the concentration of compound added.

FIG 3

T3SS-mediated secretion by selected mutants is resistant to inhibition by MBX 2359. (A) Concentration dependence of MBX 2359 inhibition of T3SS-mediated secretion of ExoS-BLA by the P. aeruginosa screening strain MDM1710 (△) and four selected mutants, MDM1739 [PscF(R75C)] (●), MDM1749 [PscF(R75H)] (■), MDM1750 (PscF(V62I)] (▲), and MDM1770 [PscF(G80D)] (◆). Secretion was measured by the rate of hydrolysis of nitrocefin by ExoS-BLA. Results are plotted as a fraction of that of the uninhibited control versus the concentration of T3SS inhibitor MBX 2359 added. (B) Inhibition by MBX 2359 of T3SS-mediated secretion of native effector ExoS and ExoS-BLA fusion proteins into the culture medium was measured by immunoblot analysis. Supernatants (normalized to cell number) of the screening strain MDM1710, the highly resistant mutant MDM1749 [PscF(R75H)], and the moderately resistant mutant MDM1750 [PscF(V62I)] were analyzed. Detection was by primary anti-ExoS antibody and LiCor goat anti-rabbit as the secondary antibody. Blots were imaged using the LiCor imager. (C) Image-Studio (LiCor) was used to perform densitometry on each band. Densities were normalized to the no-inhibitor value and are shown graphically for MDM1710 (△), MDM1749 [PscF(R75H)] (■), and MDM1750 [PscF(V62I)] (▲) for ExoS (upper) and ExoS-BLA (lower).

FIG 4

Mutant pscF alleles provide T3SS secretion resistance to five phenoxyacetamide analogs. The original screening hit, MBX 1641 (◊), and analogs MBX 2359 (□), MBX 2164 (△), MBX 2614 (○), and MBX 2727 (✖) were tested for inhibition of T3SS-mediated secretion of ExoS-BLA carried out by ΔpscF PAO1 cells complemented with lac-promoted pscEFG on extrachromosomal plasmid pUCP24. The strains and pscF alleles were wild-type MDM1912 (A), MDM1967 [PscF(V62I)] (B), and MDM1886 [PscF(R75H)] (C). For reference, the MBX 2359 concentration response versus the wild-type pscF allele from panel A is shown in panels B and C as a dotted line.

FIG 5

Mutant pscF alleles provide T3SS translocation resistance to phenoxyacetamide MBX 2359. Inhibitor or diluent (dimethylsulfoxide) was tested at the indicated concentration for inhibition of T3SS-mediated translocation detected by the β-lactamase-sensitive fluorescent substrate CCF2-AM and carried out by ΔpscF P. aeruginosa PAO1 cells complemented with lac-promoted pscEFG on extrachromosomal plasmid pUCP24. The strains and pscF alleles were wild-type MDM1912 (A), MDM1967 [PscF(V62I)] (B), and MDM1886 [PscF(R75H)] (C). The J774 bar indicates the addition of CCF2-AM to J774 cells in the absence of bacteria. The WT and ΔpscF bars indicate the addition of CCF2-AM to J774 cells infected with wild-type P. aeruginosa and with uncomplemented ΔpscF P. aeruginosa, respectively. *, P < 0.005 by two-tailed paired Student t test.

FIG 6

Mutant pscF alleles fail to provide T3SS secretion resistance to three nonphenoxyacetamide inhibitors. The sensitivity of secretion to a concentration dilution series of T3SS inhibitors INP0007 (◊), CBG-5376648 (△), CBG-6594330 (○), and the phenoxyacetamide MBX 2359 (■) is shown versus ΔpscF PAO1 cells complemented with lac-promoted pscEFG on extrachromosomal plasmid pUCP24. The strains and pscF alleles were wild-type MDM1912 (A), MDM1967 [PscF(V62I)] (B), and MDM1886 [PscF(R75H)] (C).

FIG 7

Comparative homology model of the P. aeruginosa T3SS needle complex. Ribbon representations show different subunits. (A) Side view. (B) Top view with residues Val62 (red), Arg75 (green), and Gly80 (yellow) shown in space-filling format for three different putative inhibitor interaction sites (circled). Loops joining α helices are shown in purple, as are extended chains positioned on the surface of the needle complex representing the N-terminal domains. Cyan helices represent the α1 and α2 helical structures. Closeup view of three PscF subunits shown in ribbon format, with residues Val62, Arg75, and Gly80 highlighted in stick format (C) and with inter-α-carbon distances shown (D). Each residue is in a different PscF subunit, and the alpha-helical ribbons are colored differently to represent the three different subunits.