A Structure-Function-Inhibition Analysis of the Pseudomonas aeruginosa Type III Secretion Needle Protein PscF

Abstract

The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.IMPORTANCEP. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40 to 69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the PscF needle in T3SS secretion and translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. The results also support a model of direct interaction of the needle with PhA and suggest that, with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.

Keywords: P. aeruginosa; phenoxyacetamides; translocation; type III needle protein; type III secretion.

FIG 1

Coexpression of the pscF(Q83H) allele and the WT allele. (A) Expression of the pscF(Q83H) allele from a nonnative locus and promoter in the presence of native expression of the pscF(WT) allele in strain MDM2544 fails to alter EGTA-regulated T3SS induction (ExoS-Bla ± EGTA). (B) Expression of that same pscF(Q83H) allele from a nonnative locus and promoter in the absence of a pscF(WT) allele in strain MDM2545 causes constitutive T3SS expression that is independent of Ca²⁺ levels. (C) Increasing IPTG-induced expression of the pscF(Q83H) allele in strain MDM2546 under constant high arabinose levels of induction of the WT allele, both at nonnative loci, reduces the T3SS induction ratio to the constitutive range. (D) In the same strain, MDM2546, increasing arabinose-induced expression of the pscF(WT) allele under constant high IPTG levels of induction of the pscF(Q83H) allele, both at nonnative loci, increases the T3SS induction ratio to the normal range. (E) Relative levels of luminescence are shown for P. aeruginosa strains expressing P. luminescens luxCDABE from the same promoters (lac promoter, strain MDM1157; araBAD promoter, strain MDM1922), vectors, and inducer levels as in panels A to D. Values are n = 4 for panels A to D and n = 8 for panel E, along with the standard errors of the mean. Arabinose levels: high, 0.25%; medium, 0.125%. IPTG levels: high, 0.25 mM; medium, 0.125 mM. EGTA, 3 mM. *, P < 0.05 (panels A to D, as determined by one-way analysis of variance analysis with Tukey’s multiple comparison test [GraphPad Prism], as are all pairwise comparisons in panel E).

FIG 2

Effects of PscF(R75H) and PscF(WT) on phenoxyacetamide resistance in P. aeruginosa strains lacking a pscF copy at the native chromosomal locus. Expression of mini-Tn7-inserted pscF(WT) from the arabinose promoter lowered MBX-2359 IC₅₀ values with increased arabinose for strain MDM2537 when lac-promoted pscF(R75H) on pUCP24 was held constant with 0.25 mM IPTG (circles). The expression of mini-Tn7-inserted pscF(R75H) from the arabinose promoter raised MBX-2359 IC₅₀ values with increased arabinose for strain MDM2551 when lac-promoted pscF(WT) was held constant at 0.25 mM IPTG (boxes).

FIG 3

Comparison of the effect of T3SS inhibitor MBX-2359 on secretion and translocation by P. aeruginosa cells carrying various pscF alleles. Secretion of T3SS effector was measured by SDS-PAGE (left panel of each pair). Translocation of ExoS-Bla was measured by using the FRET substrate CCF2/AM (right panel of each pair). The blue/green fluorescence ratio (response ratio) was calculated as follows: response ratio = (RFU_460 nm – RFU_{460 nm background})/(RFU_530 nm – RFU_{530 nm background}), where RFU indicates the relative fluorescence units. Background fluorescence is fluorescence emission from cells that were infected but not loaded with CCF2-AM. Controls were as follows: strain MDM2423 is PA99::miniCTX-exoS-bla, and strain MDM2424 is ΔpscF PA99::miniCTX-exoS-bla. The results for PA99 carrying WT pscF allele (A) and PhA-resistant mutant alleles (B to J) are shown.

FIG 4

Location of amino acid residues in PscF that can mutate to provide PhA resistance based on a homology structural model of 29 assembled PscF subunits derived from the Salmonella PrgI needle structure (PDB 2LPZ) (see the “PscF forward” model of Lombardi et al. [36]). (A) Alpha carbon backbone structures of three adjacent subunits (A, G, and H) of PscF extracted from the assembled 29 subunit needle homology model; the needle lumen is “northeast” in this view. Looking down the C-terminal alpha-helix of subunit A (green), side chains are shown for all residues identified as capable of mutating to provide PhA resistance in P. aeruginosa (red, high-level resistance; orange, moderate resistance). All except the side chains of residues N11 and I63 extend into the lumen-facing hemisphere. (B) A longitudinal view (Biovia Discovery Studio) of a slice through the center of the PscF needle model reveals a repeated pattern of clustered residues capable of mutating to cause PhA resistance (dark green, high-level resistance; light green, moderate resistance) amid residues only associated with PhA sensitivity or loss of secretion function (dark blue) or not tested by mutagenesis (light blue). Residues in each cluster fall within 9 Å of each other, although they derive from five different adjacent PscF subunits and may constitute a binding site for PhA analogs.