Discovery of bacterial fatty acid synthase type II inhibitors using a novel cellular bioluminescent reporter assay

Abstract

Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII.

FIG 1

Summary of expression profiling of P. aeruginosa ΔaccD cells complemented by lac-promoted accD in limited versus excess IPTG inducer levels. (A) Abundance of genes upregulated ≥4-fold (gray bars) and ≥8-fold (black bars) as a percentage of genes in each COG category. (B) Log₂ fold upregulation of FASII-related genes from the lipid metabolism COG category. Error bars represent standard deviations (n = 5).

FIG 2

Luminescence reports from wild-type and regulated complemented ΔaccD strains. (A) Kinetics of growth (OD₆₀₀ [open symbols, dotted lines]) and luminescence (RLU [closed symbols, solid lines]) for the ΔaccD/pUCP24-lacI^qlacPO-accD strain MDM1440 and the wild-type PAO1 strain MDM1421, both carrying mini-Tn7-P_fabD-luxCDABE reporter constructs (circles, WT; triangles, ΔaccD strain in 0.05 mM IPTG; diamonds, ΔaccD strain in 0.025 mM IPTG). (B) Luminescence normalized to cell number (RLU/OD₆₀₀) at 270 min of growth in LB for the complemented ΔaccD strains (black) MDM1439, MDM1440, and MDM1441 in 0.025 mM IPTG and wild-type strains (gray) MDM1420, MDM1421, and MDM1422 carrying three different FASII depletion reporters as indicated. Error bars represent standard deviations (n = 3).

FIG 3

Sensitivity, kinetics, and specificity of luminescent response of reporter strains to known FASII inhibitors. (A) Concentration dependence of RLU response of the efflux-deficient FASII reporter strain MDM1460 (P_fabD-lux) at 20 h to two FASII inhibitors: a pyridopyrimidine AccC inhibitor (PYP) and a thiolactomycin FabB inhibitor (TLM). Also included are several non-FASII inhibitor negative controls: a phenoxyacetamide inhibitor of P. aeruginosa type III secretion (PhAA), the gyrase inhibitor ciprofloxacin (CIP), and a benzoyl aminobenzoate (BAB) inhibitor of E. faecalis and S. pyogenes FabH. Two active analogs of the SABA screening hit series, SABA-1 and SABA-2, and one inactive analog are shown. (B) RLU values versus time of incubation in the presence of sub-MICs of FabB inhibitor TLM (1.25 μM, closed symbols) or DMSO (open symbols) for efflux-deficient P. aeruginosa strains carrying luxCDABE driven by the accC (blue squares) (MDM1480) or fabD (red circles) (MDM1460) promoter. (C) Z score of RLU values after 20 h of incubation of efflux-deficient reporter strains in the presence of PYP (2 μM), TLM (1.25 μM), BAB (20 μM), a PhAA T3SS inhibitor (20 μM), and ciprofloxacin (CIP) (2 ng/ml) compared to DMSO. Promoter elements driving luxCDABE and their inhibitory response pathways are given in the key and were integrated into PAO397 to generate strains MDM1460, MDM1473, MDM1505, MDM1419, MDM1520, and MDM1522. Error bars represent standard deviations (n = 3).

FIG 4

Identification of the SABA mechanism of action. (A) Effect of three SABA analogs at 50 μM on the synthesis of cellular macromolecules in P. aeruginosa PAO397 as measured by incorporation of radiolabeled precursors (see Materials and Methods). (B) Effect of three SABA analogs on the activity of purified E. coli acetyl-CoA carboxylase (ACC).

FIG 5

Effect of a SABA-resistant accC allele on the bioluminescent response of a P. aeruginosa FASII reporter strain. PAO397 reporter (P_fabD-luxCDABE) cells were engineered to carry the E. coli wild-type accC gene (A) or the E. coli SABA-resistant accC(F193V) allele (B) on the lac-regulated pUCP24GW-lacI^qlacPO plasmid in place of the chromosomal copy of the accC gene and incubated with a concentration range of SABA-1, TLM, or diluent (DMSO) as shown. Luminescence (RLU) was measured after 20 h. Error bars represent standard deviations (n = 3).

FIG 6

Effect of wild-type and SABA-resistant accC alleles on the growth of P. aeruginosa PAO397 in the presence of inhibitors. The extent of growth was measured at 24 h for PAO397 carrying the endogenous chromosomal copy of the wild-type accC gene (A) or for PAO397 engineered to carry the wild-type P. aeruginosa accC gene (MDM2402) (B), the E. coli wild-type accC allele (MDM2363) (C), or the E. coli SABA-resistant accC(F193V) allele (MDM2365) (D), all under lac regulation, on the pUCP24GW-lacI^qlacPO plasmid in place of the chromosomal copy of the accC gene. Cells were incubated overnight in the presence of a concentration range of TLM (series 1), ciprofloxacin (series 2), or SABA-1 (series 3) in the presence of the indicated IPTG concentration (see the key in panel A2), and growth was measured as OD₆₀₀.