Identification of a copper-responsive small molecule inhibitor of uropathogenic Escherichia coli

Abstract

Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC ΔtolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli, this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

Keywords: E. coli; UPEC; antimicrobial; copper; isothiazolone.

FIG 1

Design of high throughput screening and identification of ECIN. (A) Frozen stocks of UPEC strain CFT073 ΔtolC were thawed and diluted in LB media. An Analytik Cybio media handler was used to add media, drugs, cells, and resazurin into the wells. Plates were incubated for 24 h and then fluorescence was measured. A representative image of a screening plate is pictured above. Columns 1, 2, 23, and 24 were used for controls. Each screening plate had ~320 different small molecules. Red indicates complete bacterial inhibition while blue indicates complete bacterial growth. A representative hit is indicated by the circle. (B) Structure of 5-chloro-2-(4-chlorophenyl)−4-methyl-2,3-dihydro-1,2-thiazol-3-one the hit identified in our screen was named as ECIN and further characterized in this report. ECIN meets the Lipinski’s rule of 5 for drug-like properties, and the cutoff values for these criteria are indicated in the brackets.

FIG 2

Interaction between ECIN and copper. (A) IC of ECIN against UPEC decreases in the presence of copper. Metabolic activity of UPEC treated with varying concentrations of ECIN with or without copper was determined by measuring the fluorescence of resorufin and used to calculate % growth. Mean with SEM are represented (n = 9). (B) ECIN magnifies the copper efflux response in UPEC. The expression of major copper efflux gene, cusC, was assessed in UPEC when treated with ECIN (1.3 µg/mL; 5 µM), copper (5 µM), and the two in combination (n = 9). Fold-change relative to untreated wild-type strain is presented ANOVA with Bonferroni, **P < 0.01. (C) ICP-MS revealed that ECIN increased the amount of cell-associated copper in UPEC (n = 9). ANOVA with Bonferroni, ***P < 0.001 and ****P < 0.0001.

FIG 3

ECIN is bactericidal against UPEC. (A) Optical density of UPEC CFT073 cultures containing ECIN or controls was measured over time. Chloramphenicol (MIC = 8 µg/mL) and ciprofloxacin (MIC = 32 ng/mL) were used as bacteriostatic and bactericidal controls, respectively (n = 9). Error bars represent SEM. (B) The cultures from the growth curve were spot plated on LB agar to determine the presence of viable bacteria.

FIG 4

ECIN prevents the formation of biofilms but is unable to reduce pre-existing biofilms. (A) Biofilm prevention assays. The ratio of biomass (OD₅₅₀) formed to growth (OD₆₀₀) when UPEC was inoculated with ECIN. (B) Biofilm eradication assays. The biomass to growth ratio of pre-formed biofilms after treatment with ECIN for 24 h. No statistical significance was detected (ANOVA with Bonferroni). Error bars represent SEM (n = 9).

FIG 5

ECIN induces several stress response systems and halts metabolic processes. Representation of upregulated genes (A and B) and downregulated genes (C and D). RNAseq was performed on UPEC when treated with ECIN (1.3 µg/mL; 5 µM) at MIC and subinhibitory concentrations of copper (5 µM). The 10 most upregulated and downregulated genes are shown from UPEC treated with ECIN alone, and ECIN plus copper compared to the untreated control. Venn diagrams represent the number of unique or shared genes with other treatment groups between ECIN alone or ECIN plus copper. RNA was isolated three individual times and tested one time each (N = 3). Error bars represent SEM.

FIG 6

Cysteine rescues UPEC growth in the presence of ECIN. UPEC strain CFT073, was cultured in SLM in the presence or absence of ECIN (1.3 µg/mL; 5 µM), copper sulfate (5 µM), combination (5 µM each). Optical density was measured over time (n = 9). Error bars represent SEM. Cys, L-cysteine (1 mM) and Ser, L-serine (1 mM).

FIG 7

ECIN is weakly cytotoxic to a hepatic human cell line. ECIN in the presence or absence of copper was tested against the hepatic cell line HepG2 to determine cytotoxicity. Shades of red indicate cell viability in ECIN (2.5–10 µM) treated groups while shades of purple indicate the cell viability in groups treated with ECIN plus copper. No significant difference was found between treatments (n = 9, ANOVA with Bonferroni). Error bars represent SEM.