Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections

Abstract

The lack of new antibiotics is among the most critical challenges facing medicine. The problem is particularly acute for Gram-negative bacteria. An unconventional antibiotic strategy is to target bacterial nutrition and metabolism. The metal gallium can disrupt bacterial iron metabolism because it substitutes for iron when taken up by bacteria. We investigated the antibiotic activity of gallium ex vivo, in a mouse model of airway infection, and in a phase 1 clinical trial in individuals with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa airway infections. Our results show that micromolar concentrations of gallium inhibited P. aeruginosa growth in sputum samples from patients with CF. Ex vivo experiments indicated that gallium inhibited key iron-dependent bacterial enzymes and increased bacterial sensitivity to oxidants. Furthermore, gallium resistance developed slowly, its activity was synergistic with certain antibiotics, and gallium did not diminish the antibacterial activity of host macrophages. Systemic gallium treatment showed antibiotic activity in murine lung infections. In addition, systemic gallium treatment improved lung function in people with CF and chronic P. aeruginosa lung infection in a preliminary phase 1 clinical trial. These findings raise the possibility that human infections could be treated by targeting iron metabolism or other nutritional vulnerabilities of bacterial pathogens.

Figure 1.. Expectorated Sputum from CF patients is iron limited.

A. Addition of indicated concentrations of FeCl₃ to sputum supernatants increased the growth rate and cell yield of inoculated P. aeruginosa. Results are representative of 5 sputum samples (see Figs S1 and S2); error bars indicate SEM; * indicates p<0.01 vs. no iron addition. B and C. P. aeruginosa incubated in CF sputum highly express the pyoverdine biosynthetic gene pvdA (as measured by a pvda-gfp reporter) (B), and produce high levels of pyoverdine indicating iron starvation (C). Addition of FeCl₃ suppressed both effects. Results are mean of 3 replicates and representative of 3 experiments; error bars indicate SEM; * indicates p<0.01 vs. no iron addition. Also see Fig S1 for experiments with sputum from another subject.

Figure 2.. Gallium inhibits P. aeruginosa growth in CF sputum.

Gallium inhibited the growth of P. aeruginosa inoculated into CF sputum supernatants that were not (A) and were (B) supplemented with FeCl₃. FeCl₃ addition markedly increased P. aeruginosa growth rate and cell yield, but Ga(NO₃)₃ concentrations that effectively suppressed growth in the un-supplemented condition (4 and 6 μM) remained effective at suppressing P. aeruginosa growth. Results are mean of 3 replicates and representative of 3 experiments; error bars indicate SEM; * indicates p<0.01 vs. no gallium; ^# indicates p<0.01 vs. no added iron.

Figure 3.. Gallium inhibits P. aeruginosa catalase and ribonucleotide reductase, but not SOD or aconitase activity.

P. aeruginosa (strain PAO1) was incubated overnight in an iron-rich medium (10% TSB) containing increasing concentrations of Ga(NO₃)₃. The bacteria were harvested, lysed, and enzymatic activity determined for ribonucleotide reductase (A); aconitase (B); catalase (C); and superoxide dismutase (SOD) (D). Results shown are representative of 5 experiments and are mean enzyme activity relative to bacteria not treated with gallium; error bars indicate SEM. Gallium exposure significantly inhibited ribonucleotide reductase and catalase activity at gallium concentrations ≥2.5 μM and ≥ 20 μM, respectively (p<0.05, ANOVA). No significant effect of gallium on aconitase or SOD activity was observed.

Figure 4.. Gallium sensitizes P. aeruginosa to killing by peroxides.

Bacteria were grown without and with the indicated sub-inhibitory concentrations of Ga(NO₃)₃ and then exposed to oxidants generating peroxide, including H₂O₂ (A) and tert-Butyl hydroperoxide (Tert-Butyl) (B); and superoxide, including paraquat (C) and phenazine methosulfate (PMS) (D). Sub-inhibitory Ga(NO₃)₃ increased sensitivity to killing by peroxides. Error bars indicate SEM, data are mean values of three replicate experiments and representative of three independent experiments; error bars indicate SEM; * indicates p<0.01.

Figure 5.. Continuous passaging modestly increases gallium resistance.

Twelve replicate cultures of wild type (A-C) and AhitAB P. aeruginosa (D) were passaged in in gallium (A and D), aztreonam (B), and tobramycin (C). Bacteria were initially grown in the absence of drug, then subcultured into a range of drug concentrations. Each day, cells from the highest concentration of drug that supported growth was recorded, and cells from this condition were re-inoculated into a range of drug concentrations. The mean fold change in highest drug concentration that permitted growth (of 12 replicate cultures) is plotted as a function of the passaging day. Error bars indicate SEM. The highest drug concentration that permitted growth at in these conditions at day 0 was 1 μg/ml tobramycin and aztreonam, and 64 μg/ml gallium for wild type P. aeruginosa, and 128 μg/ml gallium for ΔhitAB P. aeruginosa.

Figure 6.. Combined activity of gallium with antibiotics.

Gallium was synergistic with colistin (A) and piperacillin/tazobactam (B), but antagonistic to tobramycin (C). Photographs (top) show disc diffusion assays. Synergistic activity was indicated by the increased zone of inhibition produced by colistin and piperacillin/tazobactam in the proximity of the gallium-containing disc. Antagonistic activity was indicated by the decreased zone of inhibition produced by tobramycin in the proximity of the gallium-containing disc. The yellow dotted line represented the expected activity (in preventing P. aeruginosa growth) of the antibiotic in the absence of gallium. Graphs (middle) show time-kill assays using sub-inhibitory concentrations of gallium and inhibitory concentrations of antibiotics. Synergistic activity was indicated by the ability of sub- inhibitory gallium to enhance killing by colistin and piperacillin/tazobactam. Antagonistic activity was indicated by the ability of sub-inhibitory gallium to reduce killing by tobramycin. Error bars indicate SEM. Isobolograms (bottom) show results of checkerboard assays presented as the fractional inhibitory concentrations (FICs) of the 2 factors in combination. Calculations are described in methods. Experiments were repeated 2–4 times, each with similar results.

Figure 7.. Parenteral gallium treats murine lung infections.

A. A single dose of gallium-free vehicle (red line) or gallium (50 μl of 250 mM Ga(NO₃)₃) was administered by the intraperitoneal (IP) route 3 hours (blue line) or 12 hours after (green line) intratracheal infection with P. aeruginosa (n=7 mice for Ga and 8 for vehicle); * indicates P< 0.001 vs. vehicle control.B. P. aeruginosa were enumerated in bronchoalveolar lavage fluid and blood sampled 12 hours after mice were infected by the intratracheal route and treated with gallium (IP) or vehicle (IP) 3 hours after infection (n=4 mice for vehicle alone and 5 for Ga). * indicates P< 0.001 vs. vehicle control. C. Administering intranasal (IN) FeCl₃ (10 μl of 2 mM FeCl₃) (black line) at the time of infection reduced the protective effect of intraperitoneal gallium, as compared intranasal iron-free vehicle (blue line) * indicates P< 0.001 vs. vehicle control; # indicates P< 0.05 vs. vehicle control; p=0.055 vs. no IN FeCl₃. The red line shows mouse survival without gallium (vehicle administered IP and IN) (n=6 mice in each group).

Figure 8.. IV gallium produces sustained blood and sputum levels, and improves lung function.

Plasma (A) and sputum (B) gallium levels in CF subjects treated with IV gallium for 5 days; boxes show 25th to 75th percentiles, hatches shows means, and whiskers show minimum and maximum values. Data shown are for cohort 1 and 2 (100 mg/m² per day and 200 mg/m² per day, respectively) combined. See Fig S9 for data from each cohort separated. Mean change in lung function (as measured by FEV₁ in mls) by study day are for both cohorts pooled together (C), and cohort 1 (D) and 2 (E) separately (100 mg/m² per day and 200 mg/m² per day, respectively). Bars represent the 95% Confidence Intervals; * represents p values > 0.005.