Peptide-mimetic treatment of Pseudomonas aeruginosa in a mouse model of respiratory infection

Abstract

The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2000 deaths in the U.S. annually. While the emergence of resistant bacteria has become ominously common, identification of useful new drug classes has been limited over the past over 40 years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity in mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30-60 min in vitro, and is effective against a range of clinical isolates, including extensively drug resistant strains. In vivo, TM5 significantly reduced bacterial load in the lungs within 24 h compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

Fig. 1. Bactericidal activity of Peptoids TM1, TM5, and TM6.

Relative luminescence (A) and CFUs/mL (B) for P. aeruginosa Xen41 treated with 2-fold dilutions of TM1, TM5, and TM6. A TM1, TM5, and TM6 were all significantly different from the no treatment control with respect to their relative luminescence at concentrations of 1 μg/ml and higher. B TM1 was significantly different from the no treatment control for CFUs/mL at concentrations of 0.5 μg/mL and above, while TM5 and TM6 were significantly different at 2 and 4 μg/ml respectively. Data points are represented as means (n=replicates). Error is shown in ± standard deviation (SD). Statistics were performed using 2-way ANOVA, comparing antimicrobial to no treatment control. P values are: <0.001 = ***, between 0.001 and 0.01 = **, and between 0.01 and 0.05 = *.

Fig. 2. Bactericidal of standard antibiotics.

Relative OD₆₀₀ (A) and CFUs/ml (B) for P. aeruginosa Xen41 with 2-fold dilutions of ciprofloxacin, ceftazidime, kanamycin, and meropenem. A Ciprofloxacin was significantly different above concentrations of 0.0625 μg/ml and above. Ceftazidime and meropenem were significantly different at concentrations above 0.125 μg/ml, while kanamycin was significantly different at concentrations of 8 μg/ml and above. B Ciprofloxacin showed a significant decrease compared to the no treatment control for CFUs/ml at all concentrations, while ceftazidime and meropenem were significantly different at 0.25 μg/ml and above. Kanamycin didn’t show any significant difference at any concentrations. Data points are represented as means using four replicates. Error is shown in ± standard deviation (SD). Statistics were performed using 2-way ANOVA, comparing antimicrobial to no treatment control. P values are: <0.0001 = ****, between 0.0001 and 0.001 = ***, between 0.001 and 0.01 = **, and between 0.01 and 0.05 = *.

Fig. 3. Comparative time-kill experiments of Peptoid TM5 and ciprofloxacin.

Time course experiments for 2-fold dilutions of TM5 (A) and ciprofloxacin (B) mixed with P. aeruginosa Xen41 were measured for luminescence at the following time points: 0, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min. In addition to 2-fold dilutions, a control with PBS was also measured (n = 4 replicates).

Fig. 4. Resistance to Peptoid TM5 could not be derived through laboratory evolution.

P. aeruginosa PAO1 was serially passaged in increased concentrations of either A ciprofloxacin or B peptoids TM5. After 3 weeks of successive passaging in concentrations doubling weekly from 0.5XMIC-2XMIC, resistance of ciprofloxacin had readily emerged with the MIC increasing from 0.125 µg/mL to 8 µg/mL. In contrast, no increase in MIC was observed for peptoid TM5. To evaluate whether TM5 was effective against drug resistant strains, the newly evolved ciprofloxacin-resistant isolates were tested against peptoids TM5 (C), which was found to be equally effective with a MIC of 4 µg/mL.

Fig. 5. Cytotoxicity of Peptoids TM1, TM5, and TM6 on mammalian cells.

An Alamar Blue assay was used to determine the effects of 24 h treatment with 2-fold dilutions of each peptoid (A) or standard frontline antibiotic (B) on the viability of A549 human lung epithelial cells. All concentrations and conditions were tested in triplicate, with error is shown as standard deviation.

Fig. 6. Cytotoxicity of Peptoid TM5 in three-dimensional models of the human lung.

A549 cells were grown in three-dimensional models of the human lung in the form of A spheroid cultures or B air-liquid interface (ALI) cultures. C Culturing A549 cells under conditions which mimic an in vivo lung environment result in decreased cytotoxicity following peptoid treatment as measured by MTS assay. D Integrity of the barrier function of ALI monolayers following peptoid treatment as measured by dextran exclusion assay showing that the monolayers remain intact following peptoid treatment with no significant difference in dextran diffusion observed at 200 µM concentrations or below. Dextran Alexa FluorTM 488 was used for the 3000 Da exclusion and Dextran Alexa FluorTM 647 was used for the 10,000 Da exclusion. Statistics were performed using 2-way ANOVA, using Šídák’s multiple comparisons test to compare sample means, with standard error mean (SEM). P values represented as: <0.0001 = ****, between 0.0001 and 0.001 = ***, between 0.001 and 0.01 = **, and between 0.01 and 0.05 = *.

Fig. 7. In vivo Toxicity of Peptoid TM5 following intratracheal delivery.

A BALB/c mice were treated with 50 µL of either 40 µg/mL (▴) or 80 µg/mL (○) doses of TM5 and monitored for survival at 0, 2, 4, 6, 8, 10, 24, 30, and 48 h post-treatment (n = 2 mice per concentration). B BALB/c mice were treated with increasing doses of TM5 to determine a toxicity threshold and monitored for disease symptoms at 0, 2, 4, 6, 8, 10, 24, 30, and 48 h post-treatment as indicated below the X-axis. Error bars represent SEM across n = 4 mice, and timepoints marked with an X indicated animals humanely euthanized at that timepoint.

Fig. 8. Disease scores of mice infected with P. aeruginosa Xen41 following peptoid or antibiotic treatment.

BALB/c mice were infected with P. aeruginosa via the intratracheal route and treated with TM1, TM5, TM6, or ciprofloxacin. A modified Karnofsky score was used to score any adverse effects observed over the course of the infection. A Average overall Karnofsky scores for each group of mice tested from 0 h to 72 h, with variance represented as SD. B Average Karnofsky scores for each group of mice over time, split into various categories tested. Columns marked with an ‘X’ indicate where a mouse was sacrificed ahead of schedule.

Fig. 9. Bacterial burdens of P. aeruginosa Xen41-infected mice following treatment with peptoid or antibiotic.

CFUs taken from sacrifice of day 1 mice. A CFUs/ml of Xen41 in the lungs of mice from varying treatment groups. B CFUs/ml of Xen41 in the spleen of mice from varying treatment groups. For all graphs, p-values are compared to the average of untreated mice. The line across represents the average of all data points within the group.

Fig. 10. IVIS imaging of treated and untreated mice at 24 h post-infection.

Images were collected using the same scale of radiance for all mice tested. Each group of mice initially sacrificed were imaged together. An additional mouse in the TM6 group was imaged later using the same scale of radiance as it met the euthanization criteria.

Fig. 11. Histological analysis of treated and untreated mice.

Breakdown of histology scoring as A averages of groups with standard error for each category. C Neutrophil counts per mm² for histology sections with standard error via computational analysis. Breakdown of histology scoring as B individual scoring across several categories for each mouse sacrificed at day 1.

Fig. 12. Histology sectioning of lungs for representative mice in each treatment group.

Histology of H&E-stained tissue sections of lungs for representative mice in each group. Insets are increased magnification of the box seen in the larger image. The short bar, in the inset, is equivalent to 50 μm and the longer bar is equivalent to 500 μm.