Evolutionary loss of an antibiotic efflux pump increases Pseudomonas aeruginosa quorum sensing mediated virulence in vivo

Abstract

Antibiotic resistance is one of the most pressing threats to human health, yet recent work highlights how loss of resistance may also drive pathogenesis in some bacteria. In two recent studies, we found that β-lactam antibiotic and nutrient stresses faced during infection selected for the genetic inactivation of the Pseudomonas aeruginosa (Pa) antibiotic efflux pump mexEFoprN. Unexpectedly, efflux pump mutations increased Pa virulence during infection; however, neither the prevalence of efflux pump inactivating mutations in real human infections, nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for efflux pump mutations that drive increased virulence in Pa clinical isolates. Using genome sequencing of hundreds of Pa clinical isolates, we show that mexEFoprN efflux pump inactivating mutations are enriched in Pa cystic fibrosis isolates relative to Pa intensive care unit clinical isolates. Combining RNA-seq, metabolomics, genetic approaches, and infection models we show that efflux pump mutants have elevated expression of two key Pa virulence factors, elastase and rhamnolipids, which increased Pa virulence and lung damage during both acute and chronic infections. Increased virulence factor production was driven by higher Pseudomonas quinolone signal levels in the efflux pump mutants. Finally, genetic restoration of the efflux pump in a representative ICU clinical isolate and the notorious CF Pa Liverpool epidemic strain reduced their virulence. Together, our findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity.

Fig. 1:. Inactivating mutations in mexEFoprN are enriched among CF Pa isolates.

a Percentage of acute respiratory ICU Pa isolates with non-synonymous (NS) mutations in mexE, mexF or oprN. b Percentage of CF Pa isolates with NS mutations in mexE, mexF, or oprN. c Genomic location of mexE and mexF inactivating mutations in clinical isolates. Blue indicates prevalence of mutation among ICU Pa isolates and red indicates prevalence among CF Pa isolates.

Fig. 2:. MexEF-OprN loss of function mutants cause lung barrier damage and increased inflammation during infection.

a Schematic of acute lung infections in C57BL/6 mice. b Bacterial CFU enumerated in the lungs of PAO1 (n=21) or PAO1 ΔmexEFoprN (n=16) infected C57BL/6 mice at 24h post infection (hpi). Data show mean ± SEM. Statistical significance analyzed by unpaired t-test. c Number of PAO1 or PAO1 ΔmexEFoprN infected C57BL/6 mice with viable bacteria in the liver at 24 hpi. Statistical significance analyzed by Fisher’s exact test. d Bacterial CFU enumerated in the lungs of PAO1 pMQ72, PAO1 ΔmexEFoprN pMQ72 or PAO1 ΔmexEFoprN pMQ72::mexEFoprN infected C57BL/6 mice at 24hpi. Data show mean ± SEM. Statistical significance analyzed by ANOVA. e Number of PAO1 pMQ72 (n=5), PAO1 ΔmexEFoprN pMQ72 (n=5) or PAO1 ΔmexEFoprN pMQ72::mexEFoprN (n=4) infected C57BL/6 mice with viable bacteria in the liver at 24 hpi. Statistical significance analyzed by Fisher’s exact test.

Fig. 3:. PQS dependent increase in levels of elastase and rhamnolipids drives hypervirulence of PAO1 ΔmexEFoprN.

a Fold change in lasB gene expression in PAO1 and PAO1 ΔmexEFoprN measured by RT-qPCR. Data show mean ± SEM. Statistical significance analyzed by unpaired t-test. b Fold change in rhlA gene expression in PAO1 and PAO1 ΔmexEFoprN measured by RT-qPCR. Data show mean ± SEM. Statistical significance analyzed by unpaired t-test. c Elastase activity determined from the supernatants of PAO1 and PAO1 ΔmexEFoprN using a fluorogenic substrate. Fluorescence measured at 330 nm/460 nm (excitation/emission) is directly proportional to elastase activity and has been normalized to total protein levels in the culture supernatants. Total protein was quantified using Bicinchoninic acid (BCA). Data show mean ± SEM. Statistical significance analyzed by unpaired t-test. d Rhamnolipid production in PAO1 and PAO1 ΔmexEFoprN using cetyltrimethylammonium bromide (CTAB) methylene blue agar plates. A halo (white arrow) indicates rhamnolipid production. e Bacterial CFU enumerated in the lungs of PAO1, PAO1 ΔmexEFoprN, PAO1 ΔmexEFoprNΔlasB, PAO1 ΔmexEFoprNΔrhlA or PAO1 ΔmexEFoprNΔlasBΔrhlA infected C57BL/6 mice at 24 hpi. Data show mean ± SEM. Statistical significance analyzed by ANOVA. f Percentage of PAO1 (n=21), PAO1 ΔmexEFoprN (n=16), PAO1 ΔmexEFoprNΔlasB (n=5), PAO1 ΔmexEFoprNΔrhlA (n=7) or PAO1 ΔmexEFoprNΔlasBΔrhlA (n=6) infected C57BL/6 mice with viable bacteria in the liver at 24hpi. Statistical significance analyzed by Fisher’s exact test. g Total PQS levels quantified in PAO1 and PAO1 ΔmexEFoprN by thin-layer chromatography (TLC). Data show mean ± SEM. Statistical significance analyzed by unpaired t-test.h Intracellular PQS levels quantified in PAO1 and PAO1 ΔmexEFoprN quantified by TLC. Data show mean ± SEM. Statistical significance analyzed by unpaired t-test. i Fold change in lasB gene expression in PAO1 ΔmexEFoprN and PAO1 ΔmexEFoprNΔpqsA measured by RT-qPCR. Data show mean ± SEM. Statistical significance analyzed by unpaired t-test. j Fold change in rhlA gene expression in PAO1 ΔmexEFoprN and PAO1 ΔmexEFoprNΔpqsA measured by RT-qPCR. Data show mean ± SEM. Statistical significance analyzed by unpaired t-test.

Fig. 4:. Hypervirulence of PAO1 ΔmexEFoprN is due to increased levels of elastase and rhamnolipids.

a Schematic of CF lung barrier dysfunction assay. b Transepithelial electrical resistance (TEER) of air liquid interface (ALI) cultures derived from human cystic fibrosis (CF) airways (ΔF508/ΔF508) measured using STX2 chopstick electrodes. TEER < 330 Ω.cm² (dotted line) indicates a loss in epithelial barrier function. TEER was recorded before (0h) and at 6h after exposure to PAO1, PAO1 ΔmexEFoprN or PAO1 ΔmexEFoprNΔlasBΔrhlA supernatants. Data show mean ± SEM. Statistical significance analyzed by ANOVA. c Permeability of the CF airway epithelial barrier determined using 4kDa fluorescein isothiocyanate (FITC) labelled dextran at 6h post exposure to PAO1, PAO1 ΔmexEFoprN or PAO1 ΔmexEFoprNΔlasBΔrhlA supernatants. Data show mean ± SEM. Statistical significance analyzed by ANOVA. d Schematic of chronic lung infections in Scnn1b-Tg mice. e Bacterial CFU enumerated on day 7 from the lung homogenates of Scnn1b-Tg mice infected with PAO1 or PAO1 ΔmexEFoprN embedded in agar beads. Data show mean ± SEM. Statistical significance analyzed by unpaired t-test. f Survival curves of Scnn1b-Tg mice infected with PAO1 (n=10) or PAO1 ΔmexEFoprN (n=13) embedded in agar beads. Statistical significance analyzed by Mantel-Cox test. g-h Flow cytometry for total immune cells, monocyte, macrophages and neutrophils in the lungs of PAO1 or PAO1 ΔmexEFoprN infected mice at day 7 post infection. Data show mean ± SEM. Statistical significance analyzed by unpaired t-test.

Fig. 5:. Restoration of mexEFoprN expression reduces virulence of clinical Pa isolates.

a Fold change in lasB and rhlA gene expression in PAJSL219A pMQ72 and PAJSL219A pMQ72::mexEFoprN measured by RT-qPCR. Statistical significance analyzed by unpaired t-test. b Fold change in lasB and rhlA gene expression in LESB58 pME6032 and LESB58::mexEFoprN measured by RT-qPCR. Statistical significance analyzed by unpaired t-test. c Bacterial CFU were enumerated in the lungs of PAJSL219A pMQ72 and PAJSL219A::mexEFoprN infected C57BL/6 mice at 24 hpi. Statistical significance analyzed by unpaired t-test. d Bacterial CFU enumerated on day 7 from the lung homogenates of Scnn1b-Tg mice infected with Pa LESB58 pME6032 or Pa LESB58 pME6032::mexEFoprN embedded in agar beads. Statistical significance analyzed by unpaired t-test. e Transepithelial electrical resistance (TEER) of air liquid interface (ALI) cultures derived from human cystic fibrosis (CF) airways (ΔF508/ΔF508) measured using STX2 chopstick electrodes. TEER < 330 Ω.cm² (dotted line) indicates a loss in epithelial barrier function. TEER was recorded before (0h) and at 24h after exposure LESB58 pME6032 or LESB58::mexEFoprN supernatants. TEER < 330 Ω.cm² (dotted line) indicates a loss in epithelial barrier function. Statistical significance analyzed by ANOVA. f Permeability of the CF airway epithelial barrier determined using 4kDa fluorescein isothiocyanate (FITC) labelled dextran at 24h post exposure to LESB58 pME6032 or LESB58::mexEFoprN supernatants. Statistical significance analyzed by unpaired t-test. g Model depicting the important virulence mechanisms enhanced in Pa ΔmexEFoprN and the effect on the host during infection. Efflux pump mutants increase elastase and rhamnolipid production, leading to increased epithelial damage, replication during infection, dissemination, and lethality in vivo. In a-f, data shown are mean ± SEM.