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

Abstract

Antibiotic resistance is a threat to human health, yet recent work highlights how loss of resistance may drive pathogenesis in some bacteria. In two recent studies, we found that β-lactam antibiotics and nutrient stresses faced during infection selected for genetic inactivation of the Pseudomonas aeruginosa antibiotic efflux pump mexEFoprN. Unexpectedly, efflux pump mutations increased P. aeruginosa virulence during infection; however, neither the prevalence of mexEFoprN 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 virulence enhancing mutations. Using genome sequencing of clinical isolates, we show that mexEFoprN efflux pump inactivating mutations are enriched in P. aeruginosa isolates from cystic fibrosis infections relative to isolates from acute respiratory infections. Combining RNA-seq, metabolomics, genetic approaches, and infection models we show that efflux pump mutants have elevated quorum sensing driven expression of elastase and rhamnolipids which increase P. aeruginosa virulence during acute and chronic infections. Restoration of the efflux pump in a representative respiratory isolate and the notorious cystic fibrosis Liverpool epidemic strain reduced their virulence. These 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 P. aeruginosa isolates.

a Percentage of acute respiratory ICU P. aeruginosa isolates with inactivating mutations or nonsynonymous single nucleotide polymorphisms (NS SNPs) in mexE, mexF, or oprN. b Percentage of CF P. aeruginosa isolates with inactivating mutations or NS SNPs in mexE, mexF, or oprN. c Genomic location of mexE and mexF inactivating mutations in clinical isolates. Blue indicates prevalence of mutation among ICU P. aeruginosa isolates and red indicates prevalence among CF P. aeruginosa isolates. Circular cladograms for ICU (d) and CF (e) P. aeruginosa isolates, respectively. Strains with WT mexE, mexF, and oprN are indicated by blue circles, strains with NS SNPs are indicated by green rectangles, strains with inactivating mutations are indicated by red dots, reference strains are indicated in black.

Fig. 2. MexEF-OprN loss of function mutants cause systemic infection and increased inflammation during infection.

a Schematic representation of acute lung infections in C57BL/6 mice created using BioRender. b Bacterial CFU enumerated from the whole lung lysate of PAO1 (n = 21) or PAO1 ∆mexEFoprN (n = 16) infected C57BL/6 mice at 24 h post infection (hpi). Data show mean ± SEM. Statistical significance analyzed by two-sided unpaired t-test. c Percentage of PAO1 (n = 21) or PAO1 ∆mexEFoprN (n = 16) infected C57BL/6 mice with viable bacteria in the liver at 24 hpi. Statistical significance analyzed by Fisher’s exact test. n indicates the number of mice/group. d Bacterial CFU enumerated from the whole lung lysate of PAO1 pMQ72 (n = 5), PAO1 ∆mexEFoprN pMQ72 (n = 5) or PAO1 ∆mexEFoprN::mexEFoprN (n = 4) infected C57BL/6 mice at 24 hpi. Data show mean ± SEM. Statistical significance analyzed by ANOVA Kruskal–Wallis test. e Percentage of PAO1 pMQ72 (n = 5), PAO1 ∆mexEFoprN pMQ72 (n = 5), or PAO1 ∆mexEFoprN::mexEFoprN (n = 4) infected C57BL/6 mice with viable bacteria in the liver at 24 hpi. Statistical significance analyzed by Fisher’s exact test. n indicates the number of mice/group.

Fig. 3. QS dependent increase in levels of elastase and rhamnolipids drives hypervirulence of PAO1 ∆mexEFoprN.

a Fold change in lasB gene expression in PAO1 and PAO1 ∆mexEFoprN measured by RT-qPCR. Data show mean ± SEM, n = 3 independent experiments. Statistical significance analyzed by two-sided unpaired t-test. b Fold change in rhlA gene expression in PAO1 and PAO1 ∆mexEFoprN measured by RT-qPCR. Data show mean ± SEM, n = 3 independent experiments. Statistical significance analyzed by two-sided unpaired t-test. c Elastase activity determined from the supernatants of PAO1 and PAO1 ∆mexEFoprN using a fluorogenic substrate. Fluorescence has been normalized to total protein levels in the culture supernatants. Data show mean ± SEM, n = 6 independent experiments. Statistical significance analyzed by two-sided unpaired t-test. d Rhamnolipid production in PAO1 and PAO1 ∆mexEFoprN using cetyltrimethylammonium bromide (CTAB) methylene blue agar plates. A halo (white arrow) indicates rhamnolipid production. e Bacterial CFU enumerated from the whole lung lysate of PAO1 (n = 21), PAO1 ∆mexEFoprN (n = 16), PAO1 ∆mexEFoprN∆lasB (n = 5), PAO1 ∆mexEFoprN∆rhlA (n = 7) or PAO1 ∆mexEFoprN∆lasB∆rhlA (n = 6) infected C57BL/6 mice at 24 hpi. Data show mean ± SEM. Statistical significance analyzed by ANOVA Kruskal-Wallis test. f Percentage of PAO1 (n = 21), PAO1 ∆mexEFoprN (n = 16), PAO1 ∆mexEFoprN∆lasB (n = 5), PAO1 ∆mexEFoprN∆rhlA (n = 7) or PAO1 ∆mexEFoprN∆lasB∆rhlA (n = 6) infected C57BL/6 mice with viable bacteria in the liver at 24 hpi. Statistical significance analyzed by Fisher’s exact test. n indicates the number of mice/group. g Total PQS levels quantified in PAO1 and PAO1 ∆mexEFoprN by thin-layer chromatography (TLC). Data show mean ± SEM, n = 7 independent experiments. Statistical significance analyzed by two-sided unpaired t-test. h Intracellular PQS levels quantified in PAO1 and PAO1 ∆mexEFoprN quantified by TLC. Data show mean ± SEM, n = 7 independent experiments. Statistical significance analyzed by two-sided unpaired t-test. i Fold change in lasB gene expression in PAO1 ∆mexEFoprN and PAO1 ∆mexEFoprN∆pqsA measured by RT-qPCR. Data show mean ± SEM, n = 3 independent experiments. Statistical significance analyzed by two-sided unpaired t-test. j Fold change in rhlA gene expression in PAO1 ∆mexEFoprN and PAO1 ∆mexEFoprN∆pqsA measured by RT-qPCR. Data show mean ± SEM, n = 3 independent experiments. Statistical significance analyzed by two-sided unpaired t-test.

Fig. 4. PAO1 ∆mexEFoprN is hypervirulent in CF infection models.

a Schematic representation of CF airway barrier dysfunction assay created using Biorender. b Transepithelial electrical resistance (TEER) of air liquid interface (ALI) cultures derived from human cystic fibrosis (CF) airways (∆F508/∆F508 CFTR) measured using STX2 chopstick electrodes. TEER < 330 Ω.cm² (dotted line) indicates a loss in epithelial barrier function. TEER was recorded before (0 h) and at 6 h after exposure to PAO1, PAO1 ∆mexEFoprN, or PAO1 ∆mexEFoprN∆lasB∆rhlA supernatants. Data show mean ± SEM, n = 3 independent experiments. Statistical significance analyzed by ANOVA Fisher’s LSD test. c Permeability of the CF airway epithelial barrier determined using 4 kDa fluorescein isothiocyanate (FITC) labeled dextran at 6 h post exposure to PAO1, PAO1 ∆mexEFoprN, or PAO1 ∆mexEFoprN∆lasB∆rhlA supernatants. Data show mean ± SEM, n = 3 independent experiments. Statistical significance analyzed by ANOVA Fisher’s LSD test. d Schematic representation of chronic lung infections in Scnn1b-Tg mice created using Biorender. e Bacterial CFU enumerated on day 7 from the lung homogenates of Scnn1b-Tg mice infected with PAO1 (n = 10) or PAO1 ∆mexEFoprN (n = 13) embedded in agar beads. Data show mean ± SEM. Statistical significance analyzed by two-sided unpaired t-test. f Survival curves of Scnn1b-Tg mice infected with PAO1 (n = 10) or PAO1 ∆mexEFoprN (n = 13) embedded in agar beads. Statistical significance analyzed by Mantel-Cox test. n indicates the number of mice/group. g, h Flow cytometry for total immune cells, monocyte, macrophages, and neutrophils in the lungs of PAO1 (n = 4) or PAO1 ∆mexEFoprN (n = 3) infected mice at day 7 post infection. Data show mean ± SEM. Statistical significance analyzed by two-sided unpaired t-test.

Fig. 5. Restoration of mexEFoprN expression reduces virulence of P. aeruginosa clinical isolates.

a RT-qPCR fold-change in lasB and rhlA expression in PAJSL219A pMQ72 and PAJSL219A::mexEFoprN (mean ± SEM, n = 3; two-sided unpaired t-test). b RT-qPCR fold-change in lasB and rhlA expression in LESB58 pME6032 and LESB58::mexEFoprN (mean ± SEM, n = 3; two-sided unpaired t-test). c Total PQS in PAJSL219A pMQ72 and PAJSL219A::mexEFoprN by TLC (mean ± SEM, n = 7, two-sided unpaired t-test). d Intracellular PQS in PAJSL219A pMQ72 (n = 7) and PAJSL219A::mexEFoprN (n = 6) quantified by TLC (mean ± SEM, two-sided unpaired t-test). e Total PQS in LESB58 pME6032 and LESB58::mexEFoprN by TLC (mean ± SEM, n = 3, two-sided unpaired t-test). f Intracellular PQS in LESB58 pME6032 and LESB58::mexEFoprN by TLC (mean ± SEM, n = 3, two-sided unpaired t-test). g CFU in C57BL/6 mouse whole lung lysates at 24 hpi with PAJSL219A pMQ72 (n = 7) and PAJSL219A::mexEFoprN (n = 5; mean ± SEM, two-sided unpaired t-test). h CFU on day 7 from lung homogenates of Scnn1b-Tg mice infected with LESB58 pME6032 (n = 5) or LESB58::mexEFoprN (n = 4) embedded in agar beads (mean ± SEM, two-sided unpaired t-test). i TEER of ALI cultures of human CF airways (∆F508/∆F508) before (0 h) and 24 h after exposure to LESB58 pME6032 or LESB58::mexEFoprN supernatants. Dotted line (TEER < 330 Ω.cm²): loss in epithelial barrier function (mean ± SEM, n = 3, ANOVA Fisher’s LSD test). j CF airway epithelial barrier permeability using 4 kDa FITC labeled dextran at 24 h post exposure to LESB58 pME6032 or LESB58::mexEFoprN supernatants (mean ± SEM, n = 3, two-sided unpaired t-test). k, l Percent predicted forced expiratory volume in 1 s (FEV₁pp, k) and percent predicted forced vital capacity (FVCpp, l) of pwCF infected with P. aeruginosa strains with wild-type mexEFoprN (WT) (n = 11), non-synonymous SNPs (n = 9), inactivating mutations (In) (n = 2), or a mixed population (WT + SNP (n = 3), WT + SNP+In (n = 1), SNP+In (n = 2)). Dotted lines indicate FEV₁pp = 80% (k) and FVCpp (l, i.e., 80–100%), the lower limits of normal FEV₁pp and FVCpp (k, l: ANOVA Fisher’s LSD test). m Model: mexEFoprN efflux pump mutants increase elastase and rhamnolipid, leading to increased epithelial damage, replication during infection, dissemination, and lethality in vivo (created using Biorender).