ENaC is a host susceptibility factor to bacterial infections in cystic fibrosis context

Abstract

Cystic fibrosis (CF) is a genetic disease caused by dysfunction in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. Patients with CF are hypersusceptible to Mycobacterium abscessus infection, a fast-growing mycobacterium and harmful opportunistic pathogen. Although CFTR dysfunction is known as a host susceptibility factor for M. abscessus infection, the functional impact of the trimeric Epithelial sodium Channel (ENaC), whose activity is negatively regulated by CFTR, towards M. abscessus infection has not been explored yet. To address this issue, we took advantage of miR-263a deficient Drosophila presenting a CF-like phenotype due to ENaC hyperactivity (ENaC+ ). We observed that the ENaC+ flies were as hypersusceptible to M. abscessus infection as the Cftr-deficient flies. The hypersensitivity of ENaC+ flies to M. abscessus infection was fully rescued by blocking ENaC hyperactivity, both chemically and genetically. Furthermore, we observed that ENaC hyperactivity per se was detrimental to ENaC+ Drosophila, as they were unable to mount an efficient humoral immune response. Upon infection, ENaC+ flies failed to upregulate 20-hydroxyecdysone production, which subsequently altered the production of protective antimicrobial peptides against M. abscessus. Overall, our results show that ENaC plays a key role in host susceptibility to M. abscessus infection and, correlatively to other CF pathogens.

Fig. 1. Cftr depleted and miR-263a deficient flies are hypersusceptible to infection by CF pathogens.

A Survival curves of infected miR-263a(KO)-Gal4/+ (control), and UAS-Cftr-RNAi/+; miR-263a(KO)-Gal4/+(Cftri) flies after injection of 10 CFU of M. abscessus. B Survival curves of infected w¹¹¹⁸ (control) and bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) flies after injection of 10 CFU of M. abscessus. C Survival curves of infected miR-263a(KO)-Gal4/+ (control), and UAS-Cftr-RNAi/+; miR-263a(KO)-Gal4/+(Cftri) flies after injection of 10 CFU of M. marinum. D Survival curves of infected w¹¹¹⁸ (control) and bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) flies after injection of 10 CFU of M. marinum. E Survival curves of infected miR-263a(KO)-Gal4/+ (control), and UAS-Cftr-RNAi/+; miR-263a(KO)-Gal4/+(Cftri) flies after injection of 10 CFU of S. aureus. F Survival curves of infected w¹¹¹⁸ (control) and bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) flies after injection of 10 CFU of S. aureus. G Survival curves of infected miR-263a(KO)-Gal4/+ (control), and UAS-Cftr-RNAi/+; miR-263a(KO)-Gal4/+(Cftri) flies after injection of 10 CFU of B. cepacia. H Survival curves of infected w¹¹¹⁸ (control) and bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) flies after injection of 10 CFU of B. cepacia. The survivals were compared using a log-rank test (Mantel-Cox) on at least 40 flies (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 2. The increased susceptibility conferred by Cftr depletion is dependent on ENaC hyperactivity.

A–D Survival curves of infected miR-263a(KO)-Gal4/+ (control), UAS-Cftr-RNAi/+; miR-263a(KO)-Gal4/+(Cftri), and UAS-Cftr-RNAi/+; miR-263a(KO)-Gal4 maintained on a medium containing 10 mg/mL of amiloride (Cftri + amiloride) flies after injection of 10 CFU of M. abscessus (A), S. aureus (B), B. cepacia (C) or M. marinum (D). The survivals were compared using a log-rank test (Mantel-Cox) on at least 40 flies the bacterial loads, by student t-test (ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3. ENaC hyperactivity confers hypersusceptibility to infections by CF pathogens.

A–E (A) Survival curves of w¹¹¹⁸ (control) and bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) and bft²⁴/miR-263a(KO)-Gal4; UAS-miR-263a/+ (ENaC+ + miR-263a) flies injected with 10 CFU of M. abscessus. B Survival curves of w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) or bft²⁴/miR-263a(KO)-Gal4 supplemented with amiloride (ENaC+ + amiloride) flies injected with 10 CFU of M. abscessus. C Survival curves of w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ), bft²⁴/miR-263a(KO)-Gal4; Nach-RNAi/+ (ENaC+ + Nach-RNAi) and bft²⁴/miR-263a(KO)-Gal4; ppk28-RNAi/+ (ENaC+ + ppk28-RNAi) flies injected with 10 CFU of M. abscessus. D Survival curves of w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ), bft²⁴/miR-263a. (KO)-Gal4; Nach-RNAi/+ (ENaC+ + Nach-RNAi) and bft²⁴/miR-263a(KO)-Gal4; ppk28-RNAi/+ (ENaC + + ppk28-RNAi) flies injected with 10 CFU of S. aureus. E Survival curves of w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ), bft²⁴/miR-263a(KO)-Gal4; Nach-RNAi/+ (ENaC+ + Nach-RNAi) and bft²⁴/miR-263a(KO)-Gal4; ppk28-RNAi/+ (ENaC+ + ppk28-RNAi) flies injected with 10 CFU of B. cepacia. The survivals were compared on at least 60 flies using a log-rank test (Mantel-Cox) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4. ENaC hyperactivity alters Drosophila systemic humoral immune response to CF pathogens in a 20E-dependent manner.

(A–I) (A–C) Normalized disembodied (dib), neverland (nvd), phantom (phtm) and shade (shd) mRNA levels measured by qRT-PCR. RNA was extracted from w¹¹¹⁸ (control) and bft²⁴/miR-263a(KO)-Gal4 (ENaC+) flies on day 3 post-infection with 10 CFU of M. abscessus (A), 12 h post-infection with S. aureus (B) or B. cepacia (C). Error bars correspond to the SD. D Normalized Attacin-A and Metchnikowin mRNA levels measured by qRT-PCR. RNA was extracted on day 3 post-infection from w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+) and bft²⁴/miR-263a(KO)-Gal4 supplemented with 1 µM of 20E (ENaC+ + 20E) flies injected with 10 CFU of M. abscessus. Error bars correspond to the SD. E Normalized Defensin and Drosomycin mRNA levels measured by qRT-PCR. RNA was extracted at 12 h post-infection from w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+) and bft²⁴/miR-263a(KO)-Gal4 supplemented with 1 µM of 20E (ENaC+ + 20E) flies injected with 10 CFU of S. aureus. F Normalized Drosocin, Diptericin and Defensin mRNA levels measured by qRT-PCR. RNA was extracted at 12 hours post-infection from w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+) and bft²⁴/miR-263a(KO)-Gal4 supplemented with 1 µM of 20E (ENaC + + 20E) flies injected with 10 CFU of B. cepacia. G Survival curves of w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) or bft²⁴/miR-263a(KO)-Gal4 supplemented with 1 µM of 20E (ENaC+ + 20E) flies injected with 10 CFU of M. abscessus. H Survival curves of w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+) or bft²⁴/miR-263a(KO)-Gal4 supplemented with 1 µM of 20E (ENaC+ + 20E) flies injected with 10 CFU of S. aureus. I Survival curves of w¹¹¹⁸ (control), bft²⁴/miR-263a(KO)-Gal4 (ENaC+ ) or bft²⁴/miR-263a(KO)-Gal4 supplemented with 1 µM of 20E (ENaC+ + 20E) flies injected with 10 CFU of B. cepacia. Plots represent the ratio of expression level in infected on non-infected flies for A-C and D-F. The survivals were compared on 40 flies at least using a log-rank test (Mantel-Cox) and the expression data, by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).