PA5470 Counteracts Antimicrobial Effect of Azithromycin by Releasing Stalled Ribosome in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa causes various acute and chronic infections in humans. Treatment with azithromycin (AZM) has been shown to benefit patients with chronic P. aeruginosa infections. By binding to the exit tunnel of the 50S ribosome, AZM causes ribosome stalling and depletion of the intracellular tRNA pool. It has been shown that AZM is able to kill stationary-phase P. aeruginosa cells and repress quorum sensing-regulated virulence factors as well as swarming motility. In P. aeruginosa, the PA5470 gene encodes a putative peptide chain release factor whose expression is highly induced by macrolide antibiotics. However, its function remains unknown. Here, we found that overexpression of PA5470 increased bacterial tolerance against AZM and alleviated the repression of swarming motility. Ribosome pulldown assays revealed that PA5470 contributes to the release of ribosome stalled by AZM. We further demonstrate that overexpression of PA5470 counteracts AZM-mediated repression of the translation of the quorum sensing regulator RhlR. Overall, our results revealed a novel role of PA5470 in the bacterial response to AZM.

Keywords: PA5470; Pseudomonas aeruginosa; antibiotic resistance; azithromycin.

FIG 1

Expression levels of PA5470 under treatment with various antibiotics. Wild-type PA14 was treated with carbenicillin (37.5 μg/ml; CAR), ciprofloxacin (0.78 μg/ml; CIP), amikacin (1.25 μg/ml; AMK), and azithromycin (200 μg/ml; AZM) for 3 h. Total RNA was isolated, and the mRNA levels of PA5470 were determined by real-time PCR. Error bars represent standard deviations. **, P < 0.01; ***, P < 0.001, compared to results with untreated PA14 (by a Student's t test).

FIG 2

Increased susceptibility of the ΔPA5470 mutant to AMK and AZM. Exponential-phase bacteria were treated with 0, 2.5, 5, 10, and 20 μg/ml AMK for 1.5 h at 37°C with shaking (A and C). Stationary-phase bacteria were treated with 0, 2, 5, and 10 μg/ml AZM for 20 h at 37°C with shaking (B and D). The bacterial viabilities were determined by serial dilution and plating. Error bars represent standard deviations. *, P < 0.05; **, P < 0.01, compared to results with PA14 containing empty vector (by Student's t test).

FIG 3

Overexpression of PA5470 increases bacterial tolerance to AMK and AZM without affecting the expression of mexX and mexY. (A) Exponential-phase bacteria were treated with 0, 2.5, 5, 10, and 20 μg/ml AMK for 1.5 h at 37°C with shaking. (B) Stationary-phase bacteria were treated with 0, 2, 5, and 10 μg/ml AZM for 20 h at 37°C with shaking. (C) Bacteria of the indicated strains were grown for 3 h at 37°C with shaking. Total RNA was isolated, and the mRNA levels of mexX and mexY were determined by real-time PCR. The bacterial viabilities were determined by serial dilution and plating. Error bars represent standard deviations. *, P < 0.05; **, P < 0.01, (compared to results for PA14 containing empty vector or overexpressing PA5470); ns, not significant (compared to results with PA14 containing empty vector) (Student's t test).

FIG 4

Effect of PA5470 overexpression on swarming motility and expression of rhlA in the presence of AZM. PA14 (I), the PA5470 overexpression strain (II), and the pth overexpression strain (III) were inoculated on swarming plates containing 0.2% l-arabinose without antibiotic (A) or with 10 μg/ml AZM (B). The plates were incubated overnight at 37°C. (C) Quantification of the rhlA mRNA levels. Bacteria were grown in the absence or presence of 5 μg/ml AZM with shaking for 10 h. Total RNA was isolated, and the mRNA levels of rhlA were determined by real-time PCR. The error bars represent standard deviations. **, P < 0.01, compared to results with PA14 containing empty vector (by Student's t test).

FIG 5

Interaction between PA5470 and ribosome. (A) Structure modeling of PA5470. Molecular modeling of PA5470 was completed using the Phyre2 Web server with default parameters. The result was visualized using the UCSF Chimera program. (B) Interaction between PA5470 and the ribosome. PA14 carrying pMMB67EH-PA5470-Flag and pUCP24-rplL-His or pUCP24 was grown to an OD₆₀₀ of 0.8 and treated with 0.1 mM IPTG for 3 h. Bacteria were then lysed and subjected to chromatographic purification with Ni-NTA beads. His-tagged RplL and Flag-tagged PA5470 were detected by Western blotting. IB, immunoblotting.

FIG 6

Assessment of ribosome stalling at the ermC_leader mRNA in the PA5470 overexpression strain. (A) Structure of P_tac-ermC_leader-mApple-Flag and P_tac-ermC_leader-mApple-T0T1. The DNA sequence between the ermC_leader transcriptional start site and the ATG codon of the ermC gene was cloned under the control of P_tac, followed by the coding region of mApple-Flag or two consecutive transcription terminators, T0T1. (B) The protein level of mApple-Flag. Bacteria were treated with 0.1 mM IPTG and the indicated concentration of AZM. The protein levels of mApple-Flag were determined by Western blotting. (C) Quantification of ribosome-associated ermC_leader mRNA. Bacteria treated with 5 μg/ml AZM were lysed and subjected to Ni-NTA chromatography, followed by RNA purification. The relative levels of ermC_leader mRNA were determined by real-time PCR with the 16S ribosome RNA (PA0668.1) as an internal control. **, P < 0.01, compared with the results for PA14 containing empty vector (by a Student's t test).

FIG 7

Expression of RhlR and quantification of ribosome-associated rhlR mRNAs. (A) The protein level of RhlR-Flag and the mutated RhlR (RhlR-M-Flag) in PA14/vector or PA14/P_l-ara-PA5470 with a P_rhlR-rhlR-Flag or P_rhlR-rhlR-M-Flag integrated into the chromosome. Bacteria were treated with 2 μg/ml of AZM and grown to an OD₆₀₀ of 2.0, and the protein levels of RhlR-Flag and RhlR-M-Flag were determined by Western blotting. (B) Quantification of ribosome-associated rhlR mRNA. Stationary-phase bacteria treated with 5 μg/ml AZM were lysed and subjected to Ni-NTA chromatography, followed by RNA purification. The relative levels of rhlR mRNA were determined by real-time PCR with the 16S ribosome RNA (PA0668.1) as an internal control. **, P < 0.01, compared to results with PA14 containing empty vector (by Student's t test).