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. 2016 Sep 23;60(10):5752-64.
doi: 10.1128/AAC.00260-16. Print 2016 Oct.

RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE

Darija Viducic  1 , Keiji Murakami  2 , Takashi Amoh  2 , Tsuneko Ono  3 , Yoichiro Miyake  2
Affiliations

RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE

Darija Viducic et al. Antimicrob Agents Chemother. .

Abstract

The ability of Pseudomonas aeruginosa to rapidly modulate its response to antibiotic stress and persist in the presence of antibiotics is closely associated with the process of cell-to-cell signaling. The alternative sigma factor RpoN (σ(54)) is involved in the regulation of quorum sensing (QS) and plays an important role in the survival of stationary-phase cells in the presence of carbapenems. Here, we demonstrate that a ΔrpoN mutant grown in nutrient-rich medium has increased expression of pqsA, pqsH, and pqsR throughout growth, resulting in the increased production of the Pseudomonas quinolone signal (PQS). The link between pqsA and its role in carbapenem tolerance was studied using a ΔrpoN ΔpqsA mutant, in which the carbapenem-tolerant phenotype of the ΔrpoN mutant was abolished. In addition, we demonstrate that another mechanism leading to carbapenem tolerance in the ΔrpoN mutant is mediated through pqsE Exogenously supplied PQS abolished the biapenem-sensitive phenotype of the ΔrpoN ΔpqsA mutant, and overexpression of pqsE failed to alter the susceptibility of the ΔrpoN ΔpqsA mutant to biapenem. The mutations in the ΔrpoN ΔrhlR mutant and the ΔrpoN ΔpqsH mutant led to susceptibility to biapenem. Comparison of the changes in the expression of the genes involved in QS in wild-type PAO1 with their expression in the ΔrpoN mutant and the ΔrpoN mutant-derived strains demonstrated the regulatory effect of RpoN on the transcript levels of rhlR, vqsR, and rpoS The findings of this study demonstrate that RpoN negatively regulates the expression of PQS in nutrient-rich medium and provide evidence that RpoN interacts with pqsA, pqsE, pqsH, and rhlR in response to antibiotic stress.

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Figures

FIG 1
FIG 1
Expression of pqsA (A), pqsR (B), pqsH (C), and lasR (D) in wild-type PAO1 and the ΔrpoN mutant throughout growth. Overnight cultures of PAO1 and the ΔrpoN mutant were diluted to an OD595 of 0.01 in LB medium supplemented with 1 mM glutamine, grown at 37°C, and harvested at an OD595 of ∼0.6 (6 h), 0.9 (10 h), and 1.1 (24 h) for total RNA isolation. The levels of the pqsA, pqsR, pqsH, and lasR transcripts were measured by qRT-PCR and normalized to the level of omlA expression. All results are the averages from at least three independent experiments, and the error bars represent SDs.
FIG 2
FIG 2
PQS production in wild-type PAO1 and the ΔrpoN mutant as measured by TLC. Overnight cultures of PAO1 and the ΔrpoN mutant were diluted to an OD595 of 0.01 in LB medium supplemented with 1 mM glutamine, grown at 37°C, harvested at an OD595 of ∼0.6 (6 h), 0.9 (10 h), and 1.1 (24 h), and then assayed for PQS production. Lane 1, 2 μl (10 mM) synthetic PQS; lane 2, PAO1 (6 h of growth); lane 3, ΔrpoN mutant (6 h of growth); lane 4, PAO1 (10 h of growth); lane 5, ΔrpoN mutant (10 h of growth); lane 6, PAO1 (24 h of growth); lane 7, ΔrpoN mutant (24 h of growth). A representative photograph of TLC-separated extracts from PAO1 and the ΔrpoN mutant is shown.
FIG 3
FIG 3
Killing assays of wild-type PAO1, the ΔrpoN mutant, and the ΔrpoN mutant-derived strains in the presence of biapenem (A, C, E, G) and doripenem (B, D, F, H) at 32 μg/ml. Under the assumption that the survival at time zero was 100%, the numbers of CFU were converted to percentages. The experiment was performed in triplicate. Error bars, SDs from three experiments.
FIG 4
FIG 4
PQS production in the ΔrpoN ΔpqsE, and ΔrpoN ΔvqsR mutants as measured by TLC. Overnight cultures of the ΔrpoN ΔpqsE and ΔrpoN ΔvqsR mutants were diluted to an OD595 of 0.01 in LB medium supplemented with 1 mM glutamine, grown at 37°C, harvested at an OD595 of ∼0.6 (6 h), 0.9 (10 h), and 1.1 (24 h), and then assayed for PQS production. Lane 1, 2 μl (10 mM) synthetic PQS; lane 2, ΔrpoN ΔvqsR mutant (6 h of growth); lane 3, ΔrpoN ΔpqsE mutant (6 h of growth); lane 4, ΔrpoN ΔvqsR mutant (10 h of growth); lane 5, ΔrpoN ΔpqsE mutant (10 h of growth); lane 6, ΔrpoN ΔvqsR mutant (24 h of growth); lane 7, ΔrpoN ΔpqsE mutant (24 h of growth). A representative photograph of TLC-separated extracts from the ΔrpoN ΔvqsR and ΔrpoN ΔpqsE mutants is shown.
FIG 5
FIG 5
Biapenem (32 μg/ml) killing assay of wild-type PAO1, the ΔrpoN mutant, and the ΔrpoNΔpqsH mutant. Under the assumption that the survival at time zero was 100%, the numbers of CFU were converted to percentages. The experiment was performed in triplicate. Error bars, SDs from three experiments.
FIG 6
FIG 6
Biapenem (32 μg/ml) killing assay of wild-type PAO1 and the ΔrpoN ΔpqsA mutant in the presence of PQS (50 μM) (A) and pqsE overexpression using an arabinose-inducible pJN105-pqsE construct (B). (A) Time-kill assay for the ΔpqsA mutant in the presence of biapenem (32 μg/ml). Arabinose (0.2%) was added 30 min before the addition of biapenem. The viability of wild-type PAO1, the ΔrpoN ΔpqsA mutant, and the ΔpqsA mutant is expressed as percent survival.
FIG 7
FIG 7
Biapenem (32 μg/ml) killing assay of wild-type PAO1, the ΔrpoN mutant, and the ΔrpoNΔrhlR mutant. Under the assumption that the survival at time zero was 100%, the numbers of CFU were converted to percentages. The experiment was performed in triplicate. Error bars, SDs from three experiments.
FIG 8
FIG 8
PQS production in wild-type PAO1, the ΔrpoN mutant, and the ΔrpoN ΔrhlR mutant as measured by TLC. Overnight cultures of PAO1, the ΔrpoN mutant, and the ΔrpoN ΔrhlR mutant were diluted to an OD595 of 0.01 in LB medium supplemented with 1 mM glutamine, grown at 37°C, harvested at an OD595 of ∼1.1 (24 h), and then assayed for PQS production. Lane 1, 2 μl (10 mM) synthetic PQS; lane 2, PAO1 (24 h of growth); lane 3, ΔrpoN mutant (24 h of growth); lane 4, ΔrpoN ΔrhlR mutant (24 h of growth); lane 5, 2 μl (10 mM) synthetic PQS. A representative photograph of TLC-separated extracts from PAO1, the ΔrpoN mutant, and the ΔrpoN ΔrhlR mutant is shown.
FIG 9
FIG 9
Expression levels of rhlR (A), vqsR (B), and rpoS (C) in wild-type PAO1, the ΔrpoN mutant, and the ΔrpoN mutant-derived strains. Cultures of PAO1, the ΔrpoN mutant, and the ΔrpoN mutant-derived strains grown overnight were diluted to an OD595 of 0.01, grown at 37°C in LB medium, and harvested at an OD595 of ∼0.6 (6 h), 0.9 (10 h), and 1.1 (24 h) for total RNA preparation. Transcript levels were measured by quantitative RT-PCR and normalized to the level of omlA expression. All results are averages from at least three independent experiments, and the error bars represent SDs.
FIG 10
FIG 10
Diagram of the proposed model of RpoN regulation in P. aeruginosa. The detailed network of regulation is described in the Discussion. Solid lines with arrows and dashed lines with blunt ends, genes that are positively and negatively affected, respectively. IM, inner membrane; OM, outer membrane; PS, periplasmic space.
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