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. 2013 Dec 19;8(12):e80140.
doi: 10.1371/journal.pone.0080140. eCollection 2013.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria

Yok-Ai Que  1 , Ronen Hazan  2 , Benjamin Strobel  1 , Damien Maura  1 , Jianxin He  1 , Meenu Kesarwani  1 , Panagiotis Panopoulos  1 , Amy Tsurumi  1 , Marlyse Giddey  3 , Julie Wilhelmy  4 , Michael N Mindrinos  4 , Laurence G Rahme  1
Affiliations

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria

Yok-Ai Que et al. PLoS One. .

Abstract

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 2-AA promotes antibiotic tolerance in P. aeruginosa.
A. Survival fraction of controls PA14 (black), PA14 + 2-AA (red), and mvfR - (white), mvfR - + 2-AA (pink) and pqsBC - (green) cells grown to OD600nm= 2.0 followed by the addition of 10 μg/ml meropenem. All experiments were performed in triplicates, and results expressed as mean ± SD. Differences in persister fractions between PA14, mvfR - and pqsBC- (p-value = 0.0016), between PA14 and PA14 + 2-AA (p-value = 0.0078) and between mvfR - and mvfR - + 2-AA (p-value = 0.0062), are statistically significant (one-way ANOVA, Tukey’s HSD test). B. Persister cells are antibiotic tolerant, not antibiotic resistant. After meropenem killing (1st cycle), a single PA14 surviving colony was re-inoculated in fresh medium in the presence (red) or absence (black) of 2-AA (p-value = 0.0078, Student’s t-test), and antibiotic was applied again for 24 h (2nd cycle) (p-value = 0.0003, Student’s t-test). Additional single colonies tested gave similar results. C, D. Chemical inhibition/stimulation of 2-AA production modulates persisters formation. Inhibition of 2-AA production (C) by compound M62 (orange) added to PA14 (black) culture decreased persisters formation (D) to a level similar to mvfR - mutant (white), and stimulation of 2-AA production (C) by M29 (blue) increased PA14 persisters formation (D). The experiment was performed in triplicates, and results are expressed as mean ± SD. Differences in the persisters fractions between PA14, PA14 + M62 and PA14 + M29 are statistically significant (p-value=0.0001, one-way ANOVA, Tukey HSD). E. The accumulation of persister cells is 2-AA specific, as neither the precursor/analog AA (grey) nor the analog 3-AA (brown) increased the persister cell fraction. The experiment was performed in triplicates, and results are expressed as mean ± SD. Differences in the persisters fractions are only statistically significant between PA14 (black) and PA14 + 2-AA (red) (p-value=0.0004, one-way ANOVA, Tukey HSD).
Figure 2
Figure 2. 2-AA down-regulates the transcription of many genes involved in the translational capacity of the cell.
A. Graphical analysis of mvfR - , mvfR -+ 2-AA and pqsBC - transcriptomes to examine expression of all genes involved in translation (Table S2). Down-regulated genes are shown as red (2-fold or more reduction), or pink (1.5- to 2-fold reduction), and up regulated genes as green (>2-fold increase) or light green (1.5- to 2-fold increase). No change in gene expression is shown as grey. B. Expression of 16 rRNA (black) and 23S rRNA (red) in mvfR - and pqsBC -cells was assessed by qRT-PCR in the absence or presence (+ 2-AA) of exogenously added 2-AA. The PA14 group +/- 2-AA was used as the calibration reference. C. Western blot showing Rmf levels in PA14, mvfR -, and pqsBC - cells. Baseline Rmf levels was markedly lower in mvfR - cells than in PA14 cells; Rmf levels were increased in pqsBC -, and in both PA14 and mvfR - cell types in the presence of exogenously added 2-AA. D. rmf -- cells (purple) exhibit a reduced persister fraction compared to PA14 cells (black). Differences between both strains are statistically significant (p-value = 0.0113, Student’s t-test). Experiment was performed in triplicate. E. Exogenously added 2-AA decreases polysomes levels. Ribosomal extracts from PA14 cells grown in the absence (left panel) or presence (right panel) of exogenously added 2-AA were separated in a 25% to 5% sucrose density gradient. The ratio of translating polysomes to 70S was higher in the untreated cultures (left) than in the cultures grown in presence of exogenously added 2-AA (right) reflecting a decrease in translational activity. F. Inhibition of PA14 translational capacity increase persisters formation. PA14 and mvfR - cells were grown in absence (black) or presence (orange) of sub-MIC concentration of translational inhibiting antibiotic chloramphenicol (15 mg/L), which did not affect PA14 growth (data not shown). Persisters fraction was determined as described in Figure 1. The results are presented as log2 fold of change of the untreated PA14 persisters fraction as mean ± SD.
Figure 3
Figure 3. 2-AA increase persister cell formation in other bacteria.
Survival fraction of Acinetobacter baumannii (left panel), and Burkholderia thailandensis (right panel) cells grown to OD600nm= 2.0 in the absence (black) or presence (red) of 2-AA followed by the addition of 10 μg/ml meropenem. All experiments were performed in triplicates, and results expressed as mean ± SD. Differences in persister fractions between absence and presence of 2-AA are statistically significant for both A. baumannii (p-value = 0.0068, Student’s t-test) and B. thailandensis (p-value = 0.0203, Student’s t-test).
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