An Amino Acid Substitution in Elongation Factor EF-G1A Alters the Antibiotic Susceptibility of Pseudomonas aeruginosa LasR-Null Mutants

Abstract

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum-sensing system to increase resistance to the aminoglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism that permits the emergence of lasR-null mutants under tobramycin selection. We hypothesized that some other genetic mutations that emerge in these isolates might modulate the effects of lasR-null mutations on antibiotic resistance. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. In some of these isolates, inactivating lasR further increased resistance, compared with decreasing resistance of the wild-type ancestor. These strain-dependent effects were due to a G61A nucleotide polymorphism in the fusA1 gene encoding amino acid substitution A21T in the translation elongation factor EF-G1A. The EF-G1A mutational effects required the MexXY efflux pump and the MexXY regulator ArmZ. The fusA1 mutation also modulated ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results identify a gene mutation that can reverse the direction of the antibiotic selection of lasR mutants, a phenomenon known as sign epistasis, and provide a possible explanation for the emergence of lasR-null mutants in clinical isolates. IMPORTANCE One of the most common mutations in Pseudomonas aeruginosa clinical isolates is in the quorum sensing lasR gene. In laboratory strains, lasR disruption decreases resistance to the clinical antibiotic tobramycin. To understand how lasR mutations emerge in tobramycin-treated patients, we mutated lasR in highly tobramycin-resistant laboratory strains and determined the effects on resistance. Disrupting lasR enhanced the resistance of some strains. These strains had a single amino acid substitution in the translation factor EF-G1A. The EF-G1A mutation reversed the selective effects of tobramycin on lasR mutants. These results illustrate how adaptive mutations can lead to the emergence of new traits in a population and are relevant to understanding how genetic diversity contributes to the progression of disease during chronic infections.

Keywords: LasR; Pseudomonas aeruginosa; antibiotic resistance; evolution; quorum sensing; tobramycin.

FIG 1

Tobramycin resistance of ΔlasR mutants is strain dependent. (A) The MIC of tobramycin was determined for each strain carrying intact lasR (LasR⁺, black bars) or ΔlasR (LasR⁻, white bars). The significance of the effect of strain (wild type, T1, or T3) and lasR allele (LasR⁺ or LasR⁻) and the interaction of the two on MIC was determined by two-way ANOVA by performing pairwise comparisons of each LasR^+/− strain pair with that of PA14. The interaction was significant for T1 (F_1,8 = 156.8 and P < 0.0001) and T3 (F_1,8 = 10.67 and P < 0.05). Pairwise comparisons of LasR⁺ and LasR⁻ of each strain were determined using Sidak’s post hoc test with P-values adjusted for multiple comparisons; *, P < 0.05; ****, P < 0.0001; ns, not significant. (B) Tobramycin MIC of T1 and T1-mutated strains. Where indicated, AHL (3OC12-HSL) was added before inoculating culture tubes at the 10-μM final concentration. Statistical analysis was done by one-way ANOVA and Dunett’s multiple comparisons with T1; **, P < 0.01. For panels A and B, results are the average of three independent experiments and the vertical bars indicate standard deviation.

FIG 2

Effects of fusA1 G61A and ΔlasR on tobramycin resistance. The MIC of tobramycin was determined for each strain carrying intact lasR (LasR⁺, black bars) or ΔlasR (LasR⁻, white bars). The significance of the effect of strain (indicated along the x axis) and lasR allele (LasR⁺ or LasR⁻) and the interaction of the two on MIC was determined by 2-way ANOVA by performing pairwise comparisons of each LasR^+/− strain pair with that of PA14 (A) or PA14 Prha (B). Pair-wise comparisons of LasR⁺ and LasR⁻ of each strain were determined using Sidak’s post hoc test with P values adjusted for multiple comparisons; ***, P < 0.001; ns = not significant. (A) There was a statistically significant interaction between the effects of strain and lasR for fusA1 G61A (F_1,8 = 156.8, P < 0.0001) but not for ptsP 1547T (F_1,8 = 0.3642, P = 0.5629). An additional Sidak’s post hoc test comparing PA14 and fusA1 G61A of the LasR⁺ strains revealed no significance (P > 0.4). (B) Strains had either the empty CTX-1 Prha or the CTX-1 Prha-fusA1 cassette inserted into the neutral attB site in the genome. Rhamnose was added to all cultures at 0.1% final concentration. There was a statistically significant interaction between the effects of strain and lasR for fusA1 G61A Prha (F_1,8 = 17.41, P < 0.005) but not for fusA1 G61A + Prha-fusA1 (F_1,8 = 1.7 × 10⁻⁷, P = 0.997). For panels A and B, results are the average of three independent experiments and the vertical bars indicate standard deviation.

FIG 3

Tobramycin effects on ΔlasR proliferation in cocultures. Cocultures of wild-type PA14 and PA14 ΔlasR (left) or of fusA1 G61A and fusA1 G61A ΔlasR (right) were grown with no tobramycin (No tob, closed circles) or with tobramycin (Tob, open circles) at the highest concentration that permits growth (0.3 μg/mL for PA14 or 2 μg/mL for fusA1 G61A). In both cases, the ΔlasR mutant was started at 1% of the total coculture population. After being combined, cocultures were inoculated into casein medium and subsequently transferred to fresh medium daily for 2 days. Final population densities ranged from 1 to 5 × 10⁹ cells per mL. Each data point represents a single experiment and vertical lines represent standard deviation. Statistical analysis by two-way ANOVA was used to determine the significance of the effect of the tobramycin and the lasR allele and interaction between the two on the relative fitness of the ΔlasR mutant, and the interaction was significant (P < 0.0001 and F_1,8 = 41.26). Pair-wise comparisons of LasR⁺ and LasR⁻ for each condition (+/− tob) were performed using Sidak’s post hoc analysis with P values adjusted for multiple comparisons; ****, P < 0.0001.

FIG 4

Other fusA1 mutations and their effects on tobramycin resistance of ΔlasR mutants. (A) Ribbon diagram of the fusA1-encoded protein elongation factor-G (EF-G1A) (PDB ID: 4FN5). The protein was crystallized bound to argyrin, which is shown in red. Each one of the amino acid substitutions is indicated with green coloration in the ribbon diagram and a red circle and was identified using the UCSF ChimeraX (82) (B) The MIC of tobramycin was determined for each strain carrying the wild-type fusA1 allele (PA14) or PA14 with the indicated amino acid substitution in the encoded EF-G1A. Each strain had either an intact lasR (LasR⁺, black bars) or ΔlasR (LasR⁻, white bars). Results are the average of three independent experiments and the vertical bars indicate standard deviation. The significance of the effect of the fusA1 allele and the lasR allele and the interaction of the two on MIC was determined by 2-way ANOVA by performing pairwise comparisons of each LasR^+/− strain pair with that of PA14. Only A1366G showed a significant interaction (P < 0.05, F_1,8 = 10.25). Pairwise comparisons of LasR⁺ and LasR⁻ of each strain were determined using Sidak’s post hoc test with P values adjusted for multiple comparisons. *, P < 0.05; ns, not significant.

FIG 5

Role of MexXY on tobramycin resistance of ΔlasR and fusA1 G61A mutants. (A) mexX transcript levels were determined by droplet digital PCR and normalized to the housekeeping control gene proC. Strains were wild-type PA14 or PA14 with the fusA1 G61A substitution. Each strain had either an intact lasR (LasR⁺, filled circles) or ΔlasR (LasR⁻, open circles). Each point represents in independent experiment; horizontal lines represent the geometric mean, and the vertical lines represent the geometric standard deviation. Statistical analysis by two-way ANOVA showed a significant interaction between the effects of strain and lasR allele on mexX transcripts (P < 0.005, F_1,8 = 15.67). An additional Sidak’s post hoc test comparing PA14 and fusA1 G61A of the LasR⁺ strains revealed no significance (P > 0.6). (B and C) The MIC of tobramycin was determined for each strain carrying intact lasR (LasR⁺, black bars) or ΔlasR (LasR⁻, white bars). Results are the average of three independent experiments and the vertical bars show standard deviation. In pairwise comparisons with the LasR^+/− PA14 strains, there was a significant interaction between the effects of strain and lasR allele for T1 (P < 0.01 and F_(1,8) = 29.01 for panel B and P < 0.005 and F_(1,8) = 17.99 for panel C) but not for the other strains. In panels A to C, pairwise comparisons of LasR⁺ and LasR⁻ of each strain were determined using Sidak’s post hoc test with P-values adjusted for multiple comparisons. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant.