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. 2024 Feb 29;20(2):e1012033. doi: 10.1371/journal.ppat.1012033

Fig 3. Host RNS inactivates efflux activity of intramacrophage persisters.

Fig 3

(A) Model of intoxication of the TCA cycle by host RNS. Corruption of the α-KDH complex limits cellular respiration of persisters, which limit the proton-motive force (PMF). Consequently, host RNS limits ATP production and efflux activity of intramacrophage persisters. (B) 24 h cefotaxime (light blue) or ciprofloxacin (yellow) survival of WT or ΔsucB Salmonella in WT Mφ normalized to values after 30 min internalization. p values are indicated (ANOVA with Tukey’s correction for multiple comparisons); error bars depict means and standard deviation (SD); n = 3. (C) Persister clearance after 24 h of cefotaxime followed by 24 h of cefotaxime or ciprofloxacin treatment of ΔsucB Salmonella in WT Mφ normalized to values after 30 min internalization. p values are indicated (unpaired t test at 48h); error bars depict means and standard deviation (SD). (D) (Left) Illustration of the experimental setup. Efflux-dependent accumulation of Hoechst (H33342) was assessed in absence or in presence of RNS. (Right) Measurement of H33342 accumulation over time in WT or ΔacrB Salmonella in presence or in absence of CCCP or RNS. p values are indicated (ANOVA with Dunnett’s correction for multiple testing against the WT at 30m); error bars depict means and standard deviation (SD). (E) ATP level over time obtained by bioluminescence. Experimental conditions are the same as in panel D. p values are indicated (ANOVA with Dunnett’s correction for multiple testing against the WT at 30m); error bars depict means and standard deviation (SD). (F) (Left) Illustration of the experimental setup. After 24 h of cefotaxime exposure, production of GFP was induced in intramacrophage non-growers for 2 h to distinguish active (aNG) and inactive (iNG) non-growers. Then, the efflux activity of aNG was assessed using H33342 dye. (Right) Representative FACS plots and quantification of the efflux activity of WT, ΔacrB or ΔsucB Salmonella in Nos2-/- Mφ. As a control, WT persisters were also tested in the presence of CCCP, an inhibitor of cellular respiration. p values are indicated (ANOVA with Dunnett’s correction for multiple testing against the WT treated with 2 μM H33342 dye); error bars depict means and standard deviation (SD). (G) 48 h ciprofloxacin survival of WT, ΔacrB or ΔsucB Salmonella in WT Mφ normalized to values after 30 min internalization. WT, ΔacrB and ΔsucB strains were complemented with an empty vector (pEV) or acrAB (pacrAB). p values are indicated (ANOVA with Tukey’s correction for multiple comparisons); error bars depict means and standard deviation (SD); n = 3. DT: Detection Threshold.