Phosphotransferase-dependent accumulation of (p)ppGpp in response to glutamine deprivation in Caulobacter crescentus

The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTS(Ntr)) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EI(Ntr)) of PTS(Ntr). Decreasing intracellular glutamine concentration triggers phosphorylation of EI(Ntr) and its downstream components HPr and EIIA(Ntr). Once phosphorylated, both HPr∼P and EIIA(Ntr)∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth

Antibiotic tolerance and persistence have distinct fitness trade-offs.

Genetically susceptible bacteria can escape the action of bactericidal antibiotics through antibiotic tolerance or persistence. However, one major difference between the two phenomena is their distinct penetrance within an isogenic population. While with antibiotic persistence, susceptible and persister cells co-exist, antibiotic tolerance affects the entire bacterial population. Here, we show that antibiotic tolerance can be achieved in numerous non-specific ways in vitro and during infection. More importantly, we highlight that, due to their impact on the entire bacterial population, these tolerance-inducing conditions completely mask persistence and the action of its molecular determinants. Finally, we show that even though tolerant populations display a high survival rate under bactericidal drug treatment, this feature comes at the cost of having impaired proliferation during infection. In contrast, persistence is a risk-limiting strategy that allows bacteria to survive antibiotic treatment without reducing the ability of the population to colonize their host. Altogether, our data emphasise that the distinction between these phenomena is of utmost importance to improve the design of more efficient antibiotic therapies

Strains used in this study.

The host environment is of critical importance for antibiotic efficacy. By impacting bacterial machineries, stresses encountered by pathogens during infection promote the formation of phenotypic variants that are transiently insensitive to the action of antibiotics. It is assumed that these recalcitrant bacteria—termed persisters—contribute to antibiotic treatment failure and relapsing infections. Recently, we demonstrated that host reactive nitrogen species (RNS) transiently protect persisters against the action of β-lactam antibiotics by delaying their regrowth within host cells. Here, we discovered that RNS intoxication of persisters also collaterally sensitizing them to fluoroquinolones during infection, explaining the higher efficiency of fluoroquinolones against intramacrophage Salmonella. By reducing bacterial respiration and the proton-motive force, RNS inactivate the AcrAB efflux machinery of persisters, facilitating the accumulation of fluoroquinolones intracellularly. Our work shows that target inactivity is not the sole reason for Salmonella persisters to withstand antibiotics during infection, with active efflux being a major contributor to survival. Thus, understanding how the host environment impacts persister physiology is critical to optimize antibiotics efficacy during infection.</div

Production of RNS by host cells.

Quantification of nitric oxide production by macrophages was achieved by quantifying its stable byproduct nitrite in the infection medium. Nitrite concentration in the infection medium of unstimulated (circle) or IFN-γ-stimulated (triangle) WT and Nos2-/- (square) Mφ infected for 24 h with WT Salmonella and treated with cefotaxime. p values are indicated (ANOVA with Dunnett’s correction for multiple testing against the—IFN- γ condition); error bars depict means and standard deviation (SD); n = 3. (TIF)</p

Host RNS inactivates efflux activity of intramacrophage persisters.

(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.</p

Illustration of the experimental setups.

(A) Bone marrow-derived macrophages from WT mice were infected with WT Salmonella. Then, infected Mφ were treated with cefotaxime, ciprofloxacin or both for 24 h. Finally, persisters (in brown) were extracted and plated on LB agar plate for counting. (B) Bone marrow-derived macrophages from WT or Nos2-/- mice were cultivated in the absence or in the presence of IFN-γ and infected with WT Salmonella. Then, infected Mφ were treated with cefotaxime or ciprofloxacin for 24 h. Finally, persisters (in brown) were extracted and plated on LB agar plate for counting. (C) Infected bone marrow-derived macrophages from WT mice were treated for 24 h with cefotaxime to select persisters. Then, infected macrophages were treated with cefotaxime or ciprofloxacin for 24 additional hours. Finally, persisters (in brown) were extracted and plated on LB agar plate for counting. (TIF)</p

AcrAB-TolC efflux machinery contributes to persister survival during ciprofloxacin treatment within host cells.

(A) 48 h cefotaxime or ciprofloxacin survival of WT (gray) or ΔtolC (green) 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. DT: Detection Threshold. (B) Illustration of TolC-dependent efflux machineries of Salmonella. IM: Inner Membrane, PS: Periplasmic Space, OM: Outer Membrane. (C) 48 h ciprofloxacin survival of WT, ΔtolC, ΔacrB, ΔacrD, ΔacrF, ΔmdsB, ΔmdtC or ΔemrB Salmonella in WT Mφ normalized to values after 30 min internalization. p values are indicated (ANOVA with Dunnett’s correction for multiple testing against the WT); error bars depict means and standard deviation (SD); n = 4. DT: Detection Threshold. (D) Bacterial load of WT or ΔacrB Salmonella in WT Mφ after 30 min internalization (T0) and at 16 h of infection (T16) in the absence of antibiotics. p values are indicated (ANOVA with Tukey’s correction for multiple comparisons); error bars depict means and standard deviation (SD); n = 3. (E) (Left) Illustration of the experimental setup. After 24 h of cefotaxime exposure, infected macrophages containing non-growers (NG) were exposed to 26 h of cefotaxime or ciprofloxacin. To distinguish active (aNG) and inactive (iNG) non-growers, production of GFP was induced during 2 h prior extraction and analysis. (Right) Representative FACS plots and quantification of the level of transcriptional/translational activity in active and inactive cefotaxime or ciprofloxacine-treated intramacrophage WT or ΔacrB Salmonella in WT Mφ at 50 h of infection. p values are indicated (ANOVA with Tukey’s correction for multiple comparisons); error bars depict means and standard deviation (SD); n = 3.</p

RNS-dependent persisters are sensitive to ciprofloxacin.

(A) Representative fluorescence-activated cell sorting (FACS) plots of fluorescence dilution experiments tracking the proportion of growing (G; light gray) and non-growing (NG; dark gray) WT Salmonella recovered from WT macrophages (Mφ) in the presence (+AB) or in the absence (-AB) of cefotaxime at 16 h of infection. (B) 24 h cefotaxime and/or ciprofloxacin survival of WT 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) 24 h cefotaxime (light blue) or ciprofloxacin (yellow) survival of WT Salmonella in unstimulated (-IFN-γ) or IFN-γ-stimulated (+IFN-γ) WT or Nos2-/- Mφ normalized to values after 30 min internalization. Distinction between RNS-dependent and independent persisters was determined using the proportion of persisters in Nos2-/- Mφ. p values are indicated (ANOVA with Dunnett’s correction for multiple testing against the–IFN-γ condition); error bars depict means and standard deviation (SD); n = 3. (D) Persister clearance after 24 h of cefotaxime or ciprofloxacin treatment following 24 h of cefotaxime treatment of WT Salmonella in unstimulated (-IFN-γ) or IFN-γ-stimulated (+IFN-γ) 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). (E) 24 h cefotaxime (light blue) or ciprofloxacin (yellow) survival of the initial (Q285A) and recurrent (Q285F5) isolates of ST313 Salmonella Q285 in WT or Nos2-/- 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.</p