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Intoxication of antibiotic persisters by host RNS inactivates their efflux machinery during infection [1]

['Séverin Ronneau', 'Department Of Microbiology', 'Harvard Medical School', 'Boston', 'Massachusetts', 'United States Of America', 'Charlotte Michaux', 'Rachel T. Giorgio', 'Sophie Helaine']

Date: 2024-04

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.

By influencing the physiology of bacterial pathogens, the host environment can either limit or potentiate the effectiveness of antibiotics during infection. Recently, we demonstrated that host reactive nitrogen species (RNS), generated by macrophages in response to Salmonella infection, can transiently shield a subset of recalcitrant cells from the effects of β-lactam antibiotics by reducing their cellular respiration. Here, we showed that although bacteria intoxicated by RNS are protected from β-lactam antibiotics, they remain highly susceptible to fluoroquinolones, another class of antibiotics. We found that by reducing cellular respiration, host RNS collaterally inactivate bacterial efflux machinery, which is an essential determinant of fluoroquinolone recalcitrance. Our study explains how the modulation of bacterial respiration by the host environment can differentially impact antibiotic effectiveness during infection. Understanding how host factors influence the physiology of pathogens is essential for optimizing antibiotic use.

Previously, we have shown that internalization of Salmonella enterica serovar Typhimurium (henceforward referred to as Salmonella) by macrophages promotes the formation of phenotypic variants termed antibiotic persisters [ 18 ]. Those persisters are a subpopulation of non-growing bacteria that withstand antibiotic exposure and have the potential to repopulate their environment with antibiotic-sensitive cells when the treatment is ceased [ 19 ]. Recently, we demonstrated that following persister formation, host reactive nitrogen species (RNS) maintain intracellular Salmonella persisters in their non-growing state for an extended period of time by lowering bacterial respiration through intoxication of the tricarboxylic acid (TCA) cycle. By delaying the growth resumption of persisters, RNS transiently protect Salmonella from the action of β-lactams, which are inefficient at killing non-growing bacteria [ 19 ]. Intriguingly, a recent study reported that RNS-exposed Salmonella have a lower survival to fluoroquinolones in comparison with nonexposed cells during mouse infection [ 20 ]. This apparent contradiction led us to explore the impact of RNS on the susceptibility of Salmonella to fluoroquinolones. In our study, we find that whereas host RNS impede the action of β-lactam antibiotics, they synergize with fluoroquinolones. By limiting Salmonella cellular respiration, RNS adversely impact numerous cellular functions in bacteria, in particular, the generation and maintenance of the proton motive force (PMF) across the cell membrane. Such PMF is required to fuel many components of the bacterial efflux machineries, which have been associated with antibiotic recalcitrance [ 21 – 24 ]. We therefore assessed the downstream consequences of RNS intoxication on persister efflux activity during infection and demonstrate that RNS inactivate PMF-dependent efflux pumps, allowing fluoroquinolone accumulation in non-growing bacteria and greater antibiotic efficacy. Our findings highlight the complex role of the host environment on persister physiology during infection and its implication in antibiotic recalcitrance.

Although it is established that the host environment and the stresses it imposes on pathogens drive antibiotic recalcitrance, much is still to be learned about how it impacts antimicrobial efficacy during infection. Whereas in laboratory media, many conditions and mutations limit the action of bactericidal drugs by inactivating antibiotic targets, the physiological state adopted by bacteria surviving antibiotics during infection can be extremely different [ 10 ]. For example, bacterial populations treated with chloramphenicol, a translation inhibitor, enter a non-growing state which protects them against the action of β-lactams [ 11 ]. As opposed to these inactive cells equipped to survive laboratory conditions, recalcitrant bacteria formed in host cells often retain translational activity despite their growth arrest [ 12 – 17 ], indicating that active processes support antibiotic persistence during infection.

In contrast to resistant bacteria, antibiotic-tolerant cells do not grow in the presence of antibiotics and escape the action of bactericidal drugs without requiring any genetic changes [ 1 , 2 ]. Recent work has drawn attention to the contribution of this phenomenon in the recurrence of many bacterial infections, from life-threatening tuberculosis to common urinary tract infections [ 3 – 5 ]. By compromising the efficacy of the antibiotic treatment, these recalcitrant bacteria may lead to recurrent infections but also contribute to the selection and spreading of antibiotic resistance [ 6 – 9 ]. Therefore, understanding how these cells survive antibiotic exposure within their host should provide new insights to improve current treatments.

Results

The AcrAB-TolC efflux machinery supports Salmonella ciprofloxacin persistence in macrophages Our results suggested that RNS-intoxicated persisters that survive cefotaxime are unable to perform an essential function required to withstand ciprofloxacin. Previous studies have shown that in laboratory medium, lowering the intracellular concentration of antibiotics through the bacterial efflux systems contributes to persister survival [21,29]. We thus wondered if efflux machineries also contribute to Salmonella antibiotic persistence within macrophages. To explore this possibility, we evaluated the impact of the loss of TolC, a critical outer membrane channel required for the efflux of many toxic compounds, including fluoroquinolones [30]. We compared the ability of the WT and a tolC deletion mutant to survive 48 hours of antibiotic exposure in macrophages. We found that loss of tolC drastically reduced the number of persisters after treatment with ciprofloxacin but not cefotaxime, highlighting the critical role of the efflux machineries in the ability of persisters to survive fluoroquinolones (Fig 2A). Since TolC interacts with a large array of inner membrane transporters (Fig 2B) [30], we tested the ability of single knock-out mutants of functional partners of TolC (AcrB, AcrD, AcrF, MdsB, MdtC, and EmrB) to survive 48 hours ciprofloxacin exposure within macrophages. We found that the acrB mutant displayed a lower persister fraction than the WT strain, similar to that of the tolC mutant after ciprofloxacin exposure (Fig 2C). To exclude the possibility that the acrB deletion indirectly affects persister levels by impeding general bacterial virulence during infection, we compared the proliferation of WT and ΔacrB strains in macrophages in absence of antibiotics. We found that the overall intracellular proliferation after 16 hours of infection was similar in the WT and the acrB mutant (Fig 2D), suggesting that AcrB directly contributes to antibiotic recalcitrance rather than more generally bacterial behavior within host cells. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. 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. https://doi.org/10.1371/journal.ppat.1012033.g002 Using fluorescence accumulation [31], we previously showed that intramacrophage persisters retain transcriptional and translational activity, which maximizes their survival inside the host [12,18,19]. To investigate the role of AcrB in persister physiology, we first exposed macrophages infected with WT or the acrB mutant to 24 hours of cefotaxime to select for non-growing bacteria [11,18,19]. Then, we exposed the macrophages to cefotaxime or ciprofloxacin for an additional 24-hour period and assessed the proportion of active and inactive non-growing bacteria in the population by inducing the production of GFP, which can only be synthesized by the active subpopulation (Fig 2E). Consistent with our hypothesis that acrB contributes to persister survival during ciprofloxacin treatment, the loss of acrB strongly reduced the amount of active non-growers (aNG) and delayed GFP accumulation in the population exposed to ciprofloxacin but not cefotaxime (Fig 2E). These results reveal the critical role of the AcrAB-TolC machinery on persister survival during ciprofloxacin exposure.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012033

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