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Multisite phosphorylation drives phenotypic variation in (p)ppGpp synthetase-dependent antibiotic tolerance.

ABSTRACT: Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. Individual bacteria within a population can differ in antibiotic tolerance, but whether this variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. Here we report that a gene encoding a bacterial (p)ppGpp synthetase in Bacillus subtilis, sasA, exhibits high levels of extrinsic noise in expression. We find that sasA is regulated by multisite phosphorylation of the transcription factor WalR, mediated by a Ser/Thr kinase-phosphatase pair PrkC/PrpC, and a Histidine kinase WalK of a two-component system. This regulatory intersection is crucial for controlling the appearance of outliers; rare cells with unusually high levels of sasA expression, having increased antibiotic tolerance. We create a predictive model demonstrating that the probability of a given cell surviving antibiotic treatment increases with sasA expression. Therefore, multisite phosphorylation can be used to strongly regulate variability in antibiotic tolerance.

SUBMITTER: Libby EA

PROVIDER: S-EPMC6853874 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Multisite phosphorylation drives phenotypic variation in (p)ppGpp synthetase-dependent antibiotic tolerance.

Libby Elizabeth A EA Reuveni Shlomi S Dworkin Jonathan J

Nature communications 20191113 1

Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. Individual bacteria within a population can differ in antibiotic tolerance, but whether this variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. Here we report that a gene encoding a bacterial (p)ppGpp synthetase in Bacillus subtilis, sasA, exhibits high levels of extrinsic noise in expression. We find that sasA is regulated by mult ...[more]

PMID: 31723135

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Project description:The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventative and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stress, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile Here, we report that the C. difficile (p)ppGpp synthetase RSH is incapable of utilizing GTP or GMP as a substrate but readily synthesizes ppGpp from GDP. The enzyme also utilizes many structurally diverse metal cofactors for reaction catalysis and remains functionally stable at a wide range of environmental pHs. Transcription of rsh is stimulated by stationary-phase onset and by exposure to the antibiotics clindamycin and metronidazole. Chemical inhibition of RSH by the ppGpp analog relacin increases antibiotic susceptibility in epidemic C. difficile R20291, indicating that RSH inhibitors may be a viable strategy for drug development against C. difficile infection. Finally, transcriptional suppression of rsh also increases bacterial antibiotic susceptibility, suggesting that RSH contributes to C. difficile antibiotic tolerance and survival.IMPORTANCE Clostridioides difficile infection (CDI) is an urgent public health threat with a high recurrence rate, in part because the causative bacterium has a high rate of antibiotic survival. The (p)ppGpp-mediated bacterial stringent response plays a role in antibiotic tolerance in diverse pathogens and is a potential target for development of new antimicrobials because the enzymes that metabolize (p)ppGpp have no mammalian homologs. We report that stationary-phase onset and antibiotics induce expression of the clostridial ppGpp synthetase RSH and that both chemical inhibition and translational suppression of RSH increase C. difficile antibiotic susceptibility. This demonstrates that development of RSH inhibitors to serve as adjuvants to antibiotic therapy is a potential approach for the development of new strategies to combat CDI.

Project description:BackgroundAcute respiratory infections are the most common symptomatic reason for seeking care among patients in the US, and account for the majority of all antibiotic prescribing, yet a large fraction of antibiotic prescriptions are inappropriate.ObjectiveWe sought to identify the underlying factors driving variation in antibiotic prescribing across clinicians and settings.Design, participantsUsing electronic health data for adult ambulatory visits for acute respiratory infections to a retail clinic chain and primary care practices from an integrated healthcare system, we identified a random sample of clinicians for survey.Main measuresWe evaluated independent predictors of overall prescribing and imperfect antibiotic prescribing, controlling for clinician and site of care. We defined imperfect antibiotic prescribing as prescribing for non-antibiotic-appropriate diagnoses, failure to prescribe for an antibiotic-appropriate diagnosis, or prescribing a non-guideline-concordant antibiotic.Key resultsResponse rates were 34 % for retail clinics and 24 % for physicians' offices (N = 187). Clinicians in physicians' offices prescribed antibiotics less often than those in retail clinics (53 % versus 67 %; p < 0.01), but had a higher imperfect antibiotic prescribing rate (65 % versus 31 %; p < 0.01). Feeling rushed was associated with higher antibiotic prescribing (OR 1.34; 95 % CI 1.03, 1.75). Antibiotic prescribing was also associated with clinician disagreement that antibiotics are overused (OR 1.60, 95 % CI, 1.16, 2.20). Imperfect antibiotic prescribing was associated with receiving antibiotic prescribing feedback (OR 1.35, 95 % CI 1.04, 1.75) and disagreement that patient demand was a problem (OR 1.66, 95 % CI 1.00, 2.73). Imperfect antibiotic prescribing was less common with clinicians who perceived that they prescribed antibiotics less often than their peers (OR 0.63, 95 % CI 0.46, 0.87).ConclusionsPoor-quality antibiotic prescribing was associated with feeling rushed, believing less strongly that antibiotics were overused, and believing that patient demand was not an issue, factors that can be assessed and addressed in future interventions.

Dataset Information

Multisite phosphorylation drives phenotypic variation in (p)ppGpp synthetase-dependent antibiotic tolerance.

Publications

Multisite phosphorylation drives phenotypic variation in (p)ppGpp synthetase-dependent antibiotic tolerance.

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