Dataset Information

The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

ABSTRACT: 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.

SUBMITTER: Pokhrel A

PROVIDER: S-EPMC7484180 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

Pokhrel Astha A Poudel Asia A Castro Kory B KB Celestine Michael J MJ Oludiran Adenrele A Rinehold Alden J AJ Resek Anthony M AM Mhanna Mariam A MA Purcell Erin B EB

Journal of bacteriology 20200908 19

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 syntheta ...[more]

PMID: 32661079

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Project description:ImportanceRegulatory agencies and professional organizations recommend antibiotic stewardship programs (ASPs) in US hospitals. The optimal approach to establish robust, sustainable ASPs across diverse hospitals is unknown.ObjectiveTo assess whether the Agency for Healthcare Research and Quality Safety Program for Improving Antibiotic Use is associated with reductions in antibiotic use across US hospitals.Design, setting, and participantsA pragmatic quality improvement program was conducted and evaluated over a 1-year period in US hospitals. A total of 437 hospitals were enrolled. The study was conducted from December 1, 2017, to November 30, 2018. Data analysis was performed from March 1 to October 31, 2019.InterventionsThe Safety Program assisted hospitals with establishing ASPs and worked with frontline clinicians to improve their antibiotic decision-making. All clinical staff (eg, clinicians, pharmacists, and nurses) were encouraged to participate. Seventeen webinars occurred over 12 months, accompanied by additional durable educational content. Topics focused on establishing ASPs, the science of safety, improving teamwork and communication, and best practices for the diagnosis and management of infectious processes.Main outcomes and measuresThe primary outcome was overall antibiotic use (days of antibiotic therapy [DOT] per 1000 patient days [PD]) comparing the beginning (January-February 2018) and end (November-December 2018) of the Safety Program. Data analysis occurred using linear mixed models with random hospital unit effects. Antibiotic use from 614 hospitals in the Premier Healthcare Database from the same period was analyzed to evaluate contemporary US antibiotic trends. Quarterly hospital-onset Clostridioides difficile laboratory-identified events per 10 000 PD were a secondary outcome.ResultsOf the 437 hospitals enrolled, 402 (92%) remained in the program until its completion, including 28 (7%) academic medical centers, 122 (30%) midlevel teaching hospitals, 167 (42%) community hospitals, and 85 (21%) critical access hospitals. Adherence to key components of ASPs (ie, interventions before and after prescription of antibiotics, availability of local antibiotic guidelines, ASP leads with dedicated salary support, and quarterly reporting of antibiotic use) improved from 8% to 74% over the 1-year period (P < .01). Antibiotic use decreased by 30.3 DOT per 1000 PD (95% CI, -52.6 to -8.0 DOT; P = .008). Similar changes in antibiotic use were not observed in the Premier Healthcare Database. The incidence rate of hospital-onset C difficile laboratory-identified events decreased by 19.5% (95% CI, -33.5% to -2.4%; P = .03).Conclusions and relevanceThe Agency for Healthcare Research and Quality Safety Program appeared to enable diverse hospitals to establish ASPs and teach frontline clinicians to self-steward their antibiotic use. Safety Program content is publicly available.

Project description:The endospore-forming pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and health care. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis, and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using transcriptome sequencing (RNA-seq), we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways. IMPORTANCE C. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile life cycle, other nonantibiotic treatment options are in urgent need of development. Toward this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology: Stickland metabolism. Here, we use our CRISPR-Cas9 system to "knock in" a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology, from vegetative growth to spore formation and outgrowth postgermination.

Project description:The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630?erm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions.

Dataset Information

The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

Publications

The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

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