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C-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.

ABSTRACT: Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ?dacA mutants are highly sensitive to the ?-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ?dacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ?dacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ?dacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ?dacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ?dacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ?dacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.

SUBMITTER: Whiteley AT

PROVIDER: S-EPMC5391996 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.

Whiteley Aaron T AT Garelis Nicholas E NE Peterson Bret N BN Choi Philip H PH Tong Liang L Woodward Joshua J JJ Portnoy Daniel A DA

Molecular microbiology 20170308 2

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to gro ...[more]

PMID: 28097715

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Project description:Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger among bacteria. c-di-AMP regulates many cellular pathways through direct binding to several molecular targets in bacterial cells. c-di-AMP depletion is well known to destabilize the bacterial cell wall, resulting in increased bacteriolysis and enhanced susceptibility to cell wall targeting antibiotics. Using the human pathogen Listeria monocytogenes as a model, we found that c-di-AMP accumulation also impaired cell envelope integrity. An L. monocytogenes mutant deleted for c-di-AMP phosphodiesterases (pdeA pgpH mutant) exhibited a 4-fold increase in c-di-AMP levels and several cell wall defects. For instance, the pdeA pgpH mutant was defective for the synthesis of peptidoglycan muropeptides and was susceptible to cell wall-targeting antimicrobials. Among different muropeptide precursors, we found that the pdeA pgpH strain was particularly impaired in the synthesis of d-Ala-d-Ala, which is required to complete the pentapeptide stem associated with UDP-N-acetylmuramic acid (MurNAc). This was consistent with an increased sensitivity to d-cycloserine, which inhibits the d-alanine branch of peptidoglycan synthesis. Finally, upon examining d-Ala:d-Ala ligase (Ddl), which catalyzes the conversion of d-Ala to d-Ala-d-Ala, we found that its activity was activated by K+ Based on previous reports that c-di-AMP inhibits K+ uptake, we propose that c-di-AMP accumulation impairs peptidoglycan synthesis, partially through the deprivation of cytoplasmic K+ levels, which are required for cell wall-synthetic enzymes.IMPORTANCE The bacterial second messenger c-di-AMP is produced by a large number of bacteria and conditionally essential to many species. Conversely, c-di-AMP accumulation is also toxic to bacterial physiology and pathogenesis, but its mechanisms are largely undefined. We found that in Listeria monocytogenes, elevated c-di-AMP levels diminished muropeptide synthesis and increased susceptibility to cell wall-targeting antimicrobials. Cell wall defects might be an important mechanism for attenuated virulence in bacteria with high c-di-AMP levels.

Project description:The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenesIMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

Project description:Listeria monocytogenes is able to efficiently utilize glycerol as a carbon source. In a defined minimal medium, the growth rate (during balanced growth) in the presence of glycerol is similar to that in the presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed high-level transcriptional upregulation of the genes known to be involved in glycerol uptake and metabolism (glpFK and glpD) in the presence of glycerol (compared to that in the presence of glucose and/or cellobiose). Levels of expression of the genes encoding a second putative glycerol uptake facilitator (GlpF(2)) and a second putative glycerol kinase (GlpK(2)) were less enhanced under these conditions. GlpK(1) but not GlpK(2) was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while the loss of GlpK(1) affected replication in Caco-2 cells less than did the loss of GlpK(2) and GlpD. Additional genes whose transcription levels were higher in the presence of glycerol than in the presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression control. Transcriptional downregulation in the presence of glycerol (compared to those in the presence glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N metabolism, and the biosynthesis of branched-chain amino acids. The highest level of transcriptional upregulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions, a low level of HPr-Ser-P and a high level of HPr-His-P were present in the cells, suggesting that all enzyme IIA (EIIA) (or EIIB) components of the phosphotransferase system (PTS) permeases expressed will be phosphorylated. These and other data suggest that the phosphorylation state of PTS permeases correlates with PrfA activity.

Dataset Information

C-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.

Publications

c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.

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