From magic spot ppGpp to MESH1: Stringent response from bacteria to metazoa.
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ABSTRACT:
SUBMITTER: Chi JT
PROVIDER: S-EPMC9894426 | biostudies-literature | 2023 Feb
REPOSITORIES: biostudies-literature
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From magic spot ppGpp to MESH1: Stringent response from bacteria to metazoa.
PLoS pathogens 20230202 2
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Project description:UnlabelledThe stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for "Rel SpoT homologue"; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh, relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the rsh relQ [(p)ppGpp(0)] strain, suggesting that a lack of basal (p)ppGpp places the cell in a "transcriptionally relaxed" state. Alterations in the fermentation profile and increased production of H2O2 in the (p)ppGpp(0) strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions.ImportanceDrug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogen Enterococcus faecalis as a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.
| S-EPMC3781836 | biostudies-literature
Project description:MESH1 is the metazoan homolog of bacterial SpoT, the main phosphatase that dephosphorylates and degrades (p)ppGpp, the alarmone involved in the bacterial stringent response. The functional role of MESH1 in human cells is unknown. To define the global transcriptional response to MESH1 knockdown, we employed microarrays to perform transcriptome analysis of H1975 when the MESH1 was knocked down using three independent siRNAs targeting MESH1. The changes of each gene were derived by zero-transformation, followed by filtering to derive the genes affected by MESH1 knockdown. These datasets showed the transcriptional features of the mammalian stringent response and identified a prominent TAZ repression. Thus, we performed a second experiment to determine the contribution of TAZ repression to the transcriptional response of MESH1 knockdown by comparing the effects of MESH1-knockdown gene signatures in H1975 cells transduced with control or constitutive active TAZ (TAZS89A). The transcriptional response of these two cells to MESH1 was derived by zero transformation, followed by the effects of TAZ restoration to define the contribution of TAZ repression to the transcriptome features of human stringent response. The transcriptome data will be useful for the mechanistic understanding of the functional role of MESH1 in human cancer cells.
| S-EPMC9905932 | biostudies-literature
Project description:The stringent response, originally identified in Escherichia coli as a signal that leads to reprogramming of gene expression under starvation or nutrient deprivation, is now recognized as ubiquitous in all bacteria, and also as part of a broader survival strategy in diverse, other stress conditions. Much of our insight into this phenomenon derives from the role of hyperphosphorylated guanosine derivatives (pppGpp, ppGpp, pGpp; guanosine penta-, tetra- and tri-phosphate, respectively) that are synthesized on starvation cues and act as messengers or alarmones. These molecules, collectively referred to here as (p)ppGpp, orchestrate a complex network of biochemical steps that eventually lead to the repression of stable RNA synthesis, growth, and cell division, while promoting amino acid biosynthesis, survival, persistence, and virulence. In this analytical review, we summarize the mechanism of the major signaling pathways in the stringent response, consisting of the synthesis of the (p)ppGpp, their interaction with RNA polymerase, and diverse factors of macromolecular biosynthesis, leading to differential inhibition and activation of specific promoters. We also briefly touch upon the recently reported stringent-like response in a few eukaryotes, which is a very disparate mechanism involving MESH1 (Metazoan SpoT Homolog 1), a cytosolic NADPH phosphatase. Lastly, using ppGpp as an example, we speculate on possible pathways of simultaneous evolution of alarmones and their multiple targets.
| S-EPMC9965611 | biostudies-literature
Project description:All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main metabolic stress-coping mechanism is the stringent response, which is triggered by the accumulation of "alarmone" (p)ppGpp to arrest proliferation and reprogram the transcriptome. The level of (p)ppGpp is regulated by its synthetase RelA and its hydrolase SpoT. MESH1 is the metazoan homolog of bacterial SpoT that regulates the bacterial stringent response by degrading the alarmone (p)ppGpp. While MESH1, like SpoT, can also dephosphorylate (p)ppGpp, mammalian cells do not have significant levels of this metabolite, and the relevant enzymatic activities and function of MESH1 have remained a mystery. Through genetic and biochemical analyses, we have solved the long-held mystery and identified MESH1 as the first mammalian cytosolic NADPH phosphatase involved in ferroptosis. Furthermore, we discovered that MESH1 removal leads to proliferation arrest, translation inhibition, and a prominent transcriptional and metabolic response. Therefore, MESH1 knockdown triggers a novel stress response with phenotypic conservation with the bacterial stringent response via distinct substrates and molecular pathways. Here, we summarize the background of the MESH1, illustrate the striking conservation of phenotypes in different organisms during evolution and discuss remaining questions in the field.
| S-EPMC9166373 | biostudies-literature
Project description:Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.
| S-EPMC2620808 | biostudies-literature
Project description:Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.
| S-EPMC7666150 | biostudies-literature
Project description:Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.
| S-EPMC5444170 | biostudies-literature
Project description:The stringent response to amino acid starvation, whereby stable RNA synthesis is curtailed in favour of transcription of amino acid biosynthetic genes, is controlled by the alarmone ppGpp. To elucidate the extent of gene expression effected by ppGpp, we designed an experimental system based on starvation for isoleucine, which could be applied to both wild-type Escherichia coli and the multiauxotrophic relA spoT mutant (ppGpp(0)). We used microarrays to profile the response to amino acid starvation in both strains. The wild-type response included induction of the general stress response, downregulation of genes involved in production of macromolecular structures and comprehensive restructuring of metabolic gene expression, but not induction of amino acid biosynthesis genes en masse. This restructuring of metabolism was confirmed using kinetic Biolog assays. These responses were profoundly altered in the ppGpp(0) strain. Furthermore, upon isoleucine starvation, the ppGpp(0) strain exhibited a larger cell size and continued growth, ultimately producing 50% more biomass than the wild-type, despite producing a similar amount of protein. This mutant phenotype correlated with aberrant gene expression in diverse processes, including DNA replication, cell division, and fatty acid and membrane biosynthesis. We present a model that expands and functionally integrates the ppGpp-mediated stringent response to include control of virtually all macromolecular synthesis and intermediary metabolism.
| S-EPMC3719176 | biostudies-literature
Project description:The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.
| S-EPMC3654024 | biostudies-literature
Project description:Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent of its proposed role in the two-component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.
| S-EPMC10704370 | biostudies-literature