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Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

ABSTRACT: Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a posttranscriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

SUBMITTER: Kriel A

PROVIDER: S-EPMC3483369 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

Kriel Allison A Bittner Alycia N AN Kim Sok Ho SH Liu Kuanqing K Tehranchi Ashley K AK Zou Winnie Y WY Rendon Samantha S Chen Rui R Tu Benjamin P BP Wang Jue D JD

Molecular cell 20120913 2

Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a posttranscriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics ...[more]

PMID: 22981860

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Project description:Staphylococcus aureus is an opportunistic bacterial pathogen that often results in difficult-to-treat infections. One mechanism used by S. aureus to enhance survival during infection is the stringent response. This is a stress survival pathway that utilizes the nucleotides (p)ppGpp to reallocate bacterial resources, shutting down growth until conditions improve. Small colony variants (SCVs) of S. aureus are frequently associated with chronic infections, and this phenotype has previously been linked to a hyperactive stringent response. Here, we examine the role of (p)ppGpp in the long-term survival of S. aureus under nutrient-restricted conditions. When starved, a (p)ppGpp-null S. aureus mutant strain ((p)ppGpp0) initially had decreased viability. However, after 3 days we observed the presence and dominance of a population of small colonies. Similar to SCVs, these small colony isolates (p0-SCIs) had reduced growth but remained hemolytic and sensitive to gentamicin, phenotypes that have been tied to SCVs previously. Genomic analysis of the p0-SCIs revealed mutations arising within gmk, encoding an enzyme in the GTP synthesis pathway. We show that a (p)ppGpp0 strain has elevated levels of GTP, and that the mutations in the p0-SCIs all lower Gmk enzyme activity and consequently cellular GTP levels. We further show that in the absence of (p)ppGpp, cell viability can be rescued using the GuaA inhibitor decoyinine, which artificially lowers the intracellular GTP concentration. Our study highlights the role of (p)ppGpp in GTP homeostasis and underscores the importance of nucleotide signaling for long-term survival of S. aureus in nutrient-limiting conditions, such as those encountered during infections. IMPORTANCE Staphylococcus aureus is a human pathogen that upon invasion of a host encounters stresses, such as nutritional restriction. The bacteria respond by switching on a signaling cascade controlled by the nucleotides (p)ppGpp. These nucleotides function to shut down bacterial growth until conditions improve. Therefore, (p)ppGpp are important for bacterial survival and have been implicated in promoting chronic infections. Here, we investigate the importance of (p)ppGpp for long-term survival of bacteria in nutrient-limiting conditions similar to those in a human host. We discovered that in the absence of (p)ppGpp, bacterial viability decreases due to dysregulation of GTP homeostasis. However, the (p)ppGpp-null bacteria were able to compensate by introducing mutations in the GTP synthesis pathway that led to a reduction in GTP build-up and a rescue of viability. This study therefore highlights the importance of (p)ppGpp for the regulation of GTP levels and for long-term survival of S. aureus in restricted environments.

Project description:Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a spectrum of human disease states. Metallobiology of human pathogens is revealing the fundamental role of metals in both nutritional immunity leading to pathogen starvation and metal poisoning of pathogens by innate immune cells. Spy0980 (MntE) is a paralog of the GAS zinc efflux pump CzcD. Through use of an isogenic mntE deletion mutant in the GAS serotype M1T1 strain 5448, we have elucidated that MntE is a manganese-specific efflux pump required for GAS virulence. The 5448ΔmntE mutant had significantly lower survival following infection of human neutrophils than did the 5448 wild type and the complemented mutant (5448ΔmntE::mntE). Manganese homeostasis may provide protection against oxidative stress, explaining the observed ex vivo reduction in virulence. In the presence of manganese and hydrogen peroxide, 5448ΔmntE mutant exhibits significantly lower survival than wild-type 5448 and the complemented mutant. We hypothesize that MntE, by maintaining homeostatic control of cytoplasmic manganese, ensures that the peroxide response repressor PerR is optimally poised to respond to hydrogen peroxide stress. Creation of a 5448ΔmntE-ΔperR double mutant rescued the oxidative stress resistance of the double mutant to wild-type levels in the presence of manganese and hydrogen peroxide. This work elucidates the mechanism for manganese toxicity within GAS and the crucial role of manganese homeostasis in maintaining GAS virulence. Manganese is traditionally viewed as a beneficial metal ion to bacteria, and it is also established that most bacteria can tolerate high concentrations of this transition metal. In this work, we show that in group A Streptococcus, mutation of the mntE locus, which encodes a transport protein of the cation diffusion facilitator (CDF) family, results in accumulation of manganese and sensitivity to this transition metal ion. The toxicity of manganese is indirect and is the result of a failure of the PerR regulator to respond to oxidative stress in the presence of high intracellular manganese concentrations. These results highlight the importance of MntE in manganese homeostasis and maintenance of an optimal manganese/iron ratio in GAS and the impact of manganese on resistance to oxidative stress and virulence.

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Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

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Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

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