scholarly journals Targeting the Mycobacterium tuberculosis Stringent Response as a Strategy for Shortening Tuberculosis Treatment

2021 ◽  
Vol 12 ◽  
Author(s):  
Carina Danchik ◽  
Siqing Wang ◽  
Petros C. Karakousis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Species ◽  
Stringent Response ◽  
Therapeutic Benefit ◽  
Current Understanding ◽  
Antibiotic Tolerance ◽  
Bacterial Survival ◽  
Inorganic Polyphosphate ◽  
Regulatory Molecule ◽  
Response To Stress

The stringent response is well conserved across bacterial species and is a key pathway involved both in bacterial survival and virulence and in the induction of antibiotic tolerance in Mycobacteria. It is mediated by the alarmone (p)ppGpp and the regulatory molecule inorganic polyphosphate in response to stress conditions such as nutrient starvation. Efforts to pharmacologically target various components of the stringent response have shown promise in modulating mycobacterial virulence and antibiotic tolerance. In this review, we summarize the current understanding of the stringent response and its role in virulence and tolerance in Mycobacteria, including evidence that targeting this pathway could have therapeutic benefit.

scholarly journals Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to IsoniazidIn Vivo

2016 ◽  
Vol 60 (11) ◽  
pp. 6460-6470 ◽  
Author(s):  
Yu-Min Chuang ◽  
Noton K. Dutta ◽  
Chien-Fu Hung ◽  
T.-C. Wu ◽  
Harvey Rubin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biofilm Formation ◽  
Tricarboxylic Acid Cycle ◽  
Antibiotic Sensitivity ◽  
Stringent Response ◽  
Antibiotic Tolerance ◽  
Inorganic Polyphosphate ◽  
Response Factors ◽  
Content Type

ABSTRACTMycobacterium tuberculosisremains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence ofM. tuberculosispersisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in theM. tuberculosisstringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis inM. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulatingM. tuberculosismutant strains deficient inppx1orppk2had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. Theppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency ofppk2was associated with increased sensitivity to plumbagin and meropenem and deficiency ofppx1led to enhanced susceptibility to clofazimine. A DNA vaccine expressingppx1andppk2, together with two other members of theM. tuberculosisstringent response,M. tuberculosisrelandsigE, did not show protective activity against aerosol challenge withM. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of theM. tuberculosisstringent response play an important role inM. tuberculosismetabolism, biofilm formation, and antibiotic sensitivityin vivo.

scholarly journals Structural characterisation of a polyphosphate kinase

2014 ◽  
Vol 70 (a1) ◽  
pp. C466-C466
Author(s):  
Alice Loasby ◽  
Peter Roach ◽  
Petra Oyston
Keyword(s):  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Exothermic Reaction ◽  
Stringent Response ◽  
Titration Calorimetry ◽  
Polyphosphate Kinase ◽  
Inorganic Polyphosphate ◽  
Structural Characterisation ◽  
Signalling Molecules ◽  
Expression Of Genes

When deprived of nutrients bacteria undergo what is referred to as the `stringent response'. During the stringent response, the cell induces the expression of genes to cope with stress and starvation and diverts resources away from cell growth and division. This involves altering the cellular levels of the signalling molecules: ppGpp, pppGpp and inorganic polyphosphate (polyP)[1]. This is controlled by four enzymes; Polyphosphate kinase (Ppk), Exopolyphosphatase (Ppx), RelA and SpoT. Therefore, modulation of these enzymes is an attractive method for targeting pathogenic bacteria such as Francisella tularensis (F. tularensis), the causative agent of tularemia. FtPpk transfers phosphate from polyP to ADP to generate ATP, a reaction that is fully reversible. The importance of FtPpk in infection has been demonstrated in knockout mutants which resulted in defective growth of F. tularensis in macrophages[2]. Mutagenesis in other pathogenic bacteria has yielded attenuated mutants, suggesting an important role for Ppk in a broad spectrum of bacterial species[3]. To maximise our understanding of FtPpk, our aim was to obtain co-crystals of the enzyme and substrates. Isothermal Titration Calorimetry (ITC) was used to measure the binding of polyP and ADP to FtPpk as independent substrates. FtPpk binds ADP very weakly or not at all in the absence of polyP. FtPpk binds polyP in an exothermic reaction with a relatively high affinity (0.385 µM) in the absence of ADP. Co-crystals of FtPpk with polyP and ADP have been obtained and optimised to diffract to 2.0 Å, identifying a potential binding site for polyP. A non-hydrolysable analogue of ATP has been chemically synthesised to allow co-crystallisation experiments.

scholarly journals Salicylate Reduces Susceptibility of Mycobacterium tuberculosis to Multiple Antituberculosis Drugs

2002 ◽  
Vol 46 (8) ◽  
pp. 2636-2639 ◽  
Author(s):  
Alain Schaller ◽  
Zhonghe Sun ◽  
Yongping Yang ◽  
Akos Somoskovi ◽  
Ying Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Species ◽  
Bacterial Survival ◽  
Multiple Antibiotic Resistance ◽  
Treatment Needs ◽  
Antituberculosis Drugs ◽  
Viability Assay ◽  
Tb Treatment ◽  
Survival Assay

ABSTRACT Salicylate induces multiple antibiotic resistance in various bacterial species. Here we investigated the effect of salicylate on the susceptibility of Mycobacterium tuberculosis to a range of antituberculosis (anti-TB) drugs. In the presence of salicylate, the killing effects of isoniazid (INH), rifampin (RMP), ethambutol (EMB), streptomycin (STR), and p-aminosalicylate (PAS) were reduced, as shown with a tetrazolium redox dye viability assay and a bacterial survival assay. Salicylate-induced resistance was more pronounced for PAS, STR, and EMB but was not apparent for INH and RMP when salicylate and the anti-TB agents were incorporated into 7H11 plates. The significance of these findings for TB treatment needs to be further evaluated in vivo.

scholarly journals Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

2015 ◽  
Vol 197 (7) ◽  
pp. 1146-1156 ◽  
Author(s):  
Anthony O. Gaca ◽  
Cristina Colomer-Winter ◽  
José A. Lemos
Keyword(s):  
Bacterial Species ◽  
Cell Formation ◽  
Stringent Response ◽  
Survival Strategies ◽  
Cell Physiology ◽  
Bacterial Survival ◽  
Stress Survival ◽  
Rapid Accumulation ◽  
Relationship Of

In nearly all bacterial species examined so far, amino acid starvation triggers the rapid accumulation of the nucleotide second messenger (p)ppGpp, the effector of the stringent response. While for years the enzymes involved in (p)ppGpp metabolism and the significance of (p)ppGpp accumulation to stress survival were considered well defined, a recent surge of interest in the field has uncovered an unanticipated level of diversity in how bacteria metabolize and utilize (p)ppGpp to rapidly synchronize a variety of biological processes important for growth and stress survival. In addition to the classic activation of the stringent response, it has become evident that (p)ppGpp exerts differential effects on cell physiology in an incremental manner rather than simply acting as a biphasic switch that controls growth or stasis. Of particular interest is the intimate relationship of (p)ppGpp with persister cell formation and virulence, which has spurred the pursuit of (p)ppGpp inhibitors as a means to control recalcitrant infections. Here, we present an overview of the enzymes responsible for (p)ppGpp metabolism, elaborate on the intricacies that link basal production of (p)ppGpp to bacterial homeostasis, and discuss the implications of targeting (p)ppGpp synthesis as a means to disrupt long-term bacterial survival strategies.

scholarly journals Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in Mycobacterium tuberculosis

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Yu-Min Chuang ◽  
Nirmalya Bandyopadhyay ◽  
Dalin Rifat ◽  
Harvey Rubin ◽  
Joel S. Bader ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Stringent Response ◽  
Inorganic Polyphosphate ◽  
Content Type ◽  
P Accumulation ◽  
Wall Permeability ◽  
Altered Cell ◽  
Cell Wall Permeability ◽  
Long Chain

ABSTRACTMycobacterium tuberculosiscan persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here thatM. tuberculosispoly(P) accumulation is temporally linked to bacillary growth restriction. We also identifyM. tuberculosisRv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression ofRv1026(ppx2), we found thatM. tuberculosispoly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, theppx2knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of theppx2knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role inM. tuberculosisgrowth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability.IMPORTANCEThe stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediateMycobacterium tuberculosispersistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in theppx2gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.

scholarly journals Phosphorylation on PstP regulates cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

2019 ◽  
Author(s):  
Farah Shamma ◽  
Kadamba Papavinasasundaram ◽  
Samantha Y. Quintanilla ◽  
Aditya Bandekar ◽  
Christopher Sassetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Control Cell ◽  
Model Organism ◽  
Mycolic Acid ◽  
Regulatory Function ◽  
Antibiotic Tolerance ◽  
Bacterial Survival ◽  
Cell Wall Metabolism

AbstractMycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.ImportanceRegulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

scholarly journals Inorganic polyphosphate accumulation suppresses the dormancy response and virulence in Mycobacterium tuberculosis

2019 ◽  
Vol 294 (28) ◽  
pp. 10819-10832 ◽  
Author(s):  
Prabhakar Tiwari ◽  
Tannu Priya Gosain ◽  
Mamta Singh ◽  
Gaurav D. Sankhe ◽  
Garima Arora ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Transcriptional Profiling ◽  
Critical Role ◽  
Stringent Response ◽  
Growth Conditions ◽  
Therapeutic Interventions ◽  
Polyphosphate Kinase ◽  
Low Oxygen ◽  
Inorganic Polyphosphate ◽  
Double Knockout

Stringent response pathways involving inorganic polyphosphate (PolyP) play an essential role in bacterial stress adaptation and virulence. The intracellular levels of PolyP are modulated by the activities of polyphosphate kinase-1 (PPK1), polyphosphate kinase-2 (PPK2), and exopolyphosphatases (PPXs). The genome of Mycobacterium tuberculosis encodes two functional PPXs, and simultaneous deletion of ppx1 and ppx2 results in a defect in biofilm formation. We demonstrate here that these PPXs cumulatively contribute to the ability of M. tuberculosis to survive in nutrient-limiting, low-oxygen growth conditions and also in macrophages. Characterization of single (Δppx2) and double knockout (dkppx) strains of M. tuberculosis indicated that PPX-mediated PolyP degradation is essential for establishing bacterial infection in guinea pigs. RNA-Seq–based transcriptional profiling revealed that relative to the parental strain, the expression levels of DosR regulon–regulated dormancy genes were significantly reduced in the dkppx mutant strain. In concordance, we also provide evidence that PolyP inhibits the autophosphorylation activities associated with DosT and DosS sensor kinases. The results in this study uncover that enzymes involved in PolyP homeostasis play a critical role in M. tuberculosis physiology and virulence and are attractive targets for developing more effective therapeutic interventions.

scholarly journals Phosphorylation on PstP regulates cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Farah Shamma ◽  
Kadamba Papavinasasundaram ◽  
Samantha Y. Quintanilla ◽  
Aditya Bandekar ◽  
Christopher Sassetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Control Cell ◽  
Model Organism ◽  
Mycolic Acid ◽  
Regulatory Function ◽  
Antibiotic Tolerance ◽  
Bacterial Survival ◽  
Cell Wall Metabolism

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis. Importance Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

scholarly journals Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

2021 ◽  
Vol 22 (14) ◽  
pp. 7521
Author(s):  
Marko Nedeljković ◽  
Diego Emiliano Sastre ◽  
Eric John Sundberg
Keyword(s):  
Immune Responses ◽  
Basal Body ◽  
Vaccine Development ◽  
Bacterial Species ◽  
High Variability ◽  
Bacterial Survival ◽  
Bacterial Flagellum ◽  
Capping Proteins ◽  
Flagellar Filament ◽  
Flagellar Assembly

The bacterial flagellum is a complex and dynamic nanomachine that propels bacteria through liquids. It consists of a basal body, a hook, and a long filament. The flagellar filament is composed of thousands of copies of the protein flagellin (FliC) arranged helically and ending with a filament cap composed of an oligomer of the protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases are even completely absent. Flagellar assembly is a complex and energetically costly process triggered by environmental stimuli and, accordingly, highly regulated on transcriptional, translational and post-translational levels. Apart from its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction and pathogenicity, such as adhesion to surfaces, secretion of virulence factors and formation of biofilms. Additionally, due to its ability to provoke potent immune responses, flagellins have a role as adjuvants in vaccine development. In this review, we summarize the latest knowledge on the structure of flagellins, capping proteins and filaments, as well as their regulation and role during the colonization and infection of the host.

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