RESUMEN
The alarmone (p)ppGpp serves as the signalling molecule for the bacterial universal stringent response and plays a crucial role in bacterial virulence, persistence, and stress adaptation. Consequently, there is a significant focus on developing new drugs that target and modulate the levels of (p)ppGpp as a potential strategy for controlling bacterial infections. However, despite the availability of various methods for detecting (p)ppGpp, a simple and straightforward detection method is needed. In this study, we demonstrated that malachite green, a well-established compound used for phosphate detection, can directly detect (p)ppGpp and its analogues esp., pGpp. By utilizing malachite green, we identified three new inhibitors of the hydrolase activity of SpoT, one of the two RelA-SpoT homolog (RSH) proteins responsible for making and hydrolyzing (p)ppGpp in Escherichia coli. These findings highlight the convenience and practicality of malachite green, which can be widely employed in high-throughput studies to investigate (pp)pGpp in vitro and discover novel regulators of RSH proteins.
RESUMEN
Upregulation of MYC is a hallmark of cancer, wherein MYC drives oncogenic gene expression and elevates total RNA synthesis across cancer cell transcriptomes. Although this transcriptional anabolism fuels cancer growth and survival, the consequences and metabolic stresses induced by excess cellular RNA are poorly understood. Herein, we discover that RNA degradation and downstream ribonucleotide catabolism is a novel mechanism of MYC-induced cancer cell death. Combining genetics and metabolomics, we find that MYC increases RNA decay through the cytoplasmic exosome, resulting in the accumulation of cytotoxic RNA catabolites and reactive oxygen species. Notably, tumor-derived exosome mutations abrogate MYC-induced cell death, suggesting excess RNA decay may be toxic to human cancers. In agreement, purine salvage acts as a compensatory pathway that mitigates MYC-induced ribonucleotide catabolism, and inhibitors of purine salvage impair MYC+ tumor progression. Together, these data suggest that MYC-induced RNA decay is an oncogenic stress that can be exploited therapeutically. Significance: MYC is the most common oncogenic driver of poor-prognosis cancers but has been recalcitrant to therapeutic inhibition. We discovered a new vulnerability in MYC+ cancer where MYC induces cell death through excess RNA decay. Therapeutics that exacerbate downstream ribonucleotide catabolism provide a therapeutically tractable approach to TNBC (Triple-negative Breast Cancer) and other MYC-driven cancers.
Asunto(s)
Neoplasias de la Mama , Proteínas Proto-Oncogénicas c-myc , Estabilidad del ARN , Ribonucleótidos , Humanos , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ribonucleótidos/farmacología , Línea Celular Tumoral , Ratones , Regulación Neoplásica de la Expresión Génica , AnimalesRESUMEN
Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.
Asunto(s)
Guanosina Trifosfato , IMP Deshidrogenasa , IMP Deshidrogenasa/metabolismo , IMP Deshidrogenasa/química , IMP Deshidrogenasa/antagonistas & inhibidores , Regulación Alostérica , Guanosina Trifosfato/metabolismo , Microscopía por Crioelectrón , Dominio Catalítico , Modelos Moleculares , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Guanosina Pentafosfato/metabolismo , Inosina Monofosfato/metabolismo , Inosina Monofosfato/química , Unión Proteica , Adenosina Trifosfato/metabolismo , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/metabolismoRESUMEN
Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 µM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 µM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.
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Diseño de Fármacos , Inhibidores Enzimáticos , Pentosiltransferasa , Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Relación Estructura-Actividad , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Estructura Molecular , Dominio CatalíticoRESUMEN
Burn injuries are a significant global health concern, with more than 11 million people requiring medical intervention each year and approximately 180,000 deaths annually. Despite progress in health and social care, burn injuries continue to result in socioeconomic burdens for victims and their families. The management of severe burn injuries involves preventing and treating burn shock and promoting skin repair through a two-step procedure of covering and closing the wound. Currently, split-thickness/full-thickness skin autografts are the gold standard for permanent skin substitution. However, deep burns treated with split-thickness skin autografts may contract, leading to functional and appearance issues. Conversely, defects treated with full-thickness skin autografts often result in more satisfactory function and appearance. The development of tissue-engineered dermal templates has further expanded the scope of wound repair, providing scar reductive and regenerative properties that have extended their use to reconstructive surgical interventions. Although their interactions with the wound microenvironment are not fully understood, these templates have shown potential in local infection control. This narrative review discusses the current state of wound repair in burn injuries, focusing on the progress made from wound cover to wound closure and local infection control. Advancements in technology and therapies hold promise for improving the outcomes for burn injury patients. Understanding the underlying mechanisms of wound repair and tissue regeneration may provide new insights for developing more effective treatments in the future.
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Quemaduras , Humanos , Quemaduras/cirugía , Quemaduras/patología , Piel/patología , Cicatrización de Heridas , Trasplante de Piel/métodos , Cicatriz/etiología , Cicatriz/prevención & control , Cicatriz/cirugíaRESUMEN
In the past few decades, society has faced rapid development and spreading of antimicrobial resistance due to antibiotic misuse and overuse and the immense adaptability of bacteria. Difficulties in obtaining effective antimicrobial molecules from natural sources challenged scientists to develop synthetic molecules with antimicrobial effect. We developed modular molecules named LEGO-Lipophosphonoxins (LEGO-LPPO) capable of inducing cytoplasmic membrane perforation. In this structure-activity relationship study we focused on the role of the LEGO-LPPO hydrophobic module directing the molecule insertion into the cytoplasmic membrane. We selected three LEGO-LPPO molecules named C9, C8 and C7 differing in the length of their hydrophobic chain and consisting of an alkenyl group containing one double bond. The molecule with the long hydrophobic chain (C9) was shown to be the most effective with the lowest MIC and highest perforation rate both in vivo and in vitro. We observed high antimicrobial activity against both G+ and G- bacteria with significant differences in LEGO-LPPOs mechanism of action on these two cell types. We observed a highly cooperative mechanism of LEGO-LPPO action on G- bacteria as well as on liposomes resembling G- bacteria. LEGO-LPPO action on G- bacteria was significantly slower compared to G+ bacteria suggesting the role of the outer membrane in affecting the LEGO-LPPOs perforation rate. This notion was supported by the higher sensitivity of the E. coli strain with a compromised outer membrane. Finally, we noted that the composition of the cytoplasmic membrane affects the activity of LEGO-LPPOs since the presence of phosphatidylethanolamine increases their membrane disrupting activity.
RESUMEN
Lipophosphonoxins (LPPOs) represent a new group of membrane-targeting antibiotics. Three generations of LPPOs have been described: First-generation LPPOs, second-generation LPPOs, and LEGO-LPPOs. All three generations have a similar mode of bactericidal action of targeting and disrupting the bacterial cytoplasmic membrane of prokaryotic cells, with limited effect on eukaryotic cells. First-generation LPPOs showed excellent bactericidal activity against Gram-positive species, including multiresistant strains. Second-generation LPPOs broaden the antibiotic effect also against Gram-negative bacteria. However, both first- and second-generation LPPOs lose their antibacterial activity in the presence of serum albumin. LEGO-LPPOs were found to be active against both Gram-positive and Gram-negative bacteria, have better selectivity as compared to first- and second-generation resistance to LEGO-LPPOs was also not observed, and are active even in the presence of serum albumin. Second-generation LPPOs have been studied as antimicrobial additives in bone cement and as nanofiber dressing components in the treatment of wound infections in mice. Second-generation LPPOs and LEGO-LPPOs were also tested to treat ex vivo simulated endodontic infections in dental root canals. The results of all these studies were encouraging and suggested further investigation of LPPOs in these indications. This paper aims to review and compile published data on LPPOs.
RESUMEN
The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-LPPOs, loosely based on LPPOs, consisting of a central linker module with two attached connector modules on either side. The connector modules are then decorated with polar and hydrophobic modules. We performed an extensive structure-activity relationship study by varying the length of the linker and hydrophobic modules. The best compounds were active against both Gram-negative and Gram-positive species including multiresistant strains and persisters. LEGO-LPPOs act by first depleting the membrane potential and then creating pores in the cytoplasmic membrane. Importantly, their efficacy is not affected by the presence of serum albumins. Low cytotoxicity and low propensity for resistance development demonstrate their potential for therapeutic use.
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Antibacterianos , Bacterias Grampositivas , Albúminas , Antibacterianos/química , Membrana Celular , Bacterias Gramnegativas , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
Mycobacteria express enzymes from both the de novo and purine-salvage pathways. However, the regulation of these processes and the roles of individual metabolic enzymes have not been sufficiently detailed. Both Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm) possess three guaB genes, but information is only available on guaB2, which encodes an essential inosine 5'-monophosphate dehydrogenase (IMPDH) involved in de novo purine biosynthesis. This study shows that guaB1, annotated in databases as a putative IMPDH, encodes a guanosine 5'-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine-salvage pathway and contains a cystathionine-ß-synthase domain (CBS), which is essential for enzyme activity. GMPR activity is allosterically regulated by the ATP/GTP ratio in a pH-dependent manner. Bioinformatic analysis has indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.
Asunto(s)
Cistationina , Mycobacterium tuberculosis , Adenosina Trifosfato , Cistationina betasintasa/genética , GMP-Reductasa/genética , GMP-Reductasa/metabolismo , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/metabolismo , Inosina , Inosina Monofosfato/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
(1) Background: The root canal system has complex anatomical and histological features that make it impossible to completely remove all bacteria by mechanical means only; they must be supplemented with disinfectant irrigation. Current disinfectants are unable to eliminate certain microorganisms that persist in the root canal, resulting in treatment failure. At the Institute of Organic Chemistry and Biochemistry, Prague, novel substances with the bactericidal effect, termed lipophosphonoxins (LPPOs), have been discovered. The aim of this pilot study was to investigate the ex vivo effects of second- and third-generation LPPOs on Enterococcus faecalis and compare them with 5% sodium hypochlorite (NaOCl), 0.12% chlorhexidine digluconate, and 17% ethylenediaminetetraacetic acid (EDTA). (2) Methods: The root canal's dentin was used as a carrier for biofilm formation in the extracted human mature mandibular premolars. The samples were filled with cultivation broth and 0.25% glucose with tested solutions. In control samples, only fresh cultivation broth (negative control) and cultivation broth with bacterial suspension (growth control) were used. Each sample was inoculated with E. faecalis CCM4224 except for the negative control, and cultivation was performed. To determine the number of planktonic cells, the sample content was inoculated on blood agar. To evaluate biofilm formation inhibition, samples were placed in tubes with BHI. (3) Results: LPPOs exhibited a reduction in biofilm growth and bacteria comparable to NaOCl, and they were superior to other tested disinfectants. (4) Conclusions: The study results suggest the effect of lipophosphonoxins on E. faecalis CCM 4224 reduces planktonic bacterial cells and inhibits formation of biofilm in root canal samples.
RESUMEN
Active wound dressings are attracting extensive attention in soft tissue repair and regeneration, including bacteria-infected skin wound healing. As the wide use of antibiotics leads to drug resistance we present here a new concept of wound dressings based on the polycaprolactone nanofiber scaffold (NANO) releasing second generation lipophosphonoxin (LPPO) as antibacterial agent. Firstly, we demonstrated in vitro that LPPO released from NANO exerted antibacterial activity while not impairing proliferation/differentiation of fibroblasts and keratinocytes. Secondly, using a mouse model we showed that NANO loaded with LPPO significantly reduced the Staphylococcus aureus counts in infected wounds as evaluated 7 days post-surgery. Furthermore, the rate of degradation and subsequent LPPO release in infected wounds was also facilitated by lytic enzymes secreted by inoculated bacteria. Finally, LPPO displayed negligible to no systemic absorption. In conclusion, the composite antibacterial NANO-LPPO-based dressing reduces the bacterial load and promotes skin repair, with the potential to treat wounds in clinical settings.
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Antibacterianos/administración & dosificación , Vendajes , Nanofibras , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/tratamiento farmacológico , Animales , Antibacterianos/uso terapéutico , Modelos Animales de Enfermedad , RatonesRESUMEN
While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.
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Nucleótidos de Adenina/metabolismo , Proteínas Bacterianas/metabolismo , Ligasas/metabolismo , Nucleótidos de Adenina/síntesis química , Sitio Alostérico , Bacillus subtilis , Desoxirribonucleótidos , Escherichia coli , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Unión Proteica , Conformación Proteica , Pirofosfatasas/metabolismoRESUMEN
RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.
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Toxinas Bacterianas/metabolismo , Guanosina Pentafosfato/metabolismo , Ligasas/metabolismo , ARN de Transferencia/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacología , Bacilos Grampositivos Asporogénicos/química , Bacilos Grampositivos Asporogénicos/metabolismo , Guanosina Pentafosfato/química , Ligasas/química , Ligasas/genética , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Pirofosfatasas , Ribosomas/metabolismoRESUMEN
Lipophosphonoxins (LPPOs) are small modular synthetic antibacterial compounds that target the cytoplasmic membrane. First-generation LPPOs (LPPO I) exhibit an antimicrobial activity against Gram-positive bacteria; however they do not exhibit any activity against Gram-negatives. Second-generation LPPOs (LPPO II) also exhibit broadened activity against Gram-negatives. We investigated the reasons behind this different susceptibility of bacteria to the two generations of LPPOs using model membranes and the living model bacteria Bacillus subtilis and Escherichia coli. We show that both generations of LPPOs form oligomeric conductive pores and permeabilize the bacterial membrane of sensitive cells. LPPO activity is not affected by the value of the target membrane potential, and thus they are also active against persister cells. The insensitivity of Gram-negative bacteria to LPPO I is probably caused by the barrier function of the outer membrane with LPS. LPPO I is almost incapable of overcoming the outer membrane in living cells, and the presence of LPS in liposomes substantially reduces their activity. Further, the antimicrobial activity of LPPO is also influenced by the phospholipid composition of the target membrane. A higher proportion of phospholipids with neutral charge such as phosphatidylethanolamine or phosphatidylcholine reduces the LPPO permeabilizing potential.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacillus subtilis/efectos de los fármacos , Membrana Externa Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Antibacterianos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Bacillus subtilis/química , Bacillus subtilis/citología , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Permeabilidad de la Membrana Celular , Escherichia coli/química , Escherichia coli/citología , Membrana Dobles de Lípidos , Potenciales de la Membrana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Fosfatidilcolinas/análisis , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/análisis , Fosfatidiletanolaminas/metabolismoRESUMEN
Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.
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Antibacterianos/química , Proteínas Bacterianas/metabolismo , Pentosiltransferasa/metabolismo , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Proteínas Bacterianas/química , Sitios de Unión , Cristalografía por Rayos X , Enfermedades Gastrointestinales/tratamiento farmacológico , Enfermedades Gastrointestinales/microbiología , Enfermedades Gastrointestinales/patología , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/patología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Humanos , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/metabolismo , Hipoxantinas/química , Hipoxantinas/metabolismo , Hipoxantinas/farmacología , Hipoxantinas/uso terapéutico , Cinética , Simulación de Dinámica Molecular , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacología , Organofosfonatos/uso terapéutico , Pentosiltransferasa/química , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Profármacos/uso terapéutico , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.
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Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Ligasas/genética , Staphylococcus aureus/genética , Estrés Fisiológico , Proteínas Bacterianas/metabolismo , Ligasas/metabolismo , Staphylococcus aureus/metabolismoRESUMEN
Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.
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Bacterias/crecimiento & desarrollo , Guanosina Pentafosfato/metabolismo , Sistemas Toxina-Antitoxina/fisiología , Nucleótidos de Adenina/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Bases de Datos Genéticas , Regulación Bacteriana de la Expresión Génica/genética , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligasas/metabolismo , Pirofosfatasas/metabolismo , Transducción de Señal , Estrés Fisiológico/fisiologíaRESUMEN
Successful surgeries involving orthopedic implants depend on the avoidance of biofilm development on the implant surface during the early postoperative period. Here, we investigate the potential of novel antibacterial compounds-second-generation lipophosphonoxins (LPPOs II)-as additives to surgical bone cements. We demonstrate (i) excellent thermostability of LPPOs II, which is essential to withstand elevated temperatures during exothermic cement polymerization; (ii) unchanged tensile strength and elongation at the break properties of the composite cements containing LPPOs II compared to cements without additives; (iii) convenient elution kinetics on the order of days; and (iv) the strong antibiofilm activity of the LPPO II-loaded cements even against bacteria resistant to the medicinally utilized antibiotic, gentamicin. Thus, LPPOs II display promising potential as antimicrobial additives to surgical bone cements.
RESUMEN
Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of >25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Escherichia coli K12/aislamiento & purificación , Espectrometría de Masas/métodos , Nucleótidos/química , Concentración de Iones de Hidrógeno , Límite de Detección , Solventes/químicaRESUMEN
Therapeutic treatment of tuberculosis (TB) is becoming increasingly problematic due to the emergence of drug resistant Mycobacterium tuberculosis (Mt). Thus, new targets for anti-TB drug discovery need to be identified to combat and eradicate this disease. One such target is hypoxanthine-guanine phosphoribosyltransferase (HGPRT) which synthesises the 6-oxopurine nucleoside monophosphates essential for DNA/RNA production. [3R,4R]-4-Hypoxanthin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine and [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine (compound 6) are the most potent inhibitors of MtHGPRT yet discovered having Ki values of 60â¯nM. The crystal structure of the MtHGPRT.6 complex was obtained and compared with that of human HGPRT in complex with the same inhibitor. These structures provide explanations for the 60-fold difference in the inhibition constants between these two enzymes and a foundation for the design of next generation inhibitors. In addition, crystal structures of MtHGPRT in complex with two pyrrolidine nucleoside phosphosphonate inhibitors plus pyrophosphate provide insights into the final stage of the catalytic reaction. As the first step in ascertaining if such compounds have the potential to be developed as anti-TB therapeutics, the tetra-(ethyl L-phenylalanine) tetraamide prodrug of 6 was tested in cell based assays. This compound arrested the growth of virulent Mt not only in its replicating phase (IC50 of 14 µΜ) but also in its latent phase (IC50 of 29 µΜ). Furthermore, it arrested the growth of Mt in infected macrophages (MIC50 of 85 µΜ) and has a low cytotoxicity in mammalian cells (CC50 of 132⯱â¯20⯵M). These inhibitors are therefore viewed as forerunners of new anti-TB chemotherapeutics.