Academic literature on the topic 'Intrabacterial target'

ABSTRACTEfficient iron acquisition is crucial for the pathogenesis ofMycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active againstM. tuberculosishave been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound.

The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence ofMycobacterium tuberculosis(Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival ofMtbduring the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival ofMtbon even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficientMtbcultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and β-hydroxybutyrate. These changes were indicative of a glyoxylate-induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth of MS-deficientMtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization ofMtbin both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function ofMtbmalate synthase and advances its validation as a target for drug development.

Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC50 (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC50, concentration inhibiting growth by 50% = 10 to 20 nM). Here, we investigated the mechanism underlying this potency disconnect. To confirm that TA-C targets DHFR and investigate its unusual potency pattern, we focused on resistance mechanisms. In Mtb, resistance to DHFR inhibitors is frequently associated with mutations in thymidylate synthase thyA, which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed thyA mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F420. Thus, we hypothesized that TA-C may be metabolized by Mtb F420–dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F420-dependent resistance phenotype, suggesting that F420H2-dependent reduction is required for TA-C to exert its potent antibacterial activity. Indeed, chemically synthesized TA-C-Acid, the putative product of TA-C reduction, displayed a 100-fold lower IC50 against DHFR. Screening seven recombinant Mtb FDORs revealed that at least two of these enzymes reduce TA-C. This redundancy in activation explains why no mutations in the activating enzymes were identified in the resistance screen. Analysis of the reaction products confirmed that FDORs reduce TA-C at the predicted site, yielding TA-C-Acid. This work demonstrates that intrabacterial metabolism converts TA-C, a moderately active “prodrug,” into a 100-fold-more-potent DHFR inhibitor, thus explaining the disconnect between enzymatic and whole-cell activity.

ABSTRACT Mycobacterium tuberculosis can persist in macrophage phagosomes that acidify to a pH of ∼4.5 after activation of the macrophage with gamma interferon. How the bacterium resists the low pH of the acidified phagosome is incompletely understood. A screen of 10,100 M. tuberculosis transposon mutants for mutants hypersensitive to pH 4.5 led to the discovery of 21 genes whose disruption attenuated survival of M. tuberculosis at a low pH (41). Here, we show that acid-sensitive M. tuberculosis mutants with transposon insertions in Rv2136c, Rv2224c, ponA2, and lysX were hypersensitive to antibiotics, sodium dodecyl sulfate, heat shock, and reactive oxygen and nitrogen intermediates, indicating that acid resistance can be associated with protection against other forms of stress. The Rv2136c mutant was impaired in intrabacterial pH homeostasis and unable to maintain a neutral intrabacterial pH in activated macrophages. The Rv2136c, Rv2224c, and ponA2 mutants were attenuated in mice, with the Rv2136c mutant displaying the most severe level of attenuation. Pathways utilized by M. tuberculosis for acid resistance and intrabacterial pH maintenance are potential targets for chemotherapy.

ABSTRACT Helicobacter pylori is the only neutralophile that has been able to colonize the human stomach by using a variety of acid-adaptive mechanisms. One of the adaptive mechanisms is increased buffering due to expression of an acid-activated inner membrane urea channel, UreI, and a neutral pH-optimum intrabacterial urease. To delineate other possible adaptive mechanisms, changes in gene expression in response to acid exposure were examined using genomic microarrays of H. pylori exposed to different levels of external pH (7.4, 6.2, 5.5, and 4.5) for 30 min in the absence and presence of 5 mM urea. Gene expression was correlated with intrabacterial pH measured using 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein and compared to that observed with exposure to 42°C for 30 min. Microarrays containing the 1,534 open reading frames of H. pylori strain 26695 were hybridized with cDNAs from control (pH 7.4; labeled with Cy3) and acidic (labeled with Cy5) conditions. The intrabacterial pH was 8.1 at pH 7.4, fell to 5.3 at pH 4.5, and rose to 6.2 with urea. About 200 genes were up-regulated and ∼100 genes were down-regulated at pH 4.5 in the absence of urea, and about half that number changed in the presence of urea. These genes included pH-homeostatic, transcriptional regulatory, motility, cell envelope, and pathogenicity genes. The up-regulation of some pH-homeostatic genes was confirmed by real-time PCR. There was little overlap with the genes induced by temperature stress. These results suggest that H. pylori has evolved multifaceted acid-adaptive mechanisms enabling it to colonize the stomach that may be novel targets for eliminating infection.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, is responsible for the death of 1.5 million people each year and the number of bacteria resistant to the standard regimen is constantly increasing. This highlights the need to discover molecules that act on new M. tuberculosis targets. Mycolic acids, which are very long-chain fatty acids essential for M. tuberculosis viability, are synthesized by two types of fatty acid synthase (FAS) systems. MabA (FabG1) is an essential enzyme belonging to the FAS-II cycle. We have recently reported the discovery of anthranilic acids as MabA inhibitors. Here, the structure–activity relationships around the anthranilic acid core, the binding of a fluorinated analog to MabA by NMR experiments, the physico-chemical properties and the antimycobacterial activity of these inhibitors were explored. Further investigation of the mechanism of action in bacterio showed that these compounds affect other targets than MabA in mycobacterial cells and that their antituberculous activity is due to the carboxylic acid moiety which induces intrabacterial acidification.

ABSTRACTMycobacterium tuberculosiskills more people than any other bacterial pathogen and is becoming increasingly untreatable due to the emergence of resistance. Verapamil, an FDA-approved calcium channel blocker, potentiates the effect of several antituberculosis (anti-TB) drugsin vitroandin vivo. This potentiation is widely attributed to inhibition of the efflux pumps ofM. tuberculosis, resulting in intrabacterial drug accumulation. Here, we confirmed and quantified verapamil's synergy with several anti-TB drugs, including bedaquiline (BDQ) and clofazimine (CFZ), but found that the effect is not due to increased intrabacterial drug accumulation. We show that, consistent with itsin vitropotentiating effects on anti-TB drugs that target or require oxidative phosphorylation, the cationic amphiphile verapamil disrupts membrane function and induces a membrane stress response similar to those seen with other membrane-active agents. We recapitulated these activitiesin vitrousing inverted mycobacterial membrane vesicles, indicating a direct effect of verapamil on membrane energetics. We observed bactericidal activity against nonreplicating “persister”M. tuberculosisthat was consistent with such a mechanism of action. In addition, we demonstrated a pharmacokinetic interaction whereby human-equivalent doses of verapamil caused a boost of rifampin exposure in mice, providing a potential explanation for the observed treatment-shortening effect of verapamil in mice receiving first-line drugs. Our findings thus elucidate the mechanistic basis for verapamil's potentiation of anti-TB drugsin vitroandin vivoand highlight a previously unrecognized role for the membrane ofM. tuberculosisas a pharmacologic target.

AbstractThe Salmonella enterica SseK1 protein is a type three secretion system effector that glycosylates host proteins during infection on specific arginine residues with N-acetyl glucosamine (GlcNAc). SseK1 also Arg-glycosylates endogenous bacterial proteins and we thus hypothesized that SseK1 activities might be integrated with regulating the intrabacterial abundance of UPD-GlcNAc, the sugar-nucleotide donor used by this effector. After searching for new SseK1 substrates, we found that SseK1 glycosylates arginine residues in the dual repressor-activator protein NagC, leading to increased DNA-binding affinity and enhanced expression of the NagC-regulated genes glmU and glmS. SseK1 also glycosylates arginine residues in GlmR, a protein that enhances GlmS activity. This Arg-glycosylation improves the ability of GlmR to enhance GlmS activity. We also discovered that NagC is a direct activator of glmR expression. Salmonella lacking SseK1 produce significantly reduced amounts of UDP-GlcNAc as compared with Salmonella expressing SseK1. Overall, we conclude that SseK1 up-regulates UDP-GlcNAc synthesis both by enhancing the DNA-binding activity of NagC and by increasing GlmS activity through GlmR glycosylation. Such regulatory activities may have evolved to maintain sufficient levels of UDP-GlcNAc for both bacterial cell wall precursors and for SseK1 to modify other bacterial and host targets in response to environmental changes and during infection.

2011/2012
Bac7(1-35) is the smallest fragment of the proline-rich cathelicidin Bac7 that shows the same antibacterial activity as the whole natural peptide of 60 residues. In this work, we remarked that the unique gene whose deletion can confer resistance to E. coli against Bac7(1-35) is sbmA, coding for an inner membrane protein involved in the penetration of this peptide into bacterial cells. Moreover, we provided evidence that SbmA is also involved in the transmembrane transport of a fragment of another proline-rich antimicrobial peptide, arasin1(1-23), isolated from the spider crab. These findings suggest a general role of this membrane protein in the uptake of proline-rich antimicrobial peptides (PR-AMPs) into Gram-negative bacteria. We then measured the intrabacterial concentration reached by Bac7(1-35) in E. coli, and observed that this increases from micromolar in the medium to millimolar within the bacterial cell, suggesting that it may bind to cytosolic structures. For this reason, we looked for possible interactions between Bac7(1-35) and macromolecules involved in viable processes of bacteria. These studies showed that Bac7(1-35) completely inhibits in vitro the transcription/translation process starting from a concentration of 50 μM. Then we demonstrated that inhibition is: i) specific for Bac7(1-35), since it is not exerted by other cathelicidin-derived AMPs not belonging to the Prorich group, and ii) not stereo-specific, since it is exerted at the same level by the all-D isomer of Bac7(1-35). We also demonstrated the ability of Bac7(1-35) to bind DNA in vitro, but we excluded that this binding may represent the primary mechanism of bactericidal action. We also showed that the peptide does not significantly affect in vitro the transcription process, deducing that the inhibition of the transcription/translation targets primarily the translation process. We verified these data in vivo on E. coli cells measuring the incorporation of radioactive precursors of bacterial macromolecules. We observed that the exposure of bacteria to Bac7(1-35) inhibited the incorporation of radioactive leucine, but not of radioactive thymidine and uridine, indicating a specific block at the protein synthesis level and not of DNA and RNA synthesis. In the near future, a clearer definition of the intrabacterial target(s) of Bac7(1-35) would hopefully lead to the experimentation of this molecule or of its derivatives as a new generation antibiotic drug.
Bac7(1-35) è il più breve frammento del peptide Bac7, una catelicidina ricca in prolina di 60 residui, dotato della stessa attività battericida del peptide intero. In questo lavoro di tesi, abbiamo rimarcato che sbmA è l’unico gene la cui delezione conferisce ad E. coli una resistenza a Bac7(1- 35). Tale gene codifica per una proteina della membrana interna coinvolta nell’ingresso del peptide nel citoplasma della cellula batterica. Inoltre, abbiamo dimostrato che SbmA è coinvolta anche nel trasporto transmembrana di un frammento di un altro peptide antimicrobico, l’arasina1(1-23). Tali risultati suggeriscono che questa proteina giochi un ruolo generale nell’internalizzazione di peptidi antimicrobici ricchi in prolina nei batteri Gram-negativi. Abbiamo quindi misurato la concentrazione intrabatterica raggiunta da Bac7(1-35) in E. coli e abbiamo osservato che questa aumenta da valori micromolari nel terreno di coltura a millimolari nel citosol batterico, suggerendo un suo legame a strutture interne della cellula. Per questo abbiamo cercato possibili interazioni tra il peptide e macromolecole coinvolte in processi vitali del batterio. Con questi studi abbiamo appurato che Bac7(1-35) in vitro inibisce completamente il processo di trascrizione/traduzione a partire da una concentrazione di 50 μM. Successivamente abbiamo dimostrato che questa inibizione è una peculiarità di Bac7(1-35), in quanto altri AMP derivati da catelicidine ma non ricchi in prolina non hanno dimostrato un’attività comparabile. Inoltre, questa inibizione non è stereo-specifica, in quanto anche l’isomero D di Bac7(1-35) blocca tale processo esattamente come il suo isomero L. Abbiamo inoltre dimostrato la capacità di Bac7(1-35) di legare in vitro il DNA, ma abbiamo escluso che tale legame rappresenti il meccanismo primario della sua attività battericida. Abbiamo anche dimostrato che il peptide non interferisce in vitro in maniera significativa con il processo di trascrizione, deducendo che l’effetto osservato sul processo di trascrizione/traduzione fosse da attribuirsi prevalentemente all’inibizione della traduzione. Abbiamo verificato tali dati in vivo su cellule di E. coli misurando l’incorporazione di precursori radioattivi delle macromolecole batteriche. Abbiamo osservato che l’esposizione di batteri a Bac7(1-35) bloccava l’incorporazione di leucina radioattiva ma non di timidina ed uridina, indicando un blocco specifico della sintesi proteica ma non di quelle di DNA e RNA. In futuro, una definizione ancora più chiara del target intrabatterico di Bac7(1-35) potrebbe portare alla sperimentazione di tale molecola o di suoi analoghi come farmaci antibiotici di nuova generazione.
XXV Ciclo
1985