Gotowa bibliografia na temat „Tuberculosis ArgJ”

Mycobacterium tuberculosis has been infecting millions of people worldwide over the years, causing tuberculosis. Drugs targeting distinct cellular mechanisms including synthesis of the cell wall, lipids, proteins, and nucleic acids in Mtb are currently being used for the treatment of TB. Although extensive research is being carried out at the molecular level in the infected host and pathogen, the identification of suitable drug targets and drugs remains under explored. Pranlukast, an allosteric inhibitor of MtArgJ (Mtb ornithine acetyltransferase) has previously been shown to inhibit the survival and virulence of Mtb. The main objective of this study was to identify the altered metabolic pathways and biological processes associated with the differentially expressed metabolites by PRK in Mtb. Here in this study, metabolomics was carried out using an LC-MS/MS-based approach. Collectively, 50 metabolites were identified to be differentially expressed with a significant p-value through a global metabolomic approach using a high-resolution mass spectrometer. Metabolites downstream of argJ were downregulated in the arginine biosynthetic pathway following pranlukast treatment. Predicted human protein interactors of pranlukast-treated Mtb metabolome were identified in association with autophagy, inflammation, DNA repair, and other immune-related processes. Further metabolites including N-acetylglutamate, argininosuccinate, L-arginine, succinate, ergothioneine, and L-phenylalanine were validated by multiple reaction monitoring, a targeted mass spectrometry-based metabolomic approach. This study facilitates the understanding of pranlukast-mediated metabolic changes in Mtb and holds the potential to identify novel therapeutic approaches using metabolic pathways in Mtb.

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

Abstract Macrophages are abundant in granulomas, the hallmark lesion of tuberculosis (TB), where they engage in activities critical for mycobacterial control. The assortment of macrophage functions, ranging from being the primary anti-mycobacterial effector cell to the primary mycobacterial host cell, underscores the complexity of macrophages in this system. Despite their importance, the molecular phenotypes and functions of granuloma macrophages in human TB are not well understood. In this study, we examined a variety of macrophage markers in non-human primate and human granulomas to better describe macrophage diversity and spatial organization. We identified three myeloid cell markers, including CD68, CD163, and HAM56, that stained populations of macrophages occupying discrete positions in necrotic granulomas that may be relevant to granuloma function. Neutrophils expressed high levels of calprotectin, a small bacteriostatic protein sometimes associated with macrophages, and also localized to specific positions in necrotic granulomas. In addition to phenotypic markers, we identified cell-specific expression of nitric oxide synthase isoforms (iNOS and eNOS) and arginase isoforms (arg1 and arg2) expression in granulomas by biochemical, molecular and immunohistochemical techniques. Both macrophages and neutrophils were determined to express NOS and arg enzymes, including co-expression, suggesting these enzymes with opposing effects act in concert to maintain mycobacterial control.

Essential components of public health include strengthening the surveillance of infectious diseases and developing early detection and prevention policies. This is particularly important for drug-resistant tuberculosis (DR-TB), which can be explored by using wastewater-based surveillance. This study aimed to use molecular techniques to determine the occurrence and concentration of antibiotic-resistance genes (ARGs) associated with tuberculosis (TB) resistance in untreated and treated wastewater. Raw/untreated and treated (post-chlorination) wastewater samples were taken from three wastewater treatment plants (WWTPs) in South Africa. The ARGs were selected to target drugs used for first- and second-line TB treatment. Both conventional polymerase chain reaction (PCR) and the more advanced droplet digital PCR (ddPCR) were evaluated as surveillance strategies to determine the distribution and concentration of the selected ARGs. The most abundant ARG in the untreated wastewater was the rrs gene, associated with resistance to the aminoglycosides, specifically streptomycin, with median concentration ranges of 4.69–5.19 log copies/mL. In contrast, pncA gene, associated with resistance to the TB drug pyrazinamide, was the least detected (1.59 to 2.27 log copies/mL). Resistance genes associated with bedaquiline was detected, which is a significant finding because this is a new drug introduced in South Africa for the treatment of multi-drug resistant TB. This study, therefore, establishes the potential of molecular surveillance of wastewater for monitoring antibiotic resistance to TB treatment in communities.

Currently, due to abuse in the use of human antibiotics and the weak regulatory control that the authorities have over sewage discharge and manure management, antibiotic resistance genes (ARGs) have become a new type of environmental pollutant. Three different natural water bodies (Poyang Lake, Haihe River and Qingdao No.1 Bathing Beach seawater) were sampled during the same periods to conduct a longitudinal comparison of distribution. The distribution and expression of 11 ARGs in 20 species were studied, and the correlations between the expression and the distribution of time and space of the ARGs in different water bodies were also analyzed. With the exception of ermA, blaNDM-1 and vanA, which were not detected in seawater, the other ARGs could be detected in all three water bodies. Tetracycline resistance genes (tetC, tetM and tetQ) in the seawater and Haihe River had even reached 100%, and sulfa ARGs (sul1 and sul2) in the seawater and Poyang Lake, as well as sul2 and sul3 in the Haihe River, had also reached 100%. The ARG pollution in Haihe River was much more serious, since 14 and 17 of 20 ARG species were significantly higher compared with seawater and Poyang Lake, respectively. Some ARGs also had a high absolute abundance. The absolute abundance of macrolide resistance genes (ermB) in seawater was as high as 8.61 × 107 copies/L, and the anti-tuberculosis resistant genes (rpoB and katG) in the Haihe River Basin were highly abundant at 1.32 × 106 copies/L and 1.06 × 107 copies/L, respectively. This indicates that ARGs have gradually become more diverse and extensive in natural water bodies. The results of a redundancy analysis (RDA) of the three water bodies showed that although each water body is affected by different factors in space and time, overall, the presence of AGRs is closely related to the production and life of human beings and the migration of animals.

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

Abstract M. tuberculosis infects lung macrophages (MØs) and evades immune responses by a diverse array of mechanisms. We have recently published that BCG infection triggers a MyD88-dependent Arg1 induction that suppresses NO production from infected MØs. In addition, MØ-specific Arg1 conditional knockout mice were more efficient at clearing M. tuberculosis and BCG. In the present study, we have found that while MyD88 is essential for Arg1 induction following infection, MyD88-/- MØs express robust Arg1 mRNA and protein when stimulated with supernatant from BCG-infected WT MØs. Arg1 induction stimulated with BCG supernatant correlated with enhanced activation of Stat3, but not Stat1, 4, 5, or 6. Two Stat3 activators, IL-6 and IL-10, were present in the supernatants of BCG infected WT MØs. We found the combined treatment of MØs with IL-6 and IL-10 synergistically induces Arg1 in the presence or absence of BCG infection. Consequently, we propose a model by which Arg1 is induced directly by BCG infection via MyD88 signaling, and indirectly through the autocrine/paracrine IL-6/IL-10 activation of Stat3. These data suggest that mycobacteria can condition uninfected neighboring cells to suppress NO production.

ABSTRACT Mycobacterium tuberculosis remains a major cause of pulmonary infection worldwide. Attachment of M. tuberculosis organisms to alveolar macrophages (AMs) represents the earliest phase of primary infection in pulmonary tuberculosis. In this study fibronectin (Fn), an adhesive protein, is shown to bind M. tuberculosis organisms and facilitates attachment of M. tuberculosis to murine AMs. A monoclonal antibody (MAb) specific to the heparin binding domain (HBD) of Fn decreases 125I-Fn binding to M. tuberculosis; whereas MAbs specific to either the cell binding domain (CBD) or the gelatin binding domain (GBD) have no effect on Fn binding to M. tuberculosis. In the presence of exogenous Fn (10 μg/ml) M. tuberculosis attachment to AMs increased significantly from control levels (means ± standard errors of the means) of 11.5% ± 1.1% to 44.2% ± 4.2% (P < 0.05). Fn-enhanced attachment was significantly decreased from 44.2% ± 4.2% to 10.8% ± 1.2% (P < 0.05) in the presence of anti-Fn polyclonal antibodies. The attachment is also inhibited in the presence of MAbs specific for the HBD and CBD, whereas MAbs specific to GBD did not affect the attachment. Further, an Fn cell binding peptide, Arg-Gly-Asp-Ser (RGDS), decreased the attachment from 44.2% ± 4.2% to 15.3% ± 1.2% (P < 0.05), whereas addition of a control peptide, Arg-Gly-Glu-Ser (RGES) did not affect the attachment (40.5% ± 1.8%). These results suggest that Fn-mediated attachment of M. tuberculosis can occur through the binding of Fn to the AM via the CBD and to M. tuberculosis organisms via the HBD.

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis, is responsible for over 1 million deaths each year. Mycobacteria-infected dendritic cells (DCs) migrate to the lymph node to initiate adaptive immune priming, which is vital to antimycobacterial immunity. This response is intimately tied to nutrient availability – especially the amino acid L-arginine (L-ARG), metabolism of which is altered in TB patients. We have characterized a pathway utilized by immune cells to synthesize L-ARG. Loss of L-ARG synthesis in CD11c+ cells, which includes DCs, results in increased mycobacterial burden following infection in mice. To characterize the role of this pathway in DCs, we developed a co-culture system with mycobacterial-specific CD4+ T cells and bone marrow derived DCs. Using CD4+ T cells and DCs with differing capabilities of L-ARG synthesis, we found 1) DC L-ARG synthesis supports CD4+ T cell proliferation and 2) activated T cells contain DC-derived L-ARG. We hypothesize DCs “share” synthesized L-ARG to support CD4+ T cell activation when L-ARG is limiting. Our data suggest nutrient availability as a 4th signal – following antigen presentation, co-stimulation, and cytokine receptor ligation – required for T cell activation. This work expands the current model of DC-T cell interactions and provides insight into the effects of nutrient availability in immune cells.

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Determina la bioactividad sobre Mycobacterium tuberculosis H37RV de los extractos, alcaloides y flavonoides de las especies Juglans neotropica Diels., Piper aduncum L., Croton lechleri Müll. Arg., Lantana camara L., Annona cherimola Mill, Annona muricata L. y Jatropha gossypifolia L., mediante el ensayo en microplacas con azul de alamar. Se recolectó hojas de las 7 especies vegetales seleccionadas según la quimiotaxonomía vigente: Juglans neotropica Diels., Piper Aduncum L., Croton lechleri Mull. Arg., Lantana camara L., Annona cherimola Mill, Annona muricata L. y Jatropha gossypifolia L. Se preparó extractos etanólicos y clorofórmicos. Se aisló alcaloides y flavonoides. Se determinó la bioactividad sobre Mycobacterium tuberculosis H37RV de los extractos, alcaloides y flavonoides mediante el screening antimicobacteriano basado en la reducción del azul de alamar a tres concentraciones (10, 100 y 1000 μg/ml); siguiendo el protocolo original de Collins y Franzblau 64 con algunas modificaciones. Se determinó la concentración mínima inhibitoria (CMI) in vitro de los extractos con bioactividad sobre Mycobacterium tuberculosis H37RV en el rango de concentraciones de 2000-15.63 μg/ml, mediante el ensayo en microplacas con azul de alamar; siguiendo el protocolo original de Collins y Franzblau 64. El screening antimicobacteriano basado en el azul de alamar determinó que los 14 extractos estudiados (100%) presentaron bioactividad sobre Mycobacterium tuberculosis H37RV a 1000 μg/ml. El ensayo en microplacas con azul de alamar determinó que el extracto etanólico de Piper Aduncum L presentó un CMI igual a 31.5 μg/ml. El Piper aduncum L. es una buena alternativa de extractos y metabolitos antituberculosos.
Tesis

Tuberculosis (TB) is a deadly disease responsible for the death of approximately 1.5 million people each year, with the highest being from developing nations. Tuberculosis affects mostly the lungs, and other parts of the body like nerves, bones and liver. Mycobacterium tuberculosis (Mtb) is the causative agent of TB in humans. The onset of infection is via the deposition of aerosol droplets containing the pathogen, M. tuberculosis, onto the lung alveolar surfaces. About one third of the world’s population asymptomatically harbors latent M. tuberculosis bacterium with a constant risk of disease activation. Due to the emergence of drug-resistant strains and the evolution through multi-drug resistance (MDR) to extensive drug resistance (XDR), the fight against TB has become extremely challenging. Standard treatment for TB comprises four first-line antimicrobials: isoniazid, rifampicin, pyrazinamide and ethambutol. However, resistance to all of these drugs has been observed in several MDR strains of Mtb. Despite the recent advances in target identification and drug discovery, there is a relentless need for novel inhibitors against vital pathways of Mtb. The novel drug-development regimens endorse strategies wherein the pre-approved drugs for other ailments could be re-purposed, thereby cutting down the cost and time associated with the process of drug discovery. Also, the target selection strategy requires to aim at the key enzymes from the essential biosynthetic pathways, keeping an eye on their underlying dissimilarities when compared to human host. The challenges in finding a suitable target for anti-Mtb drug discovery is it’s ever evolving stride and the conserved nature of the essential proteins. Many novel small molecule inhibitors of Mtb are undermined, during the course of studies, by cross reactivity with homologs proteins in the host. Traditionally, the replication machinery has been at the heart of drug discovery and the processes associated with logarithmic growth phase are vastly exploited for drug targeting. However, targeting these vital cellular components may result in some serious non-specific effects to the host. On the other hand, the intricate network of metabolic pathways provides novel avenues for specific targeting of pathogens, precisely for three main reasons: 1. There is an acute shortage of cellular nutrients due to the constant competition between the pathogen and the host, throughout the course of infection. 2. Infectious cycles often lead to the disruption of metabolic pathways, again leading to nutrient scarcity. 3. Survival of the pathogen within the hostile niche and under oxygen starvation conditions further potentiate the demands of crucial metabolites (amino acids, nitrogen bases, carbohydrates etc.) that are used as the building blocks for cellular machinery. 4. Metabolic pathways have evolved with time, to provide the much-required specificity for exclusive targeting of the pathogen, thereby limiting the cross-reactivity with the host pathways. In order to persist and efficiently replicate in host cells, intracellular pathogens must adapt their metabolism to the available nutrients and physical conditions (mainly pH, oxygen availability and osmotic pressure) in the host. Among the major metabolic, amino acid metabolism holds great importance; as they not only serve to meet the nutritional needs of the pathogen but also play a key role in the strategies employed during pathogenesis. Although the host and the pathogen compete for many metabolites, three amino acids, Arginine, Asparagine and Tryptophan seems to be a focus of this competition because the availability of these amino acids or their derivatives influence both pathogen behavior and the immune response. Arginine constitutes a major proportion of the total proteins in the cell and arginine and its precursor ornithine are used for the biosynthesis of the most common polyamines, putrescine and spermidine. These molecules are required for optimal growth of the organism and are involved in several physiological processes. Apart from being a critical amino acid for the synthesis of cellular proteins, arginine can also be used as a nitrogen source, under conditions of nitrogen starvation, hence crucial for pathogenesis. The glutamate and glutamine are the key metabolites in the central nitrogen metabolism; both serve as endogenous nitrogen acceptor as well as nitrogen donor. However, reports demonstrate that Mtb utilizes arginine and asparagine as the key sources of nitrogen during infection in mice models of tuberculosis. Therefore, our study focuses on the process of Arginine biosynthesis in M. tuberculosis, wherein it is essential for the survival and pathogenesis. Since the arginine metabolism is essential for both the host and the pathogen, and competition for arginine may shift the balance, and thus determine the outcome of the infection. The enzymes involved in this pathway will be a promising target for anti-TB drug development. Despite the acknowledged significance of Arginine biosynthesis in the pathogens like M. tuberculosis, inhibitors to target this pathway remain to be discovered. Moreover, inhibitors of this pathway may provide novel insights to the significance of arginine biosynthesis in Mtb associated stress responses and persistence. Ornithine acetyltransferase (MtArgJ), one of the crucial enzymes during the biosynthesis of arginine in Mtb, is essential for its survival and pathogenesis. MtArgJ lacks a homolog in human genome, thereby being a good target against Mtb. We hypothesize that a targeted inhibitor against this key player of mycobacterial metabolism has the potential to combat the Mtb survival and pathogenesis. In the present thesis, we have characterized the potential of MtArgJ from M. tuberculosis as a valuable target for drug design against tuberculosis. Most importantly, the approach is to specifically target a novel allosteric site identified in this study, on the MtArgJ surface. Since we are not using the age-old approach of substrate analog as an inhibitor, we hereby further minimize or even eliminate the chances of cross-reactivity with the host cellular proteins. In the later parts, we have identified an allosteric inhibitor of MtArgJ, that significantly reduces the survival of pathogenic Mtb through the pre-clinical models of tuberculosis. Chapter 1 of this thesis gives a detailed account of the history of Tuberculosis, and its pathogenesis. The chapter further elaborates on the metabolic pathways of Mycobacterium tuberculosis, with special emphasis on the arginine biosynthesis pathway. The drug discovery regime and therapeutic challenges associated with the disease are discussed in the later parts of the chapter. All the information discussed in this chapter serves a preface for the work done throughout the thesis, and outlinesthe objectives for rest of the chapters. Chapter 2 describes the characterization and kinetic analysis of MtArgH, the last enzyme from the arginine biosynthetic pathway in M. tuberculosis. This chapter demonstrates the importance of a c-terminal cysteine residue (Cys441) in the catalysis and thermal stability of the enzyme. We further propose the existence of a product mediated feed-back inhibition of MtArgH, wherein fumarate, one of the product of MtArgH, gradually modifies the Cys441 through succination. Chapter 3 to 5 discuss about the work carried out on the enzyme Ornithine acetyltransferase (MtArgJ), a crucial enzyme for arginine biosynthesis in M. tuberculosis. We have identified a selective allosteric inhibitor against this key player of mycobacterial metabolism, employing the below-mentioned strategy. First step was to characterize the target, followed by a structure based in-silico screen. The best hits were subjected to in-vitro validation, leading to the in-vivo testing of the potential molecule, in the pre-clinical model of tuberculosis. Target characterization In-silico screen In-vitro validation Pre-clinical testing Chapter 3 starts with the characterization of the MtArgJ, wherein we identified a novel hydrophobic pocket present on the enzyme surface. We further characterized the potential of this pocket in allosterically modulating the active site. This was then followed by a structure based in-silico screen with a library of FDA approved drugs, specifically targeting this novel allosteric pocket on MtArgJ. Chapter 4 deals with the in vitro validation of the identified compounds from in-silico screen. We here identified two lead molecules, Pranlukast (PRK) and Sorafenib (SRB), to have significant affinity for the allosteric site on MtArgJ, leading to the inhibition of its enzymatic activity. We further propose the key residues involved in this interaction, thereby suggesting a potential molecular mechanism of inhibition. Chapter 5 leads us to the in-vitro and in-vivo characterization of these compounds as a potent anti-tubercular agent. We first demonstrate its efficacy in deducing the survival of the pathogenic strains of Mtb in-vitro and in the macrophage models of infection. We also tested the efficacy of these compounds in combination with the standard of care TB therapy drugs, and found PRK to work efficiently in such combinations. Finally, we evidence the potency of PRK in compromising the survival and pathogenesis of Mtb in mice models of tuberculosis infection. PRK is presently being used as a drug against chronic asthma, therefore its human safety is already assured. This will facilitate its induction into the direct clinical trials against tuberculosis. Taken together, the work done in this thesis demonstrates a novel metabolic inhibitor of Mtb pathogenesis, through the pre-clinical models of infection with the potential for development of advanced combinatorial therapy against tuberculosis.