Indole Propionic Acid Disturbs the Normal Function of Tryptophanyl-tRNA Synthetase in Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis and the second-most contagious killer after COVID-19. The emergence of drug-resistant TB has caused a great need to identify and develop new anti-TB drugs with novel targets. Indole propionic acid (IPA), a structural analog of tryptophan (Trp), is active against M. tuberculosis in vitro and in vivo. It has been verified that IPA exerts its antimicrobial effect by mimicking Trp as an allosteric inhibitor of TrpE, which is the first enzyme in the Trp synthesis pathway of M. tuberculosis. However, other Trp structural analogs, such as indolmycin, also target tryptophanyl-tRNA synthetase (TrpRS), which has two functions in bacteria: synthesis of tryptophanyl-AMP by catalyzing ATP + Trp and producing Trp-tRNATrp by transferring Trp to tRNATrp. So, we speculate that IPA may also target TrpRS. In this study, we found that IPA can dock into the Trp binding pocket of M. tuberculosis TrpRS (TrpRSMtb), which was further confirmed by isothermal titration calorimetry (ITC) assay. The biochemical analysis proved that TrpRS can catalyze the reaction between IPA and ATP to generate pyrophosphate (PPi) without Trp as a substrate. Overexpression of wild-type trpS in M. tuberculosis increased the MIC of IPA to 32-fold, and knock-down trpS in Mycolicibacterium smegmatis made it more sensitive to IPA. The supplementation of Trp in the medium abrogated the inhibition of M. tuberculosis by IPA. We demonstrated that IPA can interfere with the function of TrpRS by mimicking Trp, thereby impeding protein synthesis and exerting its anti-TB effect.

ESX-3 secretion system in Mycobacterium: An overview.

Mycobacteria are microorganisms distributed in the environment worldwide, and some of them, such as Mycobacterium tuberculosis or M. leprae, are pathogenic. The hydrophobic mycobacterial cell envelope has low permeation and bacteria need to export products across their structure. Mycobacteria possess specialized protein secretion systems, such as the Early Secretory Antigenic Target 6 secretion (ESX) system. Five ESX loci have been described in M. tuberculosis, called ESX-1 to ESX-5. The ESX-3 secretion system has been associated with mycobacterial metabolism and growth. The locus of this system is highly conserved across mycobacterial species. Metallo-proteins regulate negative ESX-3 transcription in high conditions of iron and zinc. Moreover, this secretion system is part of an antioxidant regulatory pathway linked to Zinc. EccA3, EccB3, EccC3, EccD3, and EccE3 are components of the ESX-3 secretion machinery, whereas EsxG-EsxH, PE5-PPE4, and PE15-PPE20 are proteins secreted by this system. In addition, EspG3 and MycP3 are complementary proteins involved in transport and proteolysis respectively. This system is associated to mycobacterial virulence by releasing the bacteria from the phagosome and inhibiting endomembrane damage response. Furthermore, components of this system inhibit the host immune response by reducing the recognition of M. tuberculosis-infected cells. The components of the ESX-3 secretion system play a role in drug resistance and cell wall integrity. Moreover, the expression data of this system indicated that external and internal factors affect ESX-3 locus expression. This review provides an overview of new findings on the ESX-3 secretion system, its regulation, expression, and functions.

The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both In Vitro and In Vivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using in vitro and in vivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both in vitro and in vivo and reveal an opportunity for further investigation of new antituberculosis therapies.

Beneficial or detrimental activity of regulatory T cells, indoleamine 2,3-dioxygenase, and heme oxygenase-1 in the lungs is influenced by the level of virulence of Mycobacterium tuberculosis strain infection.

Tuberculosis (TB) caused by the complex Mycobacterium tuberculosis (Mtb) is the main cause of death by a single bacterial agent. Last year, TB was the second leading infectious killer after SARS-CoV-2. Nevertheless, many biological and immunological aspects of TB are not completely elucidated, such as the complex process of immunoregulation mediated by regulatory T cells (Treg cells) and the enzymes indoleamine 2,3-dioxygenase (IDO) and heme oxygenase 1 (HO-1). In this study, the contribution of these immunoregulatory factors was compared in mice infected with Mtb strains with different levels of virulence. First Balb/c mice were infected by intratracheal route, with a high dose of mild virulence reference strain H37Rv or with a highly virulent clinical isolate (strain 5186). In the lungs of infected mice, the kinetics of Treg cells during the infection were determined by cytofluorometry and the expression of IDO and HO-1 by RT-PCR and immunohistochemistry. Then, the contribution of immune-regulation mediated by Treg cells, IDO and HO-1, was evaluated by treating infected animals with specific cytotoxic monoclonal antibodies for Treg cells depletion anti-CD25 (PC61 clone) or by blocking IDO and HO-1 activity using specific inhibitors (1-methyl-D,L-tryptophan or zinc protoporphyrin-IX, respectively). Mice infected with the mild virulent strain showed a progressive increment of Treg cells, showing this highest number at the beginning of the late phase of the infection (28 days), the same trend was observed in the expression of both enzymes being macrophages the cells that showed the highest immunostaining. Animals infected with the highly virulent strain showed lower survival (34 days) and higher amounts of Treg cells, as well as higher expression of IDO and HO-1 one week before. In comparison with non-treated animals, mice infected with strain H37Rv with depletion of Treg cells or treated with the enzymes blockers during late infection showed a significant decrease of bacilli loads, higher expression of IFN-g and lower IL-4 but with a similar extension of inflammatory lung consolidation determined by automated morphometry. In contrast, the depletion of Treg cells in infected mice with the highly virulent strain 5186 produced diffuse alveolar damage that was similar to severe acute viral pneumonia, lesser survival and increase of bacillary loads, while blocking of both IDO and HO-1 produced high bacillary loads and extensive pneumonia with necrosis. Thus, it seems that Treg cells, IDO and HO-1 activities are detrimental during late pulmonary TB induced by mild virulence Mtb, probably because these factors decrease immune protection mediated by the Th1 response. In contrast, Treg cells, IDO and HO-1 are beneficial when the infection is produced by a highly virulent strain, by regulation of excessive inflammation that produced alveolar damage, pulmonary necrosis, acute respiratory insufficiency, and rapid death.

Effect of the Deletion of icl1 Gene and icl2 Gene on the Growth Rate of Mycobacterium tuberculosis and the Specific Regulatory Mechanism Involved.

Objective: To explore the effect of the deletion of the icl1 gene and icl2 gene on the growth rate of Mycobacterium tuberculosis (Mtb) and the specific regulatory mechanism involved. Methods: H37Rv was purchased from the Tuberculosis Prevention and Control Institute, and H37Rv was grown in Middlebrook 7H9 broth. Macrophages THP-1 cells were purchased by our researchers from the Cell Bank of the Chinese Academy of Sciences, which were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), at 37°C and 5% CO2. The experiment was divided into 3 groups: the control group (H37Rv infected with THP-1 cells), the icl1/2 deletion group (H37Rv infected with icl1/2 deleted THP-1 cells) and the icl1/2 complementation group (H37Rv infected with icl1/2 deletion, icl1/2 complementary THP-1 cells). Absorbance was measured with a microplate spectrophotometer and the bacterial growth rate was calculated. The colony-forming units (CFU) obtained from the dilution was used to calculate the total number of CFU per milliliter and the percentage of survival of mycobacteria. The protein levels of isocitrate lyase 1 (ICL1), ICL2, p-mTOR and p-Akt were analyzed by Western blot. The CD4+ level was analyzed by flow cytometry. The mRNA expression levels of CCL20, CXCL2, CXCL8, interferon gamma (IFN-γ), interleukin (IL)-17 and IL-22 were analyzed using the quantitative reverse transcription polymerase chain reaction (RT-qPCR) method. Stably transformed monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP)-LC3 reporter THP-1 cells were used to monitor the aggregation of LC3B in autophagosomes and autophagolysosomes. Results: The Mtb growth rate and CFU of the icl1/2 deletion group were decreased in comparison with the control group (P < .05). When compared with the icl1/2 deletion group, however, the Mtb growth rate and CFU of the icl1/2 complementation group were associated with increased results (P < .05). The protein levels of ICL1 and ICL2 in the icl1/2 deletion group were significantly decreased compared with the control group (P < .05), which were evidently increased in the icl1/2 complementation group when compared with the icl1/2 deletion group (P < .05). In addition, compared with the control group (25.16 ± 2.18), the level of CD4+ appeared to be increased in the icl1/2 deletion group (62.37 ± 5.46) (P < .05), while it was decreased in the icl1/2 complementation group compared with the icl1/2 deletion group (28.33 ± 1.32) (P < .05). The expression levels of chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 8 (CXCL8), IL-17, IFN-γ, and IL-22 mRNA were increased in the icl1/2 deletion group compared with the control group (P < .05), which were significantly decreased in the icl1/2 complementary group compared with the icl1/2 deletion group (P < .05). A comparison between the control group and the icl1/2 deletion group showed that the latter increased the formation of autophagosomes and autophagolysosomes in H37Rv-infected cells (P < .05). However, compared with the icl1/2 deletion group, the icl1/2 complementation group decreased the formation of autophagosomes and autolysosomes in H37Rv-infected cells (P < .05). Moreover, the expression levels of phosphor-mammalian target of rapamycin (p-mTOR) and p-Akt in the icl1/2 deletion group were significantly reduced compared with the control group (P < .05), and were increased in the icl1/2 complementation group compared with the icl1/2 deletion group (P < .05). Conclusion: Loss of icl1/2 was believed to increase the expression of CD4 and CCL20, CXCL8 as well as CXCL2 in the immune system, which increased autophagy. Furthermore, it exerted potential in inhibiting the growth of intracellular Mtb in macrophages.

Polyfluorinated salicylic acid analogs do not interfere with siderophore biosynthesis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is a leading cause of infectious disease mortality. The salicylic acid derived small molecule siderophores known as mycobactins are essential in vivo for iron acquisition of Mtb where iron is restricted in the host. Herein, we synthesize and explore the mechanism of action of polyfluorinated salicylic acid derivates, which were previously reported to possess potent antimycobacterial activity. We hypothesized fluorinated salicylic acid derivates may inhibit mycobactin biosynthesis through initial bioactivation and conversion to downstream metabolites that block late steps in assembly of the mycobactins. Enzymatic studies demonstrated that some of the fluorinated salicylic acid derivatives compounds were readily activated by the bifunctional adenylating enzyme MbtA, responsible for incorporation of salicylic acid into the mycobactin biosynthetic pathway; however, they did not inhibit mycobactin biosynthesis as confirmed by LS-MS/MS using an authentic synthetic mycobactin standard. Further mechanistic analysis of the most active derivative (Sal-4) using an MbtA-overexpressing Mtb strain as well as complementation studies with iron and salicylic acid revealed Sal-4 cannot be antagonized by overexpression of MbtA or through supplementation with iron or salicylic acid. Taken together, our results indicate the observed antimycobacterial activity of polyfluorinated salicylic acid derivative is independent of mycobactin biosynthesis.

Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

A Pivotal Role for Mycobactin/mbtE in Growth and Adaptation of Mycobacterium abscessus.

Mycobacterium abscessus is an emerging pathogen that critically depends on iron for growth and pathogenesis. The acquisition of iron in Mycobacterium tuberculosis is governed by siderophores called mycobactins, synthesized by the mbt gene cluster, but the role of this gene cluster in the adaption of M. abscessus to iron limitation is not characterized. We identified an M. abscessus Tn_mutant with interruption of the mbtE gene (MAB_2248c), a central component of mycobactin biosynthesis. We tested this isolate growth characteristic, dependency on supplements, and transcriptomic response, comparing it to the response of wild-type (WT) bacteria in iron-limiting conditions. We also compare the structure of the mbt gene cluster across several mycobacteria. The Tn_mbtE mutant had a substantial, but not absolute, growth defect, which was more substantial in iron-limited media. Supplementation with mycobactin-J, hemin, blood, and surprisingly, albumin, salvaged the poor growth. Similarly, secreted mature (carboxy)-mycobactins from WT bacteria rescued the Tn_mbtE mutant during iron deprivation. The transcriptomic response of the Tn_mbtE mutant involved the upregulation of genes known to be implicated in iron homeostasis and was comparable to that of WT bacteria grown in iron-limiting conditions. Interestingly, the response was not identical to the response of M. tuberculosis to iron limitation. The mbt gene cluster and mycobactins play important roles in the physiology of M. abscessus. (Carboxy)-mycobactin is secreted from WT bacteria and can serve as "public good." The role of several iron-homeostasis related genes (like ideR) may differ between M. abscessus and Mtb. IMPORTANCE Mycobacterium abscessus is an emerging human pathogen belonging to the nontuberculous mycobacteria (NTM) family, causing severe pulmonary disease in compromised individuals. How this bacterium acquires iron is poorly understood. Here, we provide the first characterization of the role(s) the mbtE gene required for the biosynthesis of siderophore mycobactin in M. abscessus. We show that the gene mbtE is required for growth during iron deprivation and can be compensated by several supplements, including, surprisingly, albumin. We also show the transcriptomic response of the mbtE-mutant is comparable to the response of the parental strain to iron starvation and seems different from the response of M. tuberculosis. These results indicate the importance of studying mycobactin in M. abscessus and NTM strains. Understanding this pathway is central to understanding the acquisition of iron within hosts and its role in pathogenesis, which in turn may facilitate the development of antimycobacterial therapeutics.

DosR Regulates the Transcription of the Arginine Biosynthesis Gene Cluster by Binding to the Regulatory Sequences in Mycobacterium bovis Bacille Calmette-Guerin.

l-Arginine serves as a carbon and nitrogen source and is critical for Mycobacterium tuberculosis (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the argCJBDFGH gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of argC (rv1652), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in Mycobacterium bovis bacille Calmette-Guerin.

Identification of Concomitant Inhibitors against Glutamine Synthetase and Isocitrate Lyase in Mycobacterium tuberculosis from Natural Sources.

Tuberculosis (T.B.) is a disease that occurs due to infection by the bacterium, Mycobacterium tuberculosis (Mtb), which is responsible for millions of deaths every year. Due to the emergence of multidrug and extensive drug-resistant Mtb strains, there is an urgent need to develop more powerful drugs for inclusion in the current tuberculosis treatment regime. In this study, 1778 molecules from four medicinal plants, Azadirachta indica, Camellia sinensis, Adhatoda vasica, and Ginkgo biloba, were selected and docked against two chosen drug targets, namely, Glutamine Synthetase (G.S.) and Isocitrate Lyase (I.C.L.). Molecular Docking was performed using the Glide module of the SchrÓ§dinger suite to identify the best-performing ligands; the complexes formed by the best-performing ligands were further investigated for their binding stability via Molecular Dynamics Simulation of 100 ns. The present study suggests that Azadiradione from Azadirachta indica possesses the potential to inhibit Glutamine Synthetase and Isocitrate Lyase of M. tuberculosis concomitantly. The excellent docking score of the ligand and the stability of receptor-ligand complexes, coupled with the complete pharmacokinetic profile of Azadiradione, support the proposal of the small molecule, Azadiradione as a novel antitubercular agent. Further, wet lab analysis of Azadiradione may lead to the possible discovery of a novel antitubercular drug.

Evaluation of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS) as a Vulnerable Target in Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (MtbDAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that MtbDAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.

Multiple genetic paths including massive gene amplification allow Mycobacterium tuberculosis to overcome loss of ESX-3 secretion system substrates.

Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.

Interaction of Host Pattern Recognition Receptors (PRRs) with Mycobacterium Tuberculosis and Ayurvedic Management of Tuberculosis: A Systemic Approach.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), infects the lungs' alveolar surfaces through aerosol droplets. At this stage, the disease progression may have many consequences, determined primarily by the reactions of the human immune system. However, one approach will be to more actively integrate the immune system, especially the pattern recognition receptor (PRR) systems of the host, which notices pathogen-associated molecular patterns (PAMPs) of Mtb. Several types of PRRs are involved in the detection of Mtb, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), Dendritic cell (DC) -specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), Mannose receptor (MR), and NOD-like receptors (NLRs) related to inflammasome activation. In this study, we focus on reviewing the Mtb pathophysiology and interaction of host PPRs with Mtb as well as adverse drug effects of anti-tuberculosis drugs (ATDs) and systematic TB treatment via Ayurvedic medicine.

In Silico Identification of Possible Inhibitors for Protein Kinase B (PknB) of Mycobacterium tuberculosis.

With tuberculosis still being one of leading causes of death in the world and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), researchers have been seeking to find further therapeutic strategies or more specific molecular targets. PknB is one of the 11 Ser/Thr protein kinases of Mtb and is responsible for phosphorylation-mediated signaling, mainly involved in cell wall synthesis, cell division and metabolism. With the amount of structural information available and the great interest in protein kinases, PknB has become an attractive target for drug development. This work describes the optimization and application of an in silico computational protocol to find new PknB inhibitors. This multi-level computational approach combines protein-ligand docking, structure-based virtual screening, molecular dynamics simulations and free energy calculations. The optimized protocol was applied to screen a large dataset containing 129,650 molecules, obtained from the ZINC/FDA-Approved database, Mu.Ta.Lig Virtual Chemotheca and Chimiothèque Nationale. It was observed that the most promising compounds selected occupy the adenine-binding pocket in PknB, and the main interacting residues are Leu17, Val26, Tyr94 and Met155. Only one of the compounds was able to move the active site residues into an open conformation. It was also observed that the P-loop and magnesium position loops change according to the characteristics of the ligand. This protocol led to the identification of six compounds for further experimental testing while also providing additional structural information for the design of more specific and more effective derivatives.

Study of the bioenergetics to identify the novel pathways as a drug target against Mycobacterium tuberculosis using Petri net.

Tuberculosis is one of the life-threatening diseases globally, caused by the bacteria Mycobacterium tuberculosis. In order to control this epidemic globally, there is an urgent need to discover new drugs with novel mechanism of action that can help in shortening the duration of treatment for both drug resistant and drug sensitive tuberculosis. Mycobacterium essentially depends on oxidative phosphorylation for its growth and establishment of pathogenesis. This pathway is unique in Mycobacterium tuberculosis as compared to host due to the differences in some of the enzyme complexes carrying electron transfer. Hence, it serves as an important drug target area. The uncouplers which inhibit adenosine triphosphate synthesis, could play a vital role in serving as antimycobacterial agents and thus could help in eradicating this deadly disease. In this article, the bioenergetics of Mycobacterium tuberculosis are studied with and without uncouplers using Petri net. Petri net is among the most widely used mathematical and computational tools to model and study the complex biochemical networks. We first represented the bioenergetic pathway as a Petri net which is then validated and analyzed using invariant analysis techniques of Petri net. The valid mathematical models presented here are capable to explain the molecular mechanism of uncouplers and the processes occurring within the electron transport chain of Mycobacterium tuberculosis. The results explained the net behavior in agreement with the biological results and also suggested some possible processes and pathways to be studied as a drug target for developing antimycobacterials.

Tanshinones from Salvia miltiorrhiza inhibit Mycobacterium tuberculosis via disruption of the cell envelope surface and oxidative stress.

The unique structure of Mycobacterium tuberculosis cell envelope provides impermeable barrier against environmental stimuli. In the situation that this barrier is disturbed Mycobacteria react at the transcriptional and translational level to redirect metabolic processes and to maintain integrity of the cell. In this work we aimed to explore the early metabolic response of M. tuberculosis to tanshinones, which are active antimycobacterial compounds of Salvia miltiorrhiza Bunge root. The investigation of the expression of sigma factors revealed the significant shifts in the general bacterial regulatory network, whereas LC-MS metabolomics evidenced the changes in the composition of bacterial cell envelope and indicated altered metabolic pathways. Tanshinones acted via the disruption of the cell envelope surface and generation of reactive oxygen species. Bacteria responded with overproduction of inner region of outer membrane, fluctuations in the production of glycerophosphoinositolglycans, as well as changes in the levels of mycobactins, accompanied by enrichment of metabolic pathways related to redox balance and repair of damages caused by tanshinones.

Mycobacterial Populations Partly Change the Proportions of the Cells Undergoing Asymmetric/Symmetric Divisions in Response to Glycerol Levels in Growth Medium.

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister-daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.

Bioenergetic Heterogeneity in Mycobacterium tuberculosis Residing in Different Subcellular Niches.

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region.

INTRODUCTION: Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments. METHODS: We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions. Serum samples were collected from 289 participants enrolled in the Centers for Disease Control and Prevention TBTC Study 29 (NCT00694629) at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). RESULTS: After a peptide level proteome analysis utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS) and subsequent statistical analysis, a total of 183 core proteins demonstrated significant differences at both baseline and at week 8 timepoints between participants enrolled from African and non-African regions. The majority of the differentially expressed proteins were upregulated in participants from the African region, and included acute phase proteins, mediators of inflammation, as well as coagulation and complement pathways. Downregulated proteins in the African population were primarily linked to nutritional status and lipid metabolism pathways. CONCLUSIONS: We have identified differentially expressed nutrition and lipid pathway proteins by geographic region in TB patients undergoing treatment for pulmonary tuberculosis, which appear to be associated with differential treatment responses. Future TB clinical trials should collect expanded measures of nutritional status and further evaluate the relationship between nutrition and microbiologic treatment response.

A Role for Mycobacterium tuberculosis Sigma Factor C in Copper Nutritional Immunity.

Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These genes are expressed at higher levels after 48-h sigC induction, but also elevated are genes encoding copper-responsive regulator RicR and RicR-regulated copper toxicity response operon genes rv0846-rv0850, suggesting prolonged sigC induction results in excessive copper uptake. No growth and global transcriptional differences are observed between a sigC null mutant relative to its parent strain in 7H9 medium. In a copper-deficient medium, however, growth of the sigC deletion strain lags the parent, and 40 genes (including those in the nrp operon) are differentially expressed. Copper supplementation reverses the growth defect and silences most transcriptional differences. Together, these data support SigC as a transcriptional regulator of copper acquisition when the metal is scarce. Attenuation of sigC mutants in severe combined immunodeficient mice is consistent with an inability to overcome innate host defenses that sequester copper ions to deprive invading microbes of this essential micronutrient.