Exosomes in tuberculosis: Still terra incognita?

Today, diagnosis, vaccination, and treatment of tuberculosis (TB) remain major clinical challenges. Therefore, an introduction of new diagnostic measures and biomarkers is necessary to improve infection control. The ideal biomarker for TB infection can be defined as a host or pathogen-derived biomolecule, which is potent for identifying infection and determining its clinical stage. Exosomes, defined as cell-derived nanovesicles released into biological fluids, are involved in cell–cell communication and immune modulation. These vesicles have emerged as a new platform for improving the clinical diagnosis and prognosis of different infectious diseases and cancers. The role of these nanovehicles, as alternative biomarkers for the improvement of TB diagnosis and treatment, has been demonstrated in a significant body of literature. In this review, we summarized recent progress in the clinical application of exosome-based biomarkers in TB infection.     

In silico discovery of potential drug molecules to improve the treatment of isoniazid-resistant Mycobacterium tuberculosis

The emergence of multidrug-resistant Mycobacterium tuberculosis (M.tb) has become one of the major hurdles in the treatment of tuberculosis (TB). Drug-resistant M.tb has evolved with various strategies to avoid killing by the anti-tubercular drugs. Thus, there is a rising need to develop effective anti-TB drugs to improve the treatment of these strains. Traditional drug design approach has earned little success due to time and the cost involved in the process of development of anti-infective drugs. Numerous reports have demonstrated that several mutations in the drug target sites cause emergence of drug-resistant M.tb strains. In this study, we performed computational mutational analysis of M.tb inhA, fabD, and ahpC genes, which are the primary targets for first-line isoniazid (INH) drug. In silico virtual drug screening was performed to identify the potent drugs from a ChEMBL compound library to improve the treatment of INH-resistant M.tb. Further, these compounds were analyzed for their binding efficiency against active drug binding cavity of M.tb wild-type and mutant InhA, FabD and AhpC proteins. The drug efficacy of predicted lead compounds was verified by molecular docking using M.tb wild-type and mutant InhA, FabD and AhpC protein template models. Different in silico and pharmacophore analysis predicted three potent lead compounds with better drug-like properties against both M.tb wild-type and mutant InhA, FabD, and AhpC proteins as compared to INH drug, and thus may be considered as effective drugs for the treatment of INH-resistant M.tb strains. We hypothesize that this work may accelerate drug discovery process for the treatment of drug-resistant TB.

Communicated by Ramaswamy H. Sarma     

Molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of Mycobacterium tuberculosis Polyketide Synthase 13

In this work, the binding mechanism of new Polyketide Synthase 13 (Pks13) inhibitors has been studied through molecular dynamics simulation and free energy calculations. The drug Tam1 and its analogs, belonging to the benzofuran class, were submitted to 100 ns simulations, and according to the results obtained for root mean square deviation, all the simulations converged from approximately 30 ns. For the analysis of backbone flotation, the root mean square fluctuations were plotted for the Cα atoms; analysis revealed that the greatest fluctuation occurred in the residues that are part of the protein lid domain. The binding free energy value (ΔG_bind) obtained for the Tam16 lead molecule was of ?51.43 kcal/mol. When comparing this result with the ΔG_bind values for the remaining analogs, the drug Tam16 was found to be the highest ranked: this result is in agreement with the experimental results obtained by Aggarwal and collaborators, where it was verified that the IC₅₀ for Tam16 is the smallest necessary to inhibit the Pks13 (IC₅₀ = 0.19 μM). The energy decomposition analysis suggested that the residues which most interact with inhibitors are: Ser1636, Tyr1637, Asn1640, Ala1667, Phe1670, and Tyr1674, from which the greatest energy contribution to Phe1670 was particularly notable. For the lead molecule Tam16, a hydrogen bond with the hydroxyl of the phenol not observed in the other analogs induced a more stable molecular structure. Aggarwal and colleagues reported this hydrogen bonding as being responsible for the stability of the molecule, optimizing its physic-chemical, toxicological, and pharmacokinetic properties.     

Role of two-component regulatory systems in intracellular survival of Mycobacterium tuberculosis

The typical two-component regulatory systems (TCSs), consisting of response regulator and histidine kinase, play a central role in survival of pathogenic bacteria under stress conditions such as nutrient starvation, hypoxia, and nitrosative stress. A total of 11 complete paired two-component regulatory systems have been found in Mycobacterium tuberculosis, including a few isolated kinase and regulatory genes. Increasing evidence has shown that TCSs are closely associated with multiple physiological process like intracellular persistence, pathogenicity, and metabolism. This review gives the two-component signal transduction systems in M. tuberculosis and their signal transduction roles in adaption to the environment.     

The aldehyde hypothesis: metabolic intermediates as antimicrobial effectors

There are many reactive intermediates found in metabolic pathways. Could these potentially toxic molecules be exploited for an organism''s benefit? We propose that during certain microbial infections, the production of inherently reactive aldehydes by an infected host is a previously unappreciated innate immune defence mechanism. While there has been a significant focus on the effects of aldehydes on mammalian physiology, the idea that they might be exploited or purposefully induced to kill pathogens is new. Given that aldehydes are made as parts of metabolic programmes that accompany immune cell activation by the cytokine interferon-gamma (IFN-γ) during infections, we hypothesize that aldehydes are among the arsenal of IFN-γ-inducible effectors needed for pathogen control.     

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe–S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N–S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.     

Mycobacterium tuberculosis carbon and nitrogen metabolic fluxes

Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens causing tuberculosis (TB), a devastating infectious disease responsible for the highest human mortality and morbidity. The emergence of drug-resistant strains of the pathogen has increased the burden of TB tremendously and new therapeutics to overcome the problem of drug resistance are urgently needed. Metabolism of Mtb and its interactions with the host is important for its survival and virulence; this is an important topic of research where there is growing interest in developing new therapies and drugs that target these interactions and metabolism of the pathogen during infection. Mtb adapts its metabolism in its intracellular niche and acquires multiple nutrient sources from the host cell. Carbon metabolic pathways and fluxes of Mtb has been extensively researched for over a decade and is well-defined. Recently, there has been investigations and efforts to measure metabolism of nitrogen, which is another important nutrient for Mtb during infection. This review discusses our current understanding of the central carbon and nitrogen metabolism, and metabolic fluxes that are important for the survival of the TB pathogen.     

Screening differential circular RNA expression profiles reveals the regulatory role of circMARS in anti‐tuberculosis drug‐induced liver injury

Tuberculosis (TB) treatment is plagued by liver damage, which often leads to treatment interruptions. Circular RNAs (circRNAs) are a special class of non‐coding RNAs abundant in body fluids with important biological functions. However, the role of circRNA in anti‐tuberculosis drug‐induced liver injury (ADLI) is unclear. We explored ADLI‐specific circRNAs in TB patients using circRNA microarrays and verified circMARS in a cohort of 300 individuals. In addition to the value assessment of circMARS in patients using a receiver operating characteristic (ROC) curve, cell experiments were also performed under the guidance of bioinformatics analyses. In particular, we found that circMARS acts as a miRNA sponge by binding to miRNAs. Compared with the blank group, the expressions of circMARS, KMT2C gene, and EGFR protein in the ADLI group were increased, while miR‐6808‐5p, miR‐6874‐3p, and miR‐3157‐5p were decreased. Furthermore, when si‐circMARS was used in the ADLI groups, circMARS demotion manifested the opposite results. Subsequently, a self‐controlled cohort of 35 participants was used to verify the circMARS–miR‐6808‐5p/‐6874‐3p/‐3157‐5p–KMT2C–EGFR function axis. Therefore, circMARS may participate in the compensatory repair mechanism of ADLI through the function axis, and may be a potential biomarker for ADLI diagnosis in TB patients.     

Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A₂, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (V_max∼15.5 μmol/min/mg; K_m∼83 μM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall–deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.