Ferritins and bacterioferritins are iron storage proteins that represent key players in iron homeostasis. Several organisms possess both forms of ferritins, however, their relative physiological ...roles are less understood. Mycobacterium tuberculosis possesses both ferritin (BfrB) and bacterioferritin (BfrA), playing an essential role in its pathogenesis as reported by us earlier. This study provides insights into the role of these two proteins in iron homeostasis by employing M. tuberculosis bfr mutants. Our data suggests that BfrA is required for efficient utilization of stored iron under low iron conditions while BfrB plays a crucial role as the major defense protein under excessive iron conditions. We show that these two proteins provide protection against oxidative stress and hypoxia. Iron incorporation study showed that BfrB has higher capacity for storing iron than BfrA, which augurs well for efficient iron quenching under iron excess conditions. Moreover, iron release assay demonstrated that BfrA has 3 times superior ability to release stored iron emphasizing its requirement for efficient iron release under low iron conditions, facilitated by the presence of heme. Thus, for the first time, our observations suggest that the importance of BfrA or BfrB separately might vary depending upon the iron situation faced by the cell.
The transcriptome of a cell governs the overall cellular expression of proteins, hence, targeting mRNA holds promise as a therapeutic strategy in the field of drug discovery. RNA therapeutics ...especially, antisense oligonucleotide (ASO) technology, have gained tremendous pace in the last few decades with many of the ASOs being evaluated in various clinical phases and several ASO-based drugs are currently marketed after FDA approval. Several chemical modifications in the nucleic acid backbone of ASOs have led to improved properties of ASOs, such as oligonucleotide stability, enhanced affinity, and better pharmacokinetics contributing to their therapeutic success. ASOs interfere with the translation process either by RNase H-mediated mRNA cleavage or by posing steric hindrance in the translation machinery. Further, the cellular delivery of ASOs has always remained questionable, however, with the advancements in the delivery approaches, it is now feasible to achieve better target specific cellular delivery. In this review, we provide comprehensive insights into antisense oligonucleotide therapeutics, their various modifications, the mechanisms involved, different strategies for their delivery, and the utility of ASOs against several bacterial diseases.
ideR, an essential gene of Mycobacterium tuberculosis, is an attractive drug target as its conditional knockout displayed attenuated growth phenotype in vitro and in vivo. To the best of our ...knowledge, no inhibitors of IdeR are identified. We carried out virtual screening of NCI database against the IdeR DNA binding domain followed by inhibition studies using EMSA. Nine compounds exhibited potent inhibition with NSC 281033 (I-20) and NSC 12453 (I-42) exhibiting IC
values of 2 µg/ml and 1 µg/ml, respectively. We then attempted to optimize the leads firstly by structure based similarity search resulting in a class of inhibitors based on I-42 containing benzene sulfonic acid, 4-hydroxy-3-(2-hydroxy-1-naphthalenyl) azo scaffold with 4 molecules exhibiting IC
≤ 10 µg/ml. Secondly, optimization included development of energy based pharmacophore and screening of ZINC database followed by docking studies, yielding a molecule with IC
of 60 µg/ml. More importantly, a five-point pharmacophore model provided insight into the features essential for IdeR inhibition. Five molecules with promising IC
values also inhibited M. tuberculosis growth in broth culture with MIC
ranging from 17.5 µg/ml to 100 µg/ml and negligible cytotoxicity in various cell lines. We believe our work opens up avenues for further optimization studies.
Non-compliance to lengthy antituberculosis (TB) treatment regimen, associated side effects, and emergence of drug-resistant strains of
(
) emphasize the need to develop more effective anti-TB drugs. ...Here, we have evaluated the role of
dihydrodipicolinate reductase (DapB), a component of the diaminopimelate pathway, which is involved in the biosynthesis of both lysine and mycobacterial cell wall. We showed that DapB is essential for the
as well as intracellular growth of
. We further utilized
DapB, as a target for identification of inhibitors by employing
virtual screening, and conducted various
screening assays to identify inhibitors with potential to inhibit DapB activity and
and intracellular growth of
with no significant cytotoxicity against various mammalian cell lines. Altogether,
DapB serves as an important drug target and a hit molecule, namely, 4-(3-Phenylazoquinoxalin-2-yl) butanoic acid methyl ester has been identified as an antimycobacterial molecule in our study.
Pulmonary tuberculosis (TB) exhibits granulomatous inflammation, a site of controlling bacterial dissemination at the cost of host tissue damage. Intrigued by the granuloma type-dependent expression ...of inflammatory markers in TB, we sought to investigate underlying metabolic changes that drive amplification of inflammation in TB. Here, we show an association of higher inflammation in necrotic granulomas with the presence of triglyceride (TG)-rich foamy macrophages. The conspicuous absence of these macrophages in solid granulomas identified a link between the ensuing pathology and the metabolic programming of foamy macrophages. Consistent with
findings,
infection of macrophages with
(Mtb) led to increase in TG synthesis only under conditions of ~60% necrosis. Genetic and pharmacologic intervention that reduced necrosis prevented this bystander response. We further demonstrate that necrosis independent of Mtb also elicits the same bystander response in human macrophages. We identified a role for the human enzyme involved in TG synthesis, diacylglycerol
-acyltransferase (DGAT1), in this phenomenon. The increased TG levels in necrosis-associated foamy macrophages promoted the pro-inflammatory state of macrophages to infection while silencing expression of diacylglycerol
-acyltransferase (DGAT1) suppressed expression of pro-inflammatory genes. Our data thus invoke a role for storage lipids in the heightened host inflammatory response during infection-associated necrosis. Our data provide a functional role to macrophage lipid droplets in host defense and open new avenues for developing host-directed therapies against TB.
Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the ...non-compliance to 6-9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M. tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M. tuberculosis. In this study, a comparative homology model of M. tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC(50) values ranging from 20-100 µg/ml and two of these exhibited weak inhibition of M. tuberculosis growth with MIC(99) values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M. tuberculosis growth with an MIC(99) value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M. tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis.
Ferritins are recognized as key players in the iron storage and detoxification processes. Iron acquisition in the case of pathogenic bacteria has long been established as an important virulence ...mechanism. Here, we report a 3.0 Å crystal structure of a ferritin, annotated as Bacterioferritin B (BfrB), from Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis that continues to be one of the world's deadliest diseases. Similar to the other members of ferritin family, the Mtb BfrB subunit exhibits the characteristic fold of a four-helical bundle that possesses the ferroxidase catalytic centre. We compare the structure of Mtb BfrB with representatives of the ferritin family belonging to the archaea, eubacteria and eukarya. Unlike most other ferritins, Mtb BfrB has an extended C-terminus. To dissect the role of this extended C-terminus, truncated Mtb BfrB was purified and biochemical studies implicate this region in ferroxidase activity and iron release in addition to providing stability to the protein. Functionally important regions in a protein of known 3D-structure can be determined by estimating the degree of conservation of the amino-acid sites with its close homologues. Based on the comparative studies, we identify the slowly evolving conserved sites as well as the rapidly evolving variable sites and analyze their role in relation to structure and function of Mtb BfrB. Further, electrostatic computations demonstrate that although the electrostatic environment of catalytic residues is preserved within the family, extensive variability is exhibited by residues defining the channels and pores, in all likelihood keeping up with the diverse functions executed by these ferritins in varied environments.
The Mycobacterium tuberculosis genome sequence reveals remarkable absence of many nucleoid-associated proteins (NAPs), such as HNS, Hfq or DPS. In order to characterize the nucleoids of M. ...tuberculosis, we have attempted to identify NAPs, and report an interesting finding that a chaperonin-homolog, GroEL1, is nucleoid associated. We report that M. tuberculosis GroEL1 binds DNA with low specificity but high affinity, suggesting that it might have naturally evolved to bind DNA. We are able to demonstrate that GroEL1 can effectively function as a DNA-protecting agent against DNase I or hydroxyl-radicals. Moreover, Atomic Force Microscopic studies reveal that GroEL1 can condense a large DNA into a compact structure. We also provide in vivo evidences that include presence of GroEL1 in purified nucleoids, in vivo crosslinking followed by Southern hybridizations and immunofluorescence imaging in M. tuberculosis confirming that GroEL1: DNA interactions occur in natural biological settings. These findings therefore reveal that M. tuberculosis GroEL1 has evolved to be associated with nucleoids.
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