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Dermatophytosis is one of the most common superficial infections of the skin affecting nearly one-fifth of the world population at any given time. With nearly 30% of worldwide terbinafine-resistance cases in Trichophyton mentagrophytes/Trichophyton interdigitale and Trichophyton rubrum reported from India in recent years, there is a significant burden of the emerging drug resistance epidemic on India. Here, we carry out a comprehensive retrospective analysis of dermatophytosis in India using 1038 research articles pertaining to 161,245 cases reported from 1939 to 2021. We find that dermatophytosis is prevalent in all parts of the country despite variable climatic conditions in different regions. Our results show T. rubrum as the most prevalent until 2015, with a sudden change in dermatophyte spectrum towards T. mentagrophytes/T. interdigitale complex since then. We also carried out an 18S rRNA-based phylogenetics and an average nucleotide identity-and single nucleotide polymorphism-based analysis of available whole genomes and find very high relatedness among the prevalent dermatophytes, suggesting geographic specificity. The comprehensive epidemiological and phylogenomics analysis of dermatophytosis in India over the last 80 years, presented here, would help in region-specific prevention, control and treatment of dermatophyte infections, especially considering the large number of emerging resistance cases.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger required for normal physiology as well as survival under hypoxic and reductive stress conditions of mycobacterial cells. Complete degradation of c-di-GMP is necessary for signal termination and maintaining its homeostasis inside the cells. Homeostasis of c-di-GMP in mycobacteria is brought about by the bifunctional diguanylate cyclase (DGC) that synthesizes c-di-GMP from two molecules of GTP and also catalyses the asymmetric cleavage of c-di-GMP to linear pGpG through its phosphodiesterase activity. However, the mycobacterial enzyme for the last step of degradation from pGpG to GMP has not been characterized thus far. Here, we present the identification of oligoribonuclease (Orn) as the most likely phosphodiesterase to degrade pGpG to GMP through AlphaFold-empowered structural homology that exhibited in vitro phosphodiesterase activity on pGpG substrates. In order to understand the physiological role of Orn in mycobacteria, we created a deletion mutant of orn in M. smegmatis and analysed the phenotypes that are associated with c-di-GMP signaling. We find that orn plays important roles in vivo and is required not only for proper growth of M. smegmatis in normal and stress conditions but also for biofilm formation.

Angira Dasgupta,1– 3 Rahul Chakraborty,1,2 Bodhisattwa Saha,1 Himanshi Suri,1 Praveen Singh,1,2 Anurag Raj,1,2 Bhupesh Taneja,1,2 Debasis Dash,1,2 Shantanu Sengupta,1,2 Anurag Agrawal1,2 1Institute of Genomics and Integrative Biology, New Delhi, India; 2Academy of Scientific and Innovative Research, CSIR-HRDG, Ghaziabad, Uttar Pradesh, 201002, India; 3B R Singh Hospital and Centre for Medical Education and Research, Kolkata, West Bengal, IndiaCorrespondence: Angira DasguptaB R Singh Hospital and Centre for Medical Education and Research, Sealdah, Kolkata, West Bengal, IndiaTel +91 9831517266Email angiradasgupta@gmail.comBackground: Chronic mucous hypersecretion (CMH or chronic bronchitis) per se or when associated with chronic inflammatory airway diseases such as asthma or chronic obstructive pulmonary disease (COPD) has several adverse clinical consequences. The sputum fluid phase has several candidate proteins including mucins which have the potential of being therapeutic targets, but has not yet been explored in-depth. This study aimed at exploring the profile of sputum proteins in various airway diseases.Methods: Sputum from thirty-one patients with various airway diseases was collected and the fluid phase analyzed by LC-MS/MS and subsequently by sequential window acquisition of all theoretical fragments ion spectra (SWATH) (n = 15) for protein quantitation. Hierarchical clustering and functional grouping were performed.Results: A total of 185 proteins were quantitated by SWATH of which 21 proteins were identified which could distinguish between the clinical phenotypes by hierarchical clustering. Functional protein clustering revealed 4 groups: those that are inflammation related, oxidative stress related, mucin related and a cytoskeletal and calcium related group. The levels of eight proteins (Azurocidin1, Neutrophil defensin 3, Lactotransferrin, Calmodulin 3, Coronin1A, Mucin 5B, Mucin 5AC and BPI fold containing family B1) were significantly altered (relative to mean) in exacerbator prone subjects compared to nonexacerbators. Another simple but useful metric which emerged from this study was total protein concentration in sputum which was significantly higher in frequent exacerbators.Conclusion: Sputum proteins can detect the various airway disease clinical phenotypes. Total protein concentration and eight other proteins are biomarkers for frequent exacerbators. The clinical and therapeutic implications of the functional groups of proteins need further evaluation.Keywords: proteomics, chronic bronchitis, asthma, COPD, mucin, phenotyping

Emergence of multi drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis poses a serious threat to TB control as the available treatment options are less effective in these cases. Therefore, new strategies are required for identification of new drugs and drug targets. The M. tuberculosis dihydrodipicolinate synthase is a putative drug target with no potent inhibitor. Here in this study, we have used a comprehensive computational approach to identify small molecule inhibitors and validated them in vitro. As similar structures tend to have similar functions, therefore 3 approaches, namely, (a) known inhibitor-based approach, (b) substrate analogues, (c) product analogues were used for compound screening from various chemical libraries. The fourth approach we have taken is fragment library screening.In vitro studies revealed that β-hydroxypyruvic acid tartronic acid are inhibitors of Mtb-rDapA with maximum inhibition 48% and 51% at 500µM concentrations respectively. We have also observed that β-hydroxypyruvic acid is a time dependent inhibitor of Mtb-rDapA. Further, thermal shift assays showed that β-hydroxypyruvic acid interacts with Mtb-rDapA and the thermal stability of Mtb-rDapA shifted by 3˚C. Among the product analogues of Mtb-DapA, dipicolinic acid showed to bind to Mtb-rDapB shifting thermal stability of Mtb-rDapB by 0.5˚C in binding. Additionally, we have designed a new approach by combining substrate and product analogues to achieve better inhibitions. In vitro assays validated that this combination approach increased maximum inhibition up to 100%. We envision that these compounds would serve as potent leads for tuberculosis drug discovery.

Rv2966c is a highly specific methyltransferase that methylates G966 at the N2 position in 16S rRNA of mycobacterial ribosome and can be secreted inside the host cell to methylate host DNA. However, how the secreted protein retains its structure and function in the harsh environment of host cell, remains unclear. In this work, we investigate structural features of Rv2966c at pH 4.0 and pH 7.5 by far-UV- and near-UV-circular dichroism (CD) and fluorescence spectroscopy, to gain insights into its folding and stability at the acidic pH, that it is likely to encounter inside the macrophage. We show that Rv2966c exists in a compact, folded state at both pH 7.5 and pH 4.0, a result corroborated by molecular dynamics simulations as a function of pH. In fact, Rv2966c was found to be more stable at pH 4.0 than pH 7.5, as evidenced by thermal-induced CD and nanodifferential scanning fluorimetry, and urea-induced denaturation measurements. Interestingly, unlike pH 7.5 (two-state unfolding), denaturation of Rv2966c at pH 4.0 occurs in a biphasic (N ↔ X ↔ U) manner. Further spectroscopic characterization of ‘X’ state, identifies characteristics of a molten globule-like intermediate. We finally conclude that Rv2966c maintains a compact folded state at pH 4.0 akin to that at pH 7.5 but with higher stability.

Oligoribonucleases (Orns) are highly conserved DnaQ-fold 3'-5' exoribonucleases that have been found to carry out the last step of cyclic-di-GMP (c-di-GMP) degradation, that is, pGpG to GMP in several bacteria. Removal of pGpG is critical for c-di-GMP homeostasis, as excess uncleaved pGpG can have feedback inhibition on phosphodiesterases, thereby perturbing cellular signaling pathways regulated by c-di-GMP. Perturbation of c-di-GMP levels not only affects survival under hypoxic, reductive stress, or nutrient-limiting conditions but also affects pathogenicity in infection models as well as antibiotic response in mycobacteria. Here, we have determined the crystal structure of MSMEG_4724, the Orn of Mycobacterium smegmatis (Ms_orn) to 1.87 Å resolution to investigate the function of its extended C-terminal tail that is unique among bacterial Orns. Ms_orn is a homodimer with the canonical RNase-H fold of exoribonucleases and conserved catalytic residues in the active site. Further examination of the substrate-binding site with a modeled pGpG emphasized the role of a phosphate cap and "3'OH cap" in constricting a 2-mer substrate in the active site. The unique C-terminal tail of Ms_orn aids dimerization by forming a handshake-like flap over the second protomer of the dimer. Our thermal and denaturant-induced unfolding experiments suggest that it helps in higher stability of Ms_orn as compared with Escherichia coli Orn or a C-terminal deletion mutant. We also show that the C-terminal tail is required for modulating response to stress agents in vivo. These results will help in further evaluating the role of signaling and regulation by c-di-GMP in mycobacteria.

In the current study, we report the genome sequence of two different clinical isolates from India, Trichophyton indotineae UCMS-IGIB-CI12 and Trichophyton indotineae UCMS-IGIB-CI14. The resulting genome assembly achieved a 143-fold coverage in 824 contigs for T. indotineae UCMS-IGIB-CI12 and a 136-fold coverage in 904 contigs for T. indotineae UCMS-IGIB-CI14. Both the clinical isolates contain a c.1342G>A mutation corresponding to Ala448Thr amino acid substitution in erg1 and exhibit an intermittent drug response to terbinafine. Comparative genomics analysis with available genomes of Trichophyton interdigitale/Trichophyton mentagrophytes species complex revealed a similar genome architecture and identified large number of genes associated with virulence and pathogenicity, namely, lipases, proteases, LysM domain-containing factors, carbon metabolism enzymes and cytochrome P450 enzymes, in all the genomes. An analysis of single amino acid polymorphisms (SAPs) in the protein sequences of subtilisin and lipase enzyme families identified a higher frequency of SAPs in functionally important proteins, Sub3 and Sub6 and their possible use in multilocus phylogenetic analysis of T. interdigitale/T. mentagrophytes species complex. The whole genome sequences of T. indotineae clinical isolates provided in this report will, hence, serve as a key reference point for investigation of clinical strains and emerging drug resistance among dermatophytes originating from different parts of the world.

Identification of environment specific marker-features is one of the key objectives of many metagenomic studies. It aims to identify such features in microbiome datasets that may serve as markers of the contrasting or comparable states. Hypothesis testing and black-box machine learnt models which are conventionally used for identification of these features are generally not exhaustive, especially because they generally do-not provide any quantifiable relevance (context) of/between the identified features. We present MarkerML web-server, that seeks to leverage the emergence of interpretable machine learning for facilitating the contextual discovery of metagenomic features of interest. It does so through a comprehensive and automated application of the concept of Shapley Additive Explanations in companionship to the compositionality accounted hypothesis testing for the multi-variate microbiome datasets. MarkerML not only helps in identification of marker-features, but also enables insights into the role and inter-dependence of the identified features in driving the decision making of the supervised machine learnt model. Generation of high quality and intuitive visualizations spanning prediction effect plots, model performance reports, feature dependency plots, Shapley and abundance informed cladograms (Sungrams), hypothesis tested violin plots along-with necessary provisions for excluding the participant bias and ensuring reproducibility of results, further seek to make the platform a useful asset for the scientists in the field of microbiome (and even beyond). The MarkerML web-server is freely available for the academic community at https://microbiome.igib.res.in/markerml/.

Trichophyton rubrum is one of the leading causes of superficial skin infections worldwide. It is a keratinolytic fungus specialized in colonization of keratinized tissue of skin, hair and nails for long periods of time. The fungus encodes a wide repertoire of secreted proteases in its genome that not only aid in nutrient acquisition but also establishment of infection on the host. The proteases are synthesized in ‘prepro’ form that requires removal of the prosegment for activation. In order to gain insights into the structural association of the pro domain with the catalytic domain, we investigate the structural features of the pro domain of the secreted sedolisin member Sub16 of T. rubrum. Our results show that the pro domain of Sub16 may have inherent flexibility in the absence of the associated catalytic domain which is stabilized in complex with catalytic domain. This is the first report of structural investigation on a stand-alone pro domain of sedolisin family of subtilases that will help in design of further structural studies of this protein.

Owing to its central role in cellular function, ribosome is one of the most common targets of antibiotics in bacteria. Mutations in rrs gene, ribosomal protein genes, methyltransfersases or drug influx/efflux are often found to overcome the drug response. Despite modulation of methylation pattern in the ribosome through mutations in the methyltransferases as one of key modulators of drug response, rsmG (gidB) is the only conserved methyltransferase associated with low-level drug resistance in large number of mycobacterial isolates. Here, we present the first evidence of association of methylation by mycobacterial RsmE, that methylates U1498 of 16S rRNA, with low levels of drug resistance. Deletion of the RsmE-homolog of Mycobacterium smegmatis leads to at least two-fold increase in the inhibitory concentration of aminoglycosides that bind in the decoding center proximal to U1498 in the 30S subunit. The change in inhibitory concentrations was highly specific and does not show any cross-resistance to drugs of other classes. Surprisingly, Rv2372c, the RsmE-homolog of Mycobacterium tuberculosis has the largest number of mutations among conserved ribosomal methyltransfersases, after gidB, highlighting the role of mutations in the RsmE methyltransferase as a key emerging mechanism of drug resistance in clinical strains of M. tuberculosis. Our work underlies the association of methylation by the RsmE-homolog with drug resistance and lays the groundwork to tackle this emerging mechanism of drug resistane in mycobacteria.

Resistant superficial dermatophytic infections of the skin and its appendages have emerged as a major health problem in India. Mutations in Squalene epoxidase gene have led to increasing incidence of resistance to terbinafine in dermatophytic isolates. We examined six patients with recalcitrant dermatophytosis attending Dermatology OPD at a tertiary care hospital and demonstrated terbinafine resistance by molecular method. Immediate hyperitivity (IH) reaction to Trichophytin antigen was highlighted in these patients. The patients were treated with alternate antifungals after demonstration of resistance to terbinafine based on the antifungal susceptibility testing (AFST). On follow up the patients responded well to the substitute but the duration of therapy had to be prolonged beyond six weeks.

The analysis of whole genomes has revealed specific geographical distribution of Mycobacterium tuberculosis (Mtb) strains across the globe suggestive of unique niche dependent adaptive mechanisms. We provide an important correlation of a genome‐based mutation to a molecular phenotype across two predominant clinical Mtb lineages of the Indian subcontinent. We have identified a distinct lineage specific mutation‐G247C, translating into an alanine‐proline conversion in the papA2 gene of Indo‐oceanic lineage 1 (L1) Mtb strains, and restoration of cell wall sulfolipids by simple genetic complementation of papA2 from lineage 3 (L3) or from H37Rv (lineage 4‐L4) attributed the loss of this glycolipid to this specific mutation in Indo‐Oceanic L1 Mtb. The investigation of structure of Mtb PapA2 revealed a distinct nonribosomal peptide synthetase (NRPS) C domain conformation with an unconventional presence of a zinc binding motif. Surprisingly, the A83P mutation did not map to either the catalytic center in the N‐terminal subdomain or any of the substrate‐binding region of the protein. On the contrary, the inherent ability of mutant PapA2 to form insoluble aggregates and molecular simulations with the wild‐type/mutant (Wt/mut) PapA2 purports an important role for the surface associated 83rd residue in protein conformation. This study demonstrates the importance of a critical structural residue in the papA2 protein of Mtb and helps establish a link between observed genomic alteration and its molecular consequence in the successful human pathogen Mtb. Significance We demonstrate the effect of a unique SNP in PapA2 gene of Indo‐oceanic Mycobacterium tuberculosis (Mtb) strains leading to the loss of sulfolipid from these strains. By X‐ray crystallographic analysis and molecular dynamics (MD) simulations, we show the importance of this residue in the global PapA2 structure. The presence of a Zn atom has not been reported before for this class of proteins. Here, we provide an important link between genomic alteration and its molecular consequence in Mtb highlighting one of the many adaptive mechanisms that have contributed to its success as a human pathogen. A high degree of identity with PapA1, 3, or 4 would help in interpreting the structure of these PapA proteins and other acyl transferases of other biological systems.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterised by the inevitable degeneration of central and peripheral motor neurons. Aggregation of mutant SOD1 is one of the molecular mechanisms underlying the onset of the disease. There are a number of regression models designed to predict the survival of patients based on an analysis of experimental data on thermostability, heterodimerisation energy, and changes in the hydrophobicity of SOD1 mutants. Previously, we proposed regression models linking the change in the stability of hydrogen bonds in mutant SOD1 calculated using molecular dynamics and elastic networks with patients survival time. In this study, these models were improved in terms of accuracy of survival time prediction by taking into account the variance of survival time values relative to the mean, the number of patients carrying each specific mutation, and the use of random forest regression as a regression method. The accuracy of the previous models was roughly 5.2 years while the accuracy of the new ones are up to 4 years. The model is also superior to those published by other authors. It was found that the hydrogen bonds important for prediction of survival time are formed by residues at positions located in the regions of the protein responsible for aggregation as well as in structural and functionally important sites.

Staphylococcus capitis TE8 was isolated from skin surface of a healthy human foot, and exhibited a strong antibacterial activity against Gram-positive bacteria, including Staphylococcus aureus. Whole genome sequence of S. capitis TE8 was obtained by shotgun and paired-end pyrosequencing with a coverage of 109-fold. The draft genome contains 2,516,639 bp in 8 scaffolds with 209 total contigs. The genome contains 2319 protein coding sequences, 58 tRNA and 3 rRNA. Genome sequence analysis revealed 4 distinct gene loci with the ability to encode antimicrobial peptides: (i) an epidermicin gene cluster; (ii) a gallidermin gene cluster; (iii) a gene cluster encoding six phenol soluble modulin (PSM) β-type peptides (PSMβ1-β6) and (iv) an additional gene that belonged to PSMβ family and encoded a 44 residues long peptide, HTP2388. Synthetic peptides with sequence identical to seven PSMβ-like peptides i.e. PSMβ1-β6 and peptide HTP2388 showed antibacterial activity. Genome sequence also revealed genes for adhesins, intracellular adhesins, osmoadaptation, oxidative and acid stress tolerance possibly responsible for initial attachment, colonization and survival of S. capitis TE8 on human skin. Comparative genome analysis revealed presence of a gamut of genes in S. capitis strains in comparison to Staphylococcus epidermidis and Staphylococcus caprae indicating towards their possible role in better adaptation and survival on human skin.

DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3'-5' exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress.

U1498 of 16S rRNA plays an important role in translation fidelity as well as in antibiotic response. U1498 is present in a methylated form in the decoding centre of the ribosome. In this study, Rv2372c fromMycobacterium tuberculosishas been identified as an RsmE-like methyltransferase which specifically methylates U1498 of 16S rRNA at the N3 position and can complement RsmE-deletedEscherichia coli. The crystal structure of Rv2372c has been determined, and reveals that the protein belongs to a distinct class in the SPOUT superfamily and exists as a dimer. The deletion of critical residues at the C-terminus of Rv2372c leads to an inability of the protein to form stable dimers and to abolition of the methyltransferase activity. A ternary model of Rv2372c with its cofactorS-adenosylmethionine (SAM) and the 16S rRNA fragment148716S rRNA1510helps to identify binding pockets for SAM (in the deep trefoil knot) and substrate RNA (at the dimer interface) and suggests an SN2 mechanism for the methylation of N3 of U1498 in 16S rRNA.

The group of antigen 85 proteins of Mycobacterium tuberculosis is responsible for converting trehalose monomycolate to trehalose dimycolate, which contributes to cell wall stability. Here, we have used a serial enrichment approach to identify new potential inhibitors by searching the libraries of compounds using both 2D atom pair descriptors and binary fingerprints followed by molecular docking. Three different docking softwares AutoDock, GOLD, and LigandFit were used for docking calculations. In addition, we applied the criteria of selecting compounds with binding efficiency close to the starting known inhibitor and showing potential to form hydrogen bonds with the active site amino acid residues. The starting inhibitor was ethyl-3-phenoxybenzyl-butylphosphonate, which had IC(50) value of 2.0 μM in mycolyltransferase inhibition assay. Our search from more than 34 million compounds from public libraries yielded 49 compounds. Subsequently, selection was restricted to compounds conforming to the Lipinski rule of five and exhibiting hydrogen bonding to any of the amino acid residues in the active site pocket of all three proteins of antigen 85A, 85B, and 85C. Finally, we selected those ligands which were ranked top in the table with other known decoys in all the docking results. The compound NIH415032 from tuberculosis antimicrobial acquisition and coordinating facility was further examined using molecular dynamics simulations for 10 ns. These results showed that the binding is stable, although some of the hydrogen bond atom pairs varied through the course of simulation. The NIH415032 has antitubercular properties with IC(90) at 20 μg/ml (53.023 μM). These results will be helpful to the medicinal chemists for developing new antitubercular molecules for testing.

Mammalian mitochondrial cytochromes c (cyts c ) has a conserved Leu94 which is replaced by valine/isoleucine in some lower eukaryotes and prokaryotes. It is expected that nature substituted Leu94 with Val/Ile, for they have similar van der Waals volume and hydrophobicity with a difference in side chain branching only. Reports also suggested the presence of phenylalanine at position 94, which leads to questions: (i) How bulky aromatic amino acid residue fitted at position 94 in cyt c family proteins? (ii) What is the effect of L94F mutation on protein stability and folding? Here, we selected horse cyt- c as a model to answer the second question. We generated L94F mutant of horse cytochrome c and subsequently characterised using far-UV, near-UV and Soret circular dichroism, absorbance, intrinsic and extrinsic ANS (8-anilino-1-napthalenesulfonic acid) fluorescence and dynamic light scattering measurements. We observed that this mutation affects the native state and arrests the protein folding at the molten globule state. Thermal stability of L94F mutant is also measured by spectroscopic techniques and differential scanning calorimetry. The midpoint of thermal denaturation of L94F mutant is 17 °C less than wild type. Molecular dynamics simulation study also supports our in vitro observation that this mutant has stable backbone conformation.

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix-hairpin-helix [(HhH)(2)] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)(2) domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.

Nine of ten methylated nucleotides of Escherichia coli 16 S rRNA are conserved in Mycobacterium tuberculosis. All the 10 different methyltransferases are known in E. coli, whereas only TlyA and GidB have been identified in mycobacteria. Here we have identified Rv2966c of M. tuberculosis as an ortholog of RsmD protein of E. coli. We have shown that rv2966c can complement rsmD-deleted E. coli cells. Recombinant Rv2966c can use 30 S ribosomes purified from rsmD-deleted E. coli as substrate and methylate G966 of 16 S rRNA in vitro. Structure determination of the protein shows the protein to be a two-domain structure with a short hairpin domain at the N terminus and a C-terminal domain with the S-adenosylmethionine-MT-fold. We show that the N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition. Deletion of the N-terminal domain prevents binding to nucleic acid substrates, and the truncated protein fails to carry out the m(2)G966 methylation on 16 S rRNA. The N-terminal domain also binds DNA efficiently, a property that may be utilized under specific conditions of cellular growth.

gene in Indian population. The diagnosis was basedon clinical symptoms, substantial increase in plasma, verylong-chain fatty acids and typical MRI pattern. MRI of thepatient showed peritrigonal and cerebellar semioval whitematter hypodensities and hyperintense areas (T2/fluid atten-uated inversion recovery) in bilateral cerebral white mat-ter, predominantly in parieto–occipital region. The molecu-lar analysis by direct sequencing of the

SummaryTopoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.

This review discusses the progress of India from being one of the largest producers of generics to its coming of age and initiating novel drug development programs such as the Open Source Drug Discovery for tuberculosis. A few groups have also begun to emerge which focus their research on rational or structure based drug design. We discuss here some of the ongoing efforts in drug discovery in India primarily in national research institutions and academia.

Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or IB enzymes. We used real-time single-molecule micromechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type IB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type IB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type IB molecules but by using a completely different structural and mechanistic framework.

Mutations in the superoxide dismutase (SOD1) gene account for ∼15% and in the transactive response DNA binding protein (TARDBP) gene for ∼5% of familial amyotrophic lateral sclerosis (FALS) cases. These two genes were analysed in two siblings from North India with ALS and a positive family history. The coding region of SOD1 and TARDBP genes was sequenced in both siblings. Genetic variation identified in SOD1 was typed in unaffected family members (n = 11), sporadic ALS patients (n = 48) and healthy controls (n = 35). Molecular dynamic (MD) simulations were performed on wild-type (WT) and mutant monomers of SOD1 to determine structural changes due to the identified mutation. A novel heterozygous nucleotide variation (c.255G > T) was identified in exon 4 of SOD1 in the two siblings and two asymptomatic family members but not in SALS patients and healthy controls. This variation results in a known non-synonymous substitution from leucine to phenylalanine at position 84 (L84F), making it a triallelic variation. Large conformational changes were observed in the zinc loop and electrostatic loop in an L84F mutant compared to WT SOD1 in MD simulations. In conclusion, this is the first report of mutation in SOD1 associated with FALS in India. Structural perturbations in L84F SOD1 may cause dimer destabilization, with decreased metal affinity leading to oligomerization.

Trichophyton rubrum is one of the major causative agents of dermatophytosis in humans worldwide. We report the draft genome sequence of T. rubrum var. raubitschekii from Delhi, India, isolated from a patient presenting symptoms of onychomycosis. The total estimated genome size of the clinical isolate is 25.2 MB containing 8265 predicted protein-coding sequences, 91 tRNA and 15 rRNA genes. Sequence analysis of the secreted subtilases, one of the major virulence factors in dermatophytes, clusters them into three subfamilies with distinct sequence features. The genome sequence is a step in understanding diversity of dermatophytes worldwide and will aid in identification of virulence factors and dissecting mechanisms of pathogenesis among them.

Our abilities to predict three-dimensional conformation of a polypeptide, given its amino acid sequence, remain limited despite advances in structure analysis. Analysis of structures and sequences of protein families with similar secondary structural elements, but varying topologies, might help in addressing this problem. We have studied the small beta-barrel class of proteins characterized by four strands (n = 4) and a shear number of 8 (S = 8) to understand the principles of barrel formation. Multiple alignments of the various protein sequences were generated for the analysis. Positional entropy, as a measure of residue conservation, indicated conservation of non-polar residues at the core positions. The presence of a type II beta-turn among the various barrel proteins considered was another strikingly invariant feature. A conserved glycyl-aspartyl dipeptide at the beta-turn appeared to be important in guiding the protein sequence into the barrel fold. Molecular dynamics simulations of the type II beta-turn peptide suggested that aspartate is a key residue in the folding of the protein sequence into the barrel. Our study suggests that the conserved type II beta-turn and the non-polar residues in the barrel core are crucial for the folding of the protein's primary sequence into the beta-barrel conformation.

Chaperonin-60 (cpn60) and chaperonin-10 (cpn10) are essential proteins involved in ATP-dependent folding of several intracellular proteins in the bacterial cell. Folding of the nascent substrate polypeptide takes place in the large central cavity formed by each ring of the tetradecameric cpn60. This large cavity is closed upon capping by the heptameric cpn10. Cpn10s interact with cpn60s primarily through a 17-residue mobile loop and regulate the release and binding of the substrate polypeptide from the cpn60 surface. Here, the structure of M. tuberculosis cpn10 is reported at 3.5 A resolution. The overall structure of the cpn10 monomer is formed of a four-stranded beta-barrel and two long stretches of highly flexible segments: the dome loop and the mobile loop. The seven subunits in the heptamer show very little conformational difference and exhibit nearly perfect sevenfold geometry. The binding sites for metal ions in the dome loop of cpn10 have been identified, suggesting the role of metal ions in the stabilization of the protein. Comparisons with the available cpn10 structures indicate several interesting features.

An irregular, all beta-class of proteins, comprising members of the chaperonin-10, quinone oxidoreductase, glucose dehydrogenase and alcohol dehydrogenase families has earlier been classified as the GroES fold. In this communication, we present an extensive analysis of sequences and three dimensional structures of proteins belonging to this family. The individual protein structures can be superposed within 1.6 A for more than 60 structurally equivalent residues. The comparisons show a highly conserved hydrophobic core and conservation of a few key residues. A glycyl-aspartate dipeptide is suggested as being critical for the maintenance of the GroES fold. One of the surprising findings of the study is the non-conservative nature of Ile to Leu mutations in the protein core, although Ile to Val mutations are found to occur frequently.

The structure of Rv3717 determined to 1.7 Å resolution by Pt-SAD phasing reveals a unique autolysin that lacks a cell-wall-binding domain. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. Structural analysis reveals that Rv3717 utilizes a β-hairpin turn at its N-terminus to autoregulate its enzymatic activity., Bacterial N-acetylmuramoyl-l-alanine amidases are cell-wall hydrolases that hydrolyze the bond between N-acetylmuramic acid and l-alanine in cell-wall glycopeptides. Rv3717 of Mycobacterium tuberculosis has been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Å resolution by the Pt-­SAD phasing method. Rv3717 possesses an α/β-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed in Bartonella henselae amidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, an N-acetylmuramoyl-l-alanine amidase from M. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.

It is being realized that the traditional closed-door and market driven approaches for drug discovery may not be the best suited model for the diseases of the developing world such as tuberculosis and malaria, because most patients suffering from these diseases have poor paying capacity. To ensure that new drugs are created for patients suffering from these diseases, it is necessary to formulate an alternate paradigm of drug discovery process. The current model constrained by limitations for collaboration and for sharing of resources with confidentiality hampers the opportunities for bringing expertise from diverse fields. These limitations hinder the possibilities of lowering the cost of drug discovery. The Open Source Drug Discovery project initiated by Council of Scientific and Industrial Research, India has adopted an open source model to power wide participation across geographical borders. Open Source Drug Discovery emphasizes integrative science through collaboration, open-sharing, taking up multi-faceted approaches and accruing benefits from advances on different fronts of new drug discovery. Because the open source model is based on community participation, it has the potential to self-sustain continuous development by generating a storehouse of alternatives towards continued pursuit for new drug discovery. Since the inventions are community generated, the new chemical entities developed by Open Source Drug Discovery will be taken up for clinical trial in a non-exclusive manner by participation of multiple companies with majority funding from Open Source Drug Discovery. This will ensure availability of drugs through a lower cost community driven drug discovery process for diseases afflicting people with poor paying capacity. Hopefully what LINUX the World Wide Web have done for the information technology, Open Source Drug Discovery will do for drug discovery.

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.

International audience; The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

DNA is a dynamic molecule that undergoes constant changes in the cell through interactions with numerous proteins. Several classes of enzyme are specialized in promoting DNA rearrangements, including site-specific recombinases, DNA helicases, transposases and DNA topoisomerases. Recent structures of protein-DNA reaction intermediates trapped in various states of DNA remodeling, complemented by biochemical and biophysical functional studies, have enhanced our understanding of their respective mechanistic pathways.

Chaperonin-10s possess a highly flexible segment of ~10 The GroES of E.coli has recently been proposed to have a residues that covers their dome-like structure and closes metastable oligomeric structure leading to an increase in the the central cavity of the chaperonin assembly. The dome diameter of the dome orifice in solution (Timchenko et al., loop is believed to contribute to the plasticity of their 2000). In this study, we have attempted to characterize the oligomeric structure. We have exploited the presence of a flexibility of the dome loop by exploiting the presence of a single tryptophan residue occurring in the dome loop of single tryptophan residue in the dome loop of Mycobacterium Mycobacterium tuberculosis chaperonin-10 (cpn-10), and tuberculosis cpn-10. Our results indicate that the negative through intrinsic fluorescence measurements show that in charges at the dome are the likely factors responsible for the the absence of metal ions, the tryptophan is almost fully plasticity of cpn-10 structures. solvent exposed at neutral pH. The dome loop, however, assumes a closed conformation in the presence of metal ions, or at low pH. These changes are fully reversed in the Materials and methods presence of chelating agents such as EDTA, confirming the Multiple sequence alignment role of cations in modulating the metastable states of cpn-10. Sequences of cpn-10 homologues were retrieved from the