Comparative pan genome analysis of oral Prevotella species implicated in periodontitis.

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a "Por_secre_tail" domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.

Molecular dynamics simulation study of Plasmodium falciparum and Escherichia coli SufA: Exploration of conformational changes possibly involved in iron-sulfur cluster transfer.

Iron-sulfur (Fe-S) clusters are one of the earliest known metal complexes in biological molecules. Suf system is one of the Fe-S biogenesis pathways. SufA belongs to the Suf pathway. It is an A-type carrier protein that transfers Fe-S clusters from the scaffold to target proteins. Structural studies were performed for the Suf pathway protein, SufA, in order to explore the conformational changes that probably aid in the transfer of Fe-S clusters to target proteins. Three-dimensional (3D) structure of Plasmodium falciparum (Pf) SufA homodimer was obtained by homology modeling using 3D structure of Escherichia coli (Ec) SufA as template. Molecular dynamics (MD) simulation of Pf SufA and Ec SufA homodimers followed by trajectory and pocket analyses were carried out. A co-ordinated displacement of the homodimeric chains in the interfacial region, resembling a swinging trapeze-like movement was observed. Potential involvement of this swinging trapeze-like movement of the residues belonging to the interfacial region has been proposed as a probable mechanism that assists in the transfer of Fe-S cluster from SufA to apo proteins. This was substantiated by protein-protein interaction studies in Pf SufA by performing molecular docking of 3D conformations of Pf SufA obtained from MD trajectory at every 1 ns interval with Pf ferredoxin.Communicated by Ramaswamy H. Sarma.

Insights into the pH-dependent catalytic mechanism of Sulfolobus solfataricus ß-glycosidase: A molecular dynamics study.

Sulfolobus solfataricus ß-glycosidase (SS-ßGly) belongs to Glycosyl Hydrolase family1 (GH1) with broad substrate specificity. SS-ßGly catalyzes both hydrolysis and transglycosylation reactions. SS-ßGly is commonly used to synthesize variety of galacto-oligosaccharides. A comparison of SS-ßGly with bacterial and eukaryotic homologs, using DALI search, revealed unique inserts. Free enzyme molecular dynamics (MD) simulation was performed under two different pH conditions (pH 6.5 and 2.5) at a constant temperature of 65 °C using GROMACS. A probable active-site loop (residues 331-364) in SS-ßGly was identified. Dynamics of substrate binding cavity revealed that it was buried and inaccessible during most timeframes at pH 6.5 whereas open and accessible at pH 2.5. New cavities identified during both simulations may act as probable water channel or product egress path. Analyses of docked complexes of 3D structures obtained at every 1ns interval with compounds, involved in hydrolysis and tranglycosylation reactions, demonstrated that conformational states sampled by SS-ßGly during free enzyme dynamics mimic the stages in enzyme catalysis thereby providing a mechanistic perspective. Current study revealed that conformational changes were conducive for hydrolysis at pH 6.5 and multiple cycles of transglycosylation at pH 2.5. Probable role of salt-bridge interactions in determining the type of reaction mechanism was also explored.

Free enzyme dynamics of CmaA3 and CmaA2 cyclopropane mycolic acid synthases from Mycobacterium tuberculosis: Insights into residues with potential significance in cyclopropanation.

Mycolic acids are long chain alpha-alkyl beta-hydroxy fatty acids that are major constituents of the cell wall of Mycobacterium tuberculosis. M. tuberculosis produces three main types of mycolic acids, alpha mycolic acids and keto and methoxy mycolic acids. Cycloproponated mycolic acids make the cell wall less permeable, contribute to antibiotic resistance and host immunomodulation and protect from injury. Cyclopropanation is catalyzed by enzymes of the Cyclopropane Mycolic Acid Synthase (CMAS) family. In the current study, we addressed two CMAS enzymes, proximal alpha cyclopropane mycolic acid synthase (PcaA/CmaA3) and keto cyclopropane Mycolic acid synthase (CmaA2). All-atom Molecular Dynamics (MD) simulations were performed for these enzymes for a timeframe of 100ns each (in triplicate), using GROMACS. Based on the PDB structures of apo and holo states of related CMAS enzymes, we generated a framework which helped us correlate active or inactive states of the enzymes to different conformations sampled by the enzymes during MD simulations. Dynamics suggested that the free or unbound enzymes have intrinsic memory and sample different states of catalysis even in the absence of the substrate/cofactor. Additionally, we find that F200, P201 and W204 may have functional significance. MD simulation of CmaA2 was performed with the objective of gaining insights into the putative role of a loop insert. Analysis showed that acidic residues of this loop possibly play an important role during the active state by forming salt bridges. The insights gained in this study can potentially be utilized for design of effective inhibitors against CMAS enzymes.

Crowd sourcing a new paradigm for interactome driven drug target identification in Mycobacterium tuberculosis.

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative 'Connect to Decode' (C2D) to generate the first and largest manually curated interactome of Mtb termed 'interactome pathway' (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.