In-silico three dimensional structure prediction of important Neisseria meningitidis proteins.
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Pathogenic bacteria Neisseria meningitidis cause serious infection i.e. meningitis (infection of the brain) worldwide. Among five pathogenic serogroups, serogroup B causes life threatening illness as there is no effective vaccine available due to its poor immunogenicity. A total of 73 genes in N. meningitidis genome have identified that were proved to be essential for meningococcal disease and were considered as crucial drug targets. We targeted five of those proteins, which are known to involve in amino acid biosynthesis, for homology-based three dimensional structure determinations by MODELLER (v9.19) and evaluated the models by PROSA and PROCHECK programs. Detailed structural analyses of NMB0358, NMB0943, NMB1446, NMB1577 and NMB1814 proteins were carried out during the present research. Based on a high degree of sequence conservation between target and template protein sequences, excellent models were built. The overall three dimensional architectures as well as topologies of all the proteins were quite similar with that of the templates. Active site residues of all the homology models were quite conserved with respect to their respective templates indicating similar catalytic mechanisms in these orthologues. Here, we are reporting, for the first time, detailed three dimensional folds of N. meningitidis pathogenic factors involved in a crucial cellular metabolic pathway. Moreover, the three dimensional structural information of these important drug targets would be utilized in computer-aided drug designing in future.Keywords:
MODELLER
Homology
Pathogenic bacteria
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The opportunistic pathogen Klebsiella pneumoniae is a causative agent of several hospital-acquired infections. It has become resistant to a wide range of currently available antibiotics leading to high mortality rates among patients thereby demanding a novel therapeutic intervention for treating such infections. Using a series of in silico analyses the present study aims to explore novel drug/vaccine candidates from the hypothetical proteins of K. pneumoniae. A total of 540 proteins were found to be hypothetical in this organism. Analysis of these 540 hypothetical proteins revealed that 30 proteins were pathogen-specific and essential for the pathogen survival. Motifs/domain family analysis, similarity search against known proteins, gene ontology and protein-protein interaction analysis of the shortlisted 30 proteins led to functional assignment for 17 proteins. They catalogued majorly into enzymes, lipoproteins, stress-induced proteins, transporters and other proteins (viz., two-component proteins, skeletal proteins and toxins). Among the annotated proteins, 16 proteins, located in cytoplasm, periplasm and inner membrane, were considered as potential drug targets, and one extracellular protein was considered as a vaccine candidate. Druggabality analysis indicated that the identified 17 drug/vaccine candidates were 'novel'. Further, host-pathogen interaction analysis of these identified target candidates revealed a betaine/carnitine/choline transporters (BCCT) family protein showing interactions with five host proteins. Structure prediction and validation were carried out for this protein, which could aid in structure-based inhibitor design.
Human pathogen
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Neisseria meningitidis serogroup B infections are a serious health threat to the world that cannot be prevented by vaccination. Here, we report an analysis of the MC58 Neisseria meningitidis genome aimed at the identification of new potential vaccine candidates. 'Hypothetical' and 'conserved hypothetical' annotated genes, together with those with putative functions related to the cell envelope, were subjected to extensive sequence similarity searches, as well as motif, cellular location, and domain analyses complemented with manual curation. As a result, a set of 35 unchar- acterized ORFs, predicted to encode for surface exposed or virulence related proteins, was identified. The candidates were subdivided in three categories: 1) predicted outer membrane proteins (OMPs) unique of the Neisseria genus; 2) conserved OMPs from various genus and 3) proteins homologous to known OMPs or to proteins previously found to be immunogenic in animal models. Two of the final candidates, nmb1126 and nmb0181, were cloned and expressed in Escherichia coli. The resulting products were purified by Metal Chelating Chromatography and used to immunize mice. The recombinant proteins were capable of inducing antibodies against the native antigen in preparations of a panel of three strains and displayed bactericidal activity against the homologous strains.
ORFS
Neisseria
Reverse vaccinology
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Pathogenic bacteria Neisseria meningitidis cause serious infection i.e. meningitis (infection of the brain) worldwide. Among five pathogenic serogroups, serogroups B causes life threatening illness as there is no effective vaccine available due to its poor immunogenicity. N. meningitidis genome contains73 genes which have been reported to be essential for meningococcal pathogenicity and are considered as crucial drug targets. During the present study, proteins involved in transport and binding mechanisms of the pathogen, i.e.NMB1240, NMB1612, NMB2044 and NMB2045 were targeted for homology-based three dimensional structure determinations. Homology modeling of all the target proteins by MODELLER and model evaluation by PROSA and PROCHECK were carried out. The overall three dimensional folds of the models were consistent with the folds of their respective templates including the active/binding site residues. Here, it is concluded that based on high similarities between targets’ and templates’ three dimensional architectures as well as good evaluation scores, the proposed models are much authentic and can be used in drug designing studies against N. meningitidis.
MODELLER
Homology
Pathogenic bacteria
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Abstract Background Drug target identification is a fast-growing field of research in many human diseases. Many strategies have been devised in the post-genomic era to identify new drug targets for infectious diseases. Analysis of protein sequences from different organisms often reveals cases of exon/ORF shuffling in a genome. This results in the fusion of proteins/domains, either in the same genome or that of some other organism, and is termed Rosetta stone sequences. They help link disparate proteins together describing local and global relationships among proteomes. The functional role of proteins is determined mainly by domain-domain interactions and leading to the corresponding signaling mechanism. Putative proteins can be identified as drug targets by re-annotating their functional role through domain-based strategies. Results This study has utilized a bioinformatics approach to identify the putative proteins that are ideal drug targets for pneumonia infection by re-annotating the proteins through position-specific iterations. The putative proteome of two pneumonia-causing pathogens was analyzed to identify protein domain abundance and versatility among them. Common domains found in both pathogens were identified, and putative proteins containing these domains were re-annotated. Among many druggable protein targets, the re-annotation of EJJ83173 (which contains the GFO_IDH_MocA domain) showed that its probable function is glucose-fructose oxidoreduction. This protein was found to have sufficient interactor proteins and homolog in both pathogens but no homolog in the host (human), indicating it as an ideal drug target. 3D modeling of the protein showed promising model parameters. The model was utilized for virtual screening which revealed several ligands with inhibitory activity. These ligands included molecules documented in traditional Chinese medicine and currently marketed drugs. Conclusions This novel strategy of drug target identification through domain-based putative protein re-annotation presents a prospect to validate the proposed drug target to confer its utility as a typical protein targeting both pneumonia-causing species studied herewith.
Proteome
Druggability
Comparative Genomics
Human proteome project
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Urinary tract infections are a serious health concern worldwide, especially in developing countries. Escherichia coli strain CFT073 is a highly virulent pathogenic bacterial strain. CFT073 proteome contains 4897 proteins, out of which 992 have been classified as hypothetical proteins. Identification and characterization of hypothetical proteins can aid in the selection of targets for drug design. In this study, we studied the hypothetical proteins from the UPEC strain CFT073 using various computational tools. By NCBI-CDD, 376 protein sequences showed conserved domains. Based on the functional motifs in their primary sequences, we classified these 376 hypothetical proteins into 7 functional categories. Further KEGG database was used to find the roles of these hypothetical proteins in several pathways. Protein interaction network analysis of hypothetical proteins identified 53 proteins as highly interacting metabolic proteins. Virulence factor analysis of the proteins identified 8 proteins as virulent. We conducted a non-homology search for the identified proteins of UPEC in the available human proteome. We observed that 35 proteins are non-homologous to humans and hence could be selected for drug designing targets. Qualitative characterization of the selected 35 non-homologous hypothetical proteins including essentiality analysis and evaluation of druggability by similarity search against drug bank database was performed. Out of these 35 proteins, three-dimensional structures of six proteins (NP_752562.1, NP_756345.1, NP_754893.1, NP_756600.2, NP_755264.1 and NP_752994.1) could be successfully modelled. These new annotations can help to better understand disease mechanisms at the molecular level, as well as provide new targets for drug development against the UPEC strain CFT073.Communicated by Ramaswamy H. Sarma.
Proteome
KEGG
Druggability
Virulence factor
Hypothetical protein
Homology
Protein superfamily
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Meningococcal factor H binding protein (fHbp) is a promising vaccine antigen that binds the human complement downregulatory molecule factor H (fH), and this binding enhances the survival of the organism in serum. Based on sequence variability of the entire protein, fHbp has been divided into three variant groups or two subfamilies. Here, we present evidence based on phylogenetic analysis of 70 unique fHbp amino acid sequences that the molecular architecture is modular. From sequences of natural chimeras we identified blocks of two to five invariant residues that flanked five modular variable segments. Although overall, 46 % of the fHbp amino acids were invariant, based on a crystal structure, the invariant blocks that flanked the modular variable segments clustered on the membrane surface containing the amino-terminal lipid anchor, while the remaining invariant residues were located throughout the protein. Each of the five modular variable segments could be classified into one of two types, designated alpha or beta, based on homology with segments encoded by variant 1 or 3 fHbp genes, respectively. Forty of the fHbps (57 %) comprised only alpha (n=33) or beta (n=7) type segments. The remaining 30 proteins (43 %) were chimeras and could be classified into one of four modular groups. These included all 15 proteins assigned to the previously described variant 2 in subfamily A. The modular segments of one chimeric modular group had 96 % amino acid identity with those of fHbp orthologs in Neisseria gonorrhoeae. Collectively, the data suggest that recombination between Neisseria meningitidis and N. gonorrhoeae progenitors generated a family of modular, antigenically diverse meningococcal fHbps.
Neisseria
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Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens.
Corynebacterium pseudotuberculosis
Proteome
Caseous lymphadenitis
Corynebacterium
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Background
N. gonorrhoeae, a major causative agent of STI, has acquired resistance to most of the commonly used drugs. Hence there is an urgent need to look for novel drug targets and new drugs to combat this disease. Although a large number of prokaryotic genomes have been sequenced, only a small percentage is completely annotated. Structure-function annotation of hypothetical proteins (HPs) can be exploited to identify novel drug targets.Methods
Various web tools were used under stringent condition to predict the function of different HPs and to identify novel drug targets based on their cellular localization, domains, motifs and by using STRING (http://string-db.org/) to identify their potential interactions with other proteins. To predict drug targets, essential genes were identified using DEG database and BLASTed against proteome of Homo sapiens to exclude homologous proteins.Results
Using bioinformatics tools, 206 HPs were analyzed for their subcellular localization. 140 HPs were predicted as cytoplasmic proteins and 10 as extracellular, Nine proteins in the outer membrane, seven as inner membrane whereas three in the periplasmic area. Using available tools, function to 32 HPs was assigned with high confidence; 11 proteins showed signal peptide whereas 21 proteins showed transmembrane helices. We predicted 19 proteins as putative enzymes crucial for the survival of Neisseria. These 19HPs were sub-classified as DNA modification system (5), transferases (3), hydrolase (3), FAD/NAD binding enzymes (5) and others (3). Other 12 HPs were characterized as transporter proteins including autotransporter (8), TonB dependent receptor (2), members of TAT pathway (1) and branched chain amino acid transporter (1). Two transporter proteins were predicted as adhesins and further classified as drug targets whereas five were predicted as vaccine candidates. We also predict five cytoplasmic and 4HPs localized in outer membrane as potential drug targets.Conclusion
These results are expected to be helpful in the development of improved therapeutics.Disclosure
No significant relationships.Proteome
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The ability of Streptococcus pneumoniae to induce infections relies on its virulence factor machinery. A previous CRISPR interference (CRISPRi) study had identified 254 essential proteins that may be responsible towards the pathogenicity of S. pneumoniae serotype 2 strain D39. However, 39 of them were functionally and structurally uncharacterized. Hence, by using in silico approach, this study aimed to annotate the function and structure of these un-annotated proteins. Initially, all 39 proteins went through primary screening for template availability and pathogenicity. From there, 11 of them were selected and underwent further physicochemical, functional and structural categorization through integrated bioinformatics approach by means of amino acid sequence- and structure- based analyses. The obtained data revealed that 9 targeted proteins showed high possibility to be involved in either cell viability or cell pathogenicity mechanism of the bacterium, with SPD_1333 and SPD_1743 being the two most promising proteins to be further studied. Findings from this study can help in facilitating a better understanding of pathogenic ability of this microorganism and enhance drug development and target identification processes in the aim of improving pneumococcal disease control.
Virulence factor
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