RESUMO
Legionella pneumophila is the causative agent of a severe type of pneumonia (lung infection) called Legionnaires' disease. It is emerging as an antibiotic-resistant strain day by day. Hence, identifying novel drug targets and vaccine candidates is essential to fight against this pathogen. Here, attempts were taken through a subtractive genomics approach on the complete proteome of L. pneumophila to address the challenges of multidrug resistance. A total of 2,930 proteins from L. pneumophila proteome were investigated through diverse subtractive proteomics approaches, e.g., identification of human nonhomologous and pathogen-specific essential proteins, druggability and "anti-target" analysis, subcellular localization prediction, human microbiome nonhomology screening, and protein-protein interaction studies to find out effective drug and vaccine targets. Only three fulfilled these criteria and were proposed as novel drug targets against L. pneumophila. Furthermore, outer membrane protein TolB was identified as a potential vaccine target with a better antigenicity score. Antigenicity and transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis, and a molecular docking approach were adopted to generate the most potent epitopes. The final vaccine was constructed by the combination of highly immunogenic epitopes, along with suitable adjuvant and linkers. The designed vaccine construct showed higher binding interaction with different major histocompatibility complex (MHC) molecules and human immune TLR-2 receptors with minimum deformability at the molecular level. The present study aids the development of novel therapeutics and vaccine candidates for efficient treatment and prevention of L. pneumophila infections. However, further wet-lab-based phenotypic and genomic investigations and in vivo trials are highly recommended to validate our prediction experimentally. IMPORTANCE Legionella pneumophila is a human pathogen distributed worldwide, causing Legionnaires' disease (LD), a severe form of pneumonia and respiratory tract infection. L. pneumophila is emerging as an antibiotic-resistant strain, and controlling LD is now difficult. Hence, developing novel drugs and vaccines against L. pneumophila is a major research priority. Here, the complete proteome of L. pneumophila was considered for subtractive genomics approaches to address the challenge of antimicrobial resistance. Our subtractive proteomics approach identified three potential drug targets that are promising for future application. Furthermore, a possible vaccine candidate, "outer membrane protein TolB," was proposed using reverse vaccinology analysis. The constructed vaccine candidate showed higher binding interaction with MHC molecules and human immune TLR-2 receptors at the molecular level. Overall, the present study aids in developing novel therapeutics and vaccine candidates for efficient treatment of the infections caused by L. pneumophila.
Assuntos
Legionella pneumophila , Doença dos Legionários , Proteoma , Antibacterianos , Epitopos , Genômica , Humanos , Legionella pneumophila/genética , Doença dos Legionários/tratamento farmacológico , Doença dos Legionários/prevenção & controle , Simulação de Acoplamento Molecular , Receptor 2 Toll-Like , VacinologiaRESUMO
West Nile Virus (WNV) is a life threatening flavivirus that causes significant morbidity and mortality worldwide. No preventive therapeutics including vaccines against WNV are available for human use. In this study, immunoinformatics approach was performed to design a multi epitope-based subunit vaccine against this deadly pathogen. Human (HLA) and Mice (H-2) allele specific potential T-cell and B-cell epitopes were shortlisted through a stringent procedure. Molecular docking showed selected epitopes that have stronger binding affinity with human TLR-4. Molecular dynamics simulation confirmed the stable nature of the docked complex. Furthermore, in silico cloning analysis ensures efficient expression of desired gene in the microbial system. Interestingly, previous studies showed that two of our selected epitopes have strong immune response against WNV. Therefore, selected epitopes could be strong vaccine candidates to prevent WNV infections in human. However, further in vitro and in vivo investigations could be strengthening the validation of the vaccine candidate against WNV.
Assuntos
Epitopos de Linfócito T/imunologia , Simulação de Dinâmica Molecular , Vacinas , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/imunologia , Animais , Desenho de Fármacos , Humanos , Camundongos , Simulação de Acoplamento MolecularRESUMO
Clostridioides difficile is a spore-forming enteric pathogen causing life-threatening diarrhoea and colitis. Microbial disruption caused by antibiotics has been linked with susceptibility to, and transmission and relapse of, C. difficile infection. Therefore, there is an urgent need for novel therapeutics that are effective in preventing C. difficile growth, spore germination, and outgrowth. In recent years bacteriophage-derived endolysins and their derivatives show promise as a novel class of antibacterial agents. In this study, we recombinantly expressed and characterized a cell wall hydrolase (CWH) lysin from C. difficile phage, phiMMP01. The full-length CWH displayed lytic activity against selected C. difficile strains. However, removing the N-terminal cell wall binding domain, creating CWH351-656, resulted in increased and/or an expanded lytic spectrum of activity. C. difficile specificity was retained versus commensal clostridia and other bacterial species. As expected, the putative cell wall binding domain, CWH1-350, was completely inactive. We also observe the effect of CWH351-656 on preventing C. difficile spore outgrowth. Our results suggest that CWH351-656 has therapeutic potential as an antimicrobial agent against C. difficile infection.
Assuntos
Bacteriófagos , Clostridioides difficile , Endopeptidases/metabolismo , Esporos Bacterianos , Proteínas Virais/metabolismo , Bacteriófagos/enzimologia , Bacteriófagos/genética , Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Clostridioides difficile/virologia , Endopeptidases/genética , Endopeptidases/farmacologia , Enterocolite Pseudomembranosa/tratamento farmacológico , Humanos , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética , Esporos Bacterianos/virologia , Proteínas Virais/genética , Proteínas Virais/farmacologiaRESUMO
Diverse animals, including insects, harbor microbial symbionts within their gut, body cavity, or cells. The subsocial parastrachiid stinkbug Parastrachia japonensis is well-known for its peculiar ecological and behavioral traits, including its prolonged non-feeding diapause period and maternal care of eggs/nymphs in an underground nest. P. japonensis harbors a specific bacterial symbiont within the gut cavity extracellularly, which is vertically inherited through maternal excretion of symbiont-containing white mucus. Thus far, biological roles of the symbiont in the host lifecycle has been little understood. Here we sequenced the genome of the uncultivable gut symbiont "Candidatus Benitsuchiphilus tojoi." The symbiont has an 804 kb circular chromosome encoding 606 proteins and a 14.5 kb plasmid encoding 13 proteins. Phylogenetic analysis indicated that the bacterium is closely related to other obligate insect symbionts belonging to the Gammaproteobacteria, including Buchnera of aphids and Blochmannia of ants, and the most closely related to Ishikawaella, an extracellular gut symbiont of plataspid stinkbugs. These data suggested that the symbiont genome has evolved like highly reduced gamma-proteobacterial symbiont genomes reported from a variety of insects. The presence of genes involved in biosynthesis pathways for amino acids, vitamins, and cofactors in the genome implicated the symbiont as a nutritional mutualist, supplementing essential nutrients to the host. Interestingly, the symbiont's plasmid encoded genes for thiamine and carotenoid synthesis pathways, suggesting the possibility of additional functions of the symbiont for protecting the host against oxidative stress and DNA damage. Finally, possible involvement of the symbiont in uric acid metabolism during diapause is discussed.
RESUMO
The successful adaptation of cockroaches is, in part, dependent of the activity of their obligatory endosymbionts, Blattabacterium spp., which are involved in uric acid degradation, nitrogen assimilation and nutrient provisioning. Their strategic localization, within bacteriocytes in the proximities of uric acid storage cells (urocytes), highlights their importance in the recycling of nitrogen from urea and ammonia, end-products not secreted by their host insects. In this study, we present the complete genome sequence of two new Blattabacterium spp. from Periplaneta fuliginosa (BPfu) and P. japonica (BPja), and detailed comparison with other Blattabacterium strains from different cockroach species. The genomes of BPfu and BPja show a high degree of stability as showed with for other Blattabacterium representatives, only presenting a 19-kb fragment inversion between BPja and BPfu. In fact, the phylogenomics showed BPja as an ancestor species of BPfu, BPLAN (P. americana) and BBor (Blatta orientalis), in congruence with their host cockroach phylogeny. Their functional profile is similar and closest to the omnivorous strain BBge (Blattella germanica). Interesting, BPja possesses the complete set of enzymes involved sulfate assimilatory pathway only found in BBge and BMda (Mastotermes darwiniensis). The newly sequenced genomes of BPja and BPfu emphasise the remarkable stability of Blattabacterium genomes supported by their long-term coevolution and obligatory lifestyle in their host insect.
Assuntos
Bacteroidetes/genética , Blattellidae/microbiologia , Genoma Bacteriano , Periplaneta/microbiologia , Amônia/química , Animais , DNA Bacteriano/genética , Masculino , Nitrogênio/química , Filogenia , Simbiose , Ureia/química , Ácido Úrico/químicaRESUMO
Rickettsiae are obligate intracellular bacteria that have small genomes as a result of reductive evolution. Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fevers". The life cycle of SFG rickettsiae is closely associated with that of the tick, which is generally thought to act as a bacterial vector and reservoir that maintains the bacterium through transstadial and transovarial transmission. Each SFG member is thought to have adapted to a specific tick species, thus restricting the bacterial distribution to a relatively limited geographic region. These unique features of SFG rickettsiae allow investigation of how the genomes of such biologically and ecologically specialized bacteria evolve after genome reduction and the types of population structures that are generated. Here, we performed a nationwide, high-resolution phylogenetic analysis of Rickettsia japonica, an etiological agent of Japanese spotted fever that is distributed in Japan and Korea. The comparison of complete or nearly complete sequences obtained from 31 R. japonica strains isolated from various sources in Japan over the past 30 years demonstrated an extremely low level of genomic diversity. In particular, only 34 single nucleotide polymorphisms were identified among the 27 strains of the major lineage containing all clinical isolates and tick isolates from the three tick species. Our data provide novel insights into the biology and genome evolution of R. japonica, including the possibilities of recent clonal expansion and a long generation time in nature due to the long dormant phase associated with tick life cycles.
Assuntos
Variação Genética , Genoma Bacteriano , Infecções por Rickettsia/microbiologia , Rickettsia/genética , Perfilação da Expressão Gênica , Humanos , Japão , Filogenia , Rickettsia/classificação , Infecções por Rickettsia/genética , Análise de Sequência de DNARESUMO
Protein-protein interaction (PPI) and host-pathogen interactions (HPI) proteomic analysis has been successfully practiced for potential drug target identification in pathogenic infections. In this research, we attempted to identify new drug target based on PPI and HPI computation approaches and subsequently design new drug against devastating enterohemorrhagic Escherichia coli O104:H4 C277-11 (Broad), which causes life-threatening food borne disease outbreak in Germany and other countries in Europe in 2011. Our systematic in silico analysis on PPI and HPI of E. coli O104:H4 was able to identify bacterial D-galactose-binding periplasmic and UDP-N-acetylglucosamine 1-carboxyvinyltransferase as attractive candidates for new drug targets. Furthermore, computational three-dimensional structure modeling and subsequent molecular docking finally proposed [3-(5-Amino-7-Hydroxy-[1,2,3]Triazolo[4,5-D]Pyrimidin-2-Yl)-N-(3,5-Dichlorobenzyl)-Benzamide)] and (6-amino-2-[(1-naphthylmethyl)amino]-3,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one) as promising candidate drugs for further evaluation and development for E. coli O104:H4 mediated diseases. Identification of new drug target would be of great utility for humanity as the demand for designing new drugs to fight infections is increasing due to the developing resistance and side effects of current treatments. This research provided the basis for computer aided drug design which might be useful for new drug target identification and subsequent drug design for other infectious organisms.
RESUMO
Bacterial enteric infections resulting in diarrhea, dysentery, or enteric fever constitute a huge public health problem, with more than a billion episodes of disease annually in developing and developed countries. In this study, the deadly agent of hemorrhagic diarrhea and hemolytic uremic syndrome, Escherichia coli O157:H7 was investigated with extensive computational approaches aimed at identifying novel and broad-spectrum antibiotic targets. A systematic in silico workflow consisting of comparative genomics, metabolic pathways analysis, and additional drug prioritizing parameters was used to identify novel drug targets that were essential for the pathogen's survival but absent in its human host. Comparative genomic analysis of Kyoto Encyclopedia of Genes and Genomes annotated metabolic pathways identified 350 putative target proteins in E. coli O157:H7 which showed no similarity to human proteins. Further bio-informatic approaches including prediction of subcellular localization, calculation of molecular weight, and web-based investigation of 3D structural characteristics greatly aided in filtering the potential drug targets from 350 to 120. Ultimately, 44 non-homologous essential proteins of E. coli O157:H7 were prioritized and proved to have the eligibility to become novel broad-spectrum antibiotic targets and DNA polymerase III alpha (dnaE) was the top-ranked among these targets. Moreover, druggability of each of the identified drug targets was evaluated by the DrugBank database. In addition, 3D structure of the dnaE was modeled and explored further for in silico docking with ligands having potential druggability. Finally, we confirmed that the compounds N-coeleneterazine and N-(1,4-dihydro-5H-tetrazol-5-ylidene)-9-oxo-9H-xanthene-2-sulfon-amide were the most suitable ligands of dnaE and hence proposed as the potential inhibitors of this target protein. The results of this study could facilitate the discovery and release of new and effective drugs against E. coli O157:H7 and other deadly human bacterial pathogens.
RESUMO
Cholera is a severe diarrheal disease caused by Vibrio cholerae and remains as a major health risk in developing countries. The emergence and spread of multi-drug resistant V. cholerae strains during the past two decades is now a major problem in the treatment of cholera and have created the urgent need for the development of novel therapeutic agents. Targeting transcriptional factor is now a novel approach to tackle the development of multi-drug resistant strain. In the recent year virtual high throughput screening has emerged as a widely accepted powerful technology in the identification of novel and diverse lead. This study provides new insight to the search for new potent and selective inhibitors that still remains necessary to avoid the risk of possible resistance and reduce toxicity and side effects of currently available cholera drugs. The publications of high resolution X-ray structure of V. cholerae ToxT has open the way to the structure based virtual screening to identify new small molecular inhibitors which still remain necessary to avoid the risk of possible resistance and reduce toxicity and side effects of currently available cholera drugs. In this study we have performed structure based virtual screening approach using NCI diversity set-II to look for novel inhibitor of ToxT and proposed eight candidate compounds with high scoring function. Thus from complex scoring and binding ability it is elucidated that these compounds could be the promising inhibitors or could be developed as novel lead compounds for drug design against cholera.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Cólera/microbiologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Fatores de Transcrição/antagonistas & inibidores , Vibrio cholerae/efeitos dos fármacos , Antibacterianos/uso terapêutico , Proteínas de Bactérias/química , Cólera/tratamento farmacológico , Toxina da Cólera , Cristalografia por Raios X , Resistência a Múltiplos Medicamentos , Humanos , Simulação de Acoplamento Molecular , Fatores de Transcrição/química , Ativação Transcricional/efeitos dos fármacosRESUMO
MicroRNAs (miRNAs) are the group of â¼22 nucleotides long noncoding small endogenous and evolutionary conserved post-transcriptional regulatory RNAs, which show an enormous role in various biological and metabolic processes in both animals and plants. To date not a single miRNA has been identified in coffee (Coffea arabica), which is an economically important plant of Rubiaceae family. In this study a well-developed, powerful and comparative computational approach, EST-based homology search is applied to find potential miRNA of coffee. We blasted publicly available EST sequences obtained from NCBI GenBank against previously known plant miRNAs. For the first time, one potential miRNA from a large miRNA family with appropriate fold back structures was identified through a series of filtration criteria. A total of six potential target genes in Arabidopsis were identified based on their sequence complementarities. The target genes mainly encode transport inhibitor like protein, transcription factor, DNA-binding protein, and GRR1-like protein, and these genes play an important role in various biological processes like response to chitin, cold, salt stress, water deprivation etc. Overall, findings from this study will accelerate the way for further researches of miRNAs and their functions in coffee.
RESUMO
The novel swine-origin influenza A/H1N1 virus (S-OIV) first detected in April 2009 has been identified to transmit from human to human directly and is the cause of currently emerged pandemic. In this study, nucleotide and deduced amino acid sequences of hemagglutinin (HA) and neuraminidase (NA) of the S-OIV and other influenza A viruses were analyzed through bioinformatic tools for phylogenetic analysis, genetic recombination and point mutation to investigate the emergence and adaptation of the S-OIV in human. The phylogenetic analysis showed that the HA comes from triple reassortant influenza A/H1N2 and the NA from Eurasian swine influenza A/H1N1 indicating HA and NA to descend from different lineages during the genesis of the S-OIV. Recombination analysis nullified the possibility of occurrence of recombination in HA and NA denoting the role of reassortment in the outbreak. Several conservative mutations are observed in the amino acid sequences of the HA and NA and this mutated residues are identical in the S-OIV. The results reported herein suggested the notion that the recent pandemic is the result of reassortment of different genes from different lineages of two envelope proteins, HA and NA which are responsible for antigenic activity of virus. This study further suggests that the adaptive capability of the S-OIV in human is acquired by the unique mutations generated during emergence.