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 `3interactome 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.

Single nucleotide polymorphisms in genes that are common targets of luteotropin and luteolysin in primate corpus luteum: Computational exploration

Luteal insufficiency affects fertility and hence study of mechanisms that regulate corpus luteum (CL) function is of prime importance to overcome infertility problems. Exploration of human genome sequence has helped to study the frequency of single nucleotide polymorphisms (SNPs). Clinical benefits of screening SNPs in infertility are being recognized well in recent times. Examining SNPs in genes associated with maintenance and regression of CL may help to understand unexplained luteal insufficiency and related infertility. Publicly available microarray gene expression databases reveal the global gene expression patterns in primate CL during the different functional state. We intend to explore computationally the deleterious SNPs of human genes reported to be common targets of luteolysin and luteotropin in primate CL Different computational algorithms were used to dissect out the functional significance of SNPs in the luteinizing hormone sensitive genes. The results raise the possibility that screening for SNPs might be integrated to evaluate luteal insufficiency associated with human female infertility for future studies. (C) 2012 Elsevier B.V. All rights reserved,

Identification of an exported heat shock protein 70 in Plasmodium falciparum

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

Design and synthesis of positional isomers of 5 and 6-bromo-1-(phenyl)sulfonyl]-2-(4-nitrophenoxy)methyl]-1H-benzimida zoles as possible antimicrobial and antitubercular agents

In this Letter, we report the structure activity relationship (SAR) studies on series of positional isomers of 5(6)-bromo-1-(phenyl)sulfonyl]-2-(4-nitrophenoxy)methyl]-1H-benzim idazoles derivatives 7(a-j) and 8(a j) synthesized in good yields and characterized by H-1 NMR, C-13 NMR and mass spectral analyses. The crystal structure of 7a was evidenced by X-ray diffraction study. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus, (Gram-positive), Escherichia coil and Klebsiella pneumoniae (Gram-negative), antifungal activity against Candida albicans, Aspergillus flavus and Rhizopus sp. and antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis, Mycobacterium fortuitum and MDR-TB strains. The synthesized compounds displayed interesting antimicrobial activity. The compounds 7b, 7e and 7h displayed significant activity against Mycobacterium tuberculosis H37Rv strain.

RepEx: Repeat extractor for biological sequences

Genomic sequences are far from being random but are made up of systematically ordered and information rich patterns. These repeated sequence patterns have been vastly utilized for their fundamental importance in understanding the genome function and organization. To this end, a comprehensive toolkit, RepEx, has been developed which extracts repeat (inverted, everted and mirror) patterns from the given genome sequence(s) without any constraints. The toolkit can also be used to fetch the inverted repeats present in the protein sequence (s). Further, it is capable of extracting exact and degenerate repeats with a user defined spacer intervals. It is remarkably more precise and sensitive when compared to the existing tools. An example with comprehensive case studies and a performance evaluation of the proposed toolkit has been presented to authenticate its efficiency and accuracy. (C) 2013 Elsevier Inc. All rights reserved.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

Background: mIHF belongs to a subfamily of proteins, distinct from E. coli IHF. Results: Functionally important amino acids of mIHF and the mechanism(s) underlying DNA binding, DNA bending, and site-specific recombination are distinct from that of E. coli IHF. Conclusion: mIHF functions could contribute beyond nucleoid compaction. Significance: Because mIHF is essential for growth, the molecular mechanisms identified here can be exploited in drug screening efforts. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ihfA and ihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHF. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Enriching the annotation of Mycobacterium tuberculosis H37Rv proteome using remote homology detection approaches: Insights into structure and function

The availability of the genome sequence of Mycobacterium tuberculosis H37Rv has encouraged determination of large numbers of protein structures and detailed definition of the biological information encoded therein; yet, the functions of many proteins in M. tuberculosis remain unknown. The emergence of multidrug resistant strains makes it a priority to exploit recent advances in homology recognition and structure prediction to re-analyse its gene products. Here we report the structural and functional characterization of gene products encoded in the M. tuberculosis genome, with the help of sensitive profile-based remote homology search and fold recognition algorithms resulting in an enhanced annotation of the proteome where 95% of the M. tuberculosis proteins were identified wholly or partly with information on structure or function. New information includes association of 244 proteins with 205 domain families and a separate set of new association of folds to 64 proteins. Extending structural information across uncharacterized protein families represented in the M. tuberculosis proteome, by determining superfamily relationships between families of known and unknown structures, has contributed to an enhancement in the knowledge of structural content. In retrospect, such superfamily relationships have facilitated recognition of probable structure and/or function for several uncharacterized protein families, eventually aiding recognition of probable functions for homologous proteins corresponding to such families. Gene products unique to mycobacteria for which no functions could be identified are 183. Of these 18 were determined to be M. tuberculosis specific. Such pathogen-specific proteins are speculated to harbour virulence factors required for pathogenesis. A re-annotated proteome of M. tuberculosis, with greater completeness of annotated proteins and domain assigned regions, provides a valuable basis for experimental endeavours designed to obtain a better understanding of pathogenesis and to accelerate the process of drug target discovery. (C) 2014 Elsevier Ltd. All rights reserved.

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Background: The heterotrimeric M. tuberculosis RecBCD complex, or each of its individual subunits, remains uncharacterized. Results: MtRecD exists as a homodimer in solution, catalyzes ssDNA-dependent ATP hydrolysis, unwinding of DNA replication/recombination intermediates, and interacts with RecA. Conclusion: MtRecD possesses strong 5 3- and weak 3 5-helicase activities. Significance: These findings provide insights into the mechanism underlying DSB repair and homologous recombination in mycobacteria. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5 overhangs relative to the 3 overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having 18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3 overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5 overhangs, it could also catalyze significant unwinding of substrates containing 3 overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5 3 and weak 3 5 unwinding activities. The extent of unwinding of Y-shaped DNA structures was approximate to 3-fold lower compared with duplexes with 5 overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

The annotated whole-genome sequence of Mycobacterium tuberculosis indicated that Rv1388 (Mtihf) likely encodes a putative 20 kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or organization of mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF-duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg170, Arg171, and Arg173, which might be involved in DNA binding, and a conserved proline (P150) in the tight turn. The phenotypic sensitivity of Escherichia coli Delta ihfA and Delta ihfB strains to UV and methylmethanesulfonate could be complemented with the wild-type Mtihf, but not its alleles bearing mutations in the DNA-binding residues. Protein DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, bind with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHF alpha beta. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes compaction of DNA into nucleoid-like or higher-order filamentous structures. We hence propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Estudo sobre a ocorrência de enterobactérias produtoras de ESBL isoladas de pacientes internados em unidades de pós-operatório de cirurgia cardíaca de um hospital da rede pública do Rio de Janeiro

As infecções associadas aos cuidados de saúde constituem um problema grave nas unidades hospitalares bem como nos serviços de atendimento extra-hospitalares. Diferentemente de outros países, no Brasil, existe uma incidência alta dessas infecções causadas por microorganismos Gram-negativos produtores de β-lactamase de espectroestendido (ESBL). Estas enzimas hidrolisam compostos β-lactâmicos e são consideradas mundialmente como de importância clínica, pois a localização de seus genes emelementos móveis facilitam a transmissão cruzada. Este estudo foi realizado com amostras bacterianas isoladas de material clínico e de fezes de pacientes internados em um hospital da rede pública no Rio de Janeiro, Brasil. Estes pacientes estavam internados em duas unidades de terapia intensiva cardiológica, no período de janeiro a dezembro de 2007. O estudo teve por objetivo realizar a caracterização fenotípica e genotípica desses isolados associados à colonização ou infecção dos pacientes. Os testes fenotípicos e genotípicos foram realizados na Universidade do Estado do Rio de Janeiro e incluíram provas bioquímicas, teste de susceptibilidade, teste confirmatório para a expressão da produção da enzima ESBL e Reação de Polimerase em Cadeia (PCR) com iniciadores específicos para cinco genes: blaTEM, blaSHV, CTX-M1, Toho1 e AmpC. As espécies bactérianas mais frequentemente isoladas foram Escherichia coli (25%) e Klebsiella pneumoniae (30,56%), e os genes mais prevalentes foram blaTEM (41,6%), AMPC (41,6%) blaSHV (33,3%), CTX-M1 (25%), e Toho1 (19,44%). Identificamos em 25% das amostras enterobactérias que não eram E. coli, K. pneumoniae ou Proteus sp, com fenótipo para ESBL e a expressão dos mesmos genes, confirmando a necessidade de investigação nestes grupos microbianos. O substrato mais sensível para a expressão da área de sinergismo no Teste de Aproximação foi o ceftriaxone (80%). Identificamos também que 17% das amostras positivas para ESBL apresentaram co-produção para AmpC e 50% apresentaram mais de um gene para os iniciadores testados. A presença da carbapenemase foi avaliada em amostras bacterianas com susceptibilidade intermediária para ertapenem, através do Teste de Hodge modificado. Os achados do presente estudo caracterizaram a co-produção de AmpC e ESBL, bem como sugerem a necessidade da revisão e a ampliação dos métodos para a detecção de outros padrões de resistência na nossa Instituição.

Análise da prevalência de resistência aos antimicrobianos em pacientes com infecção urinária comunitária de um laboratório privado na cidade do Rio de Janeiro

Descrever a prevalência das espécies bacterianas isoladas nas infecções urinárias comunitárias. Descrever os perfis de susceptibilidade aos antibióticos de uso oral utilizado frente às bactérias isoladas nas infecções urinárias comunitárias. Avaliar a prevalência de fenótipos de resistência bacterianos através dos resultados dos testes de susceptibilidade e dos rastreamentos específicos utilizados. Amostras colhidas exclusivamente no atendimento ambulatorial com contagens de unidades formadoras de colônias entre 100.000 a ≥1.000.000 por mililitro (UCF/ml) Com ou sem piúria no exame de elementos anormais na urina e sedimentoscopia (EAS). Foram analisados retrospectivamente os resultados de urinoculturas e dos testes de susceptibilidade a antimicrobianos, realizados em um Laboratório da rede privada na cidade do Rio de janeiro, de pacientes atendidos em ambulatórios e com quadros de ITU. As amostras de urina coletadas englobavam basicamente os seguintes bairros: Botafogo, Barra da Tijuca, Ipanema, Copacabana, Tijuca e Centro. Foram analisados um total de 8.475 culturas de urina divididas em 7.286 urinas de pacientes femininos e 1.189 de pacientes masculinos entre Janeiro de 2006 a Dezembro de 2012. As amostras foram todas coletadas na Cidade do Rio de Janeiro e englobavam basicamente os seguintes bairros: Botafogo, Barra da Tijuca, Ipanema, Copacabana, Tijuca e Centro. Encontramos um percentual de resistência de 27% para ciprofloxacina frente à Escherichia coli que com 68.23% é a principal etiologia encontrada na ITU na comunidade os resultados das três fluoroquinolonas avaliadas no estudo, ciprofloxacina (2 geração), levofloxacina (3 geração) e norfloxacina (2 geração), acharemos respectivamente 27%, 25% e 20% de resistência em Escherichia coli. O uso de fluoroquinolonas em infecções urinárias comunitárias e consequentemente os achados de padrões de resistência neste estudo, reforçam o que já foi descrito em outros trabalhos. A cefalosporina de 2 geração (cefuroxima), demonstrou percentuais de resistência bastante satisfatórios frente as principais etiologias. Em Escherichia coli o percentual foi de 2%, em Klebsiella pneumoniae 3% e em Proteus mirabilis não houve nenhum achado de resistência. Uma das vantagens da cefuroxima é ser ativa quanto à produção de beta lactamase, conferindo um espectro maior frente a possíveis produtoras desta enzima. Seu esquema posológico é de 250mg duas vezes ao dia por 7 dias para infecções urinárias não complicadas. O meio mais eficaz de melhorar a administração antimicrobiana provavelmente envolverá um programa abrangente que incorpora múltiplas estratégias e colaboração entre as diversas especialidades dentro de uma determinada instituição de saúde. Neste contexto, a observação periódica da incidência bacteriana com seus respectivos índices de resistência aos antimicrobianos por sitio de infecção e correlação com os antibióticos mais comumente utilizados, é mandatória para o sucesso terapêutico.

Estudo da resistência aos antimicrobianos em enterobactérias isoladas de mãos de manipuladores de alimentos em cantinas permissionárias de uma universidade no Município do Rio de Janeiro

A contaminação dos alimentos e transmissão de patógenos via manipuladores pode ocorrer se as condições de higiene, armazenamento e preparo não estiverem satisfatórios. Procedimentos inadequados de manipulação oferecem um perigo potencial à saúde pública devido ao alto risco de ocorrência de doenças de origem alimentar por microrganismos resistentes aos antimicrobianos. Neste estudo foram coletados swabs das mãos de 49 manipuladores de alimentos, isoladas e identificadas por metodologia convencional 244 enterobactérias de 7 cantinas permissionárias em uma Universidade do Município do Rio de Janeiro. Foram utilizados discos contendo os seguintes antimicrobianos: aztreonam, tobramicina, ceftazidima, cloranfenicol, tetraciclina, sulfa, amicacina, ampicilina e sulbactam, ciprofloxacina, gentamicina, cefalotina, cefepime, cefoxitina, imipenem, ampicilina, cefotaxima. A pesquisa de genes que codificam β-lactamases foi realizada pela Reação em Cadeia da Polimerase para os genes blaTEM, blaSHV, blaCTX, blaOXA-1 e blaCMY-2. As espécies mais prevalentes identificadas foram: Enterobacter cloacae (21,3%), Citrobacter braakii (15,2%), Escherichia coli (12,7%), Klebsiella pneumoniae subesp. Pneumoniae (12,2%). O perfil de resistência aos antimicrobianos revelou que 94 (38,5%) cepas identificadas apresentaram resistência a pelo menos dois antibióticos, 55 cepas (22,5%) a pelo menos um antibiótico, 34 cepas (13,9%) a três antibióticos, 14 cepas (5,7%) mostraram-se resistentes a quatro antibióticos. Uma cepa de E. cloacae mostrou resistência múltipla a dez antibióticos e 140 cepas (57,3%) foram resistentes simultaneamente a cefalotina e cefoxitina. Nenhuma cepa foi resistente ao aztreonam e a sulfa e apenas 01 foi resistente ao ciprofloxacina, 01 ao imipenem e 02 a cefotaxima. Foi identificado a presença do gene blaSHV em uma cepa de K. pneumoniae subsp. pneumoniae. Este dado aponta para uma mudança no perfil dessas bactérias isoladas da comunidade que passam a ter características semelhantes com as de origem hospitalar. Os resultados alertam para um perigo à saúde dos consumidores e conseqüentemente à saúde pública, devido a presença de enterobactérias resistentes aos antimicrobianos nas mãos de manipuladores de alimentos.

Adaptive diversification of vomeronasal receptor 1 genes in rodents

The vomeronasal receptor 1 (V1R) are believed to be pheromone receptors in rodents. Here we used computational methods to identify 95 and 62 new putative V1R genes from the draft rat and mouse genome sequence, respectively. The rat V1R repertoire consists of 11 subfamilies, 10 of which are shared with the mouse, while rat appears to lack the H and I subfamilies found in mouse and possesses one unique subfamily (M). The estimations of the relative divergence times suggest that many subfamilies originated after the split of rodents and primates. The analysis also reveals that these clusters underwent an expansion very close to the split of mouse and rat. In addition, maximum likelihood analysis showed that the nonsynonymous and synonymous rate ratio for most of these clusters was much higher than one, suggesting the role of positive selection in the diversification of these duplicated V1R genes. Because V1R are thought to mediate the process of signal transduction in response to pheromone detection, we speculate that the V1R genes have evolved under positive Darwinian selection to maintain the ability to discriminate between large and complex pheromonal mixtures.

Mitochondrial data support an odd-nosed colobine clade

To obtain a more complete understanding of the evolutionary history of the leaf-eating monkeys we have examined the mitochondrial genome sequence of two African and six Asian colobines. Although taxonomists have proposed grouping the "odd-nosed" colobines

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. (C) 2009 Elsevier Ltd. All rights reserved.