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Search Results: 1 - 10 of 423135 matches for " Clotilde K. S. Carlow "
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Characterization of Transcription Factors That Regulate the Type IV Secretion System and Riboflavin Biosynthesis in Wolbachia of Brugia malayi
Zhiru Li, Clotilde K. S. Carlow
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0051597
Abstract: The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium Wolbachia (wBm) that is required for parasite survival. Consequently, targeting wBm is a promising approach for anti-filarial drug development. The Type IV secretion system (T4SS) plays an important role in bacteria-host interactions and is under stringent regulation by transcription factors. In wBm, most T4SS genes are contained in two operons. We show the wBm is active since the essential assembly factor virB8-1, is transcribed in adult worms and larval stages, and VirB8-1 is present in parasite lysates. We also identify two transcription factors (wBmxR1 and wBmxR2) that bind to the promoter region of several genes of the T4SS. Gel shift assays show binding of wBmxR1 to regions upstream of the virB9-2 and wBmxR2 genes, whereas wBmxR2 binds to virB4-2 and wBmxR1 promoter regions. Interestingly, both transcription factors bind to the promoter of the ribA gene that precedes virB8-1, the first gene in operon 1 of the wBm T4SS. RT-PCR reveals ribA and virB8-1 genes are co-transcribed as one operon, indicating the ribA gene and T4SS operon 1 are co-regulated by both wBmxR1 and wBmxR2. RibA encodes a bi-functional enzyme that catalyzes two essential steps in riboflavin (Vitamin B2) biosynthesis. Importantly, the riboflavin pathway is absent in B. malayi. We demonstrate the pathway is functional in wBm, and observe vitamin B2 supplementation partially rescues filarial parasites treated with doxycycline, indicating Wolbachia may supply the essential vitamin to its worm host. This is the first characterization of a transcription factor(s) from wBm and first report of co-regulation of genes of the T4SS and riboflavin biosynthesis pathway. In addition, our results demonstrate a requirement of vitamin B2 for worm health and fertility, and imply a nutritional role of the symbiont for the filarial parasite host.
Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification
Catherine B. Poole,Nathan A. Tanner,Yinhua Zhang,Thomas C. Evans Jr.,Clotilde K. S. Carlow
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001948
Abstract: In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.
Evolution of Bacterial Phosphoglycerate Mutases: Non-Homologous Isofunctional Enzymes Undergoing Gene Losses, Gains and Lateral Transfers
Jeremy M. Foster,Paul J. Davis,Sylvine Raverdy,Marion H. Sibley,Elisabeth A. Raleigh,Sanjay Kumar,Clotilde K. S. Carlow
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0013576
Abstract: The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.
A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes
Brendan D. Galvin equal contributor,Zhiru Li equal contributor,Estelle Villemaine,Catherine B. Poole,Melissa S. Chapman,Michael P. Pollastri,Paul G. Wyatt,Clotilde K. S. Carlow
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0003145
Abstract: Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC50 values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 μM and 50 μM respectively, and C. elegans (25 μM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.
Mining Predicted Essential Genes of Brugia malayi for Nematode Drug Targets
Sanjay Kumar, Kshitiz Chaudhary, Jeremy M. Foster, Jacopo F. Novelli, Yinhua Zhang, Shiliang Wang, David Spiro, Elodie Ghedin, Clotilde K. S. Carlow
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0001189
Abstract: We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B.?malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.
Structure of the Trehalose-6-phosphate Phosphatase from Brugia malayi Reveals Key Design Principles for Anthelmintic Drugs
Jeremiah D. Farelli,Brendan D. Galvin,Zhiru Li,Chunliang Liu,Miyuki Aono,Megan Garland,Olivia E. Hallett,Thomas B. Causey,Alana Ali-Reynolds,Daniel J. Saltzberg,Clotilde K. S. Carlow,Debra Dunaway-Mariano ,Karen N. Allen
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004245
Abstract: Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.
Cofactor-Independent Phosphoglycerate Mutase from Nematodes Has Limited Druggability, as Revealed by Two High-Throughput Screens
Gregory J. Crowther,Michael L. Booker,Min He,Ting Li,Sylvine Raverdy,Jacopo F. Novelli,Panqing He,Natalie R. G. Dale,Amy M. Fife,Robert H. Barker Jr,Martin L. Kramer,Wesley C. Van Voorhis,Clotilde K. S. Carlow,Ming-Wei Wang
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002628
Abstract: Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ~220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ~160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z′-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 μM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a “druggability paradox” of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.
Targeting the Wolbachia Cell Division Protein FtsZ as a New Approach for Antifilarial Therapy
Zhiru Li,Amanda L. Garner,Christian Gloeckner,Kim D. Janda,Clotilde K. Carlow
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001411
Abstract: The use of antibiotics targeting the obligate bacterial endosymbiont Wolbachia of filarial parasites has been validated as an approach for controlling filarial infection in animals and humans. Availability of genomic sequences for the Wolbachia (wBm) present in the human filarial parasite Brugia malayi has enabled genome-wide searching for new potential drug targets. In the present study, we investigated the cell division machinery of wBm and determined that it possesses the essential cell division gene ftsZ which was expressed in all developmental stages of B. malayi examined. FtsZ is a GTPase thereby making the protein an attractive Wolbachia drug target. We described the molecular characterization and catalytic properties of Wolbachia FtsZ. We also demonstrated that the GTPase activity was inhibited by the natural product, berberine, and small molecule inhibitors identified from a high-throughput screen. Furthermore, berberine was also effective in reducing motility and reproduction in B. malayi parasites in vitro. Our results should facilitate the discovery of selective inhibitors of FtsZ as a novel anti-symbiotic approach for controlling filarial infection. Note The nucleotide sequences reported in this paper are available in GenBank? Data Bank under the accession number wAlB-FtsZ (JN616286).
Computational prediction of essential genes in an unculturable endosymbiotic bacterium, Wolbachia of Brugia malayi
Alexander G Holman, Paul J Davis, Jeremy M Foster, Clotilde KS Carlow, Sanjay Kumar
BMC Microbiology , 2009, DOI: 10.1186/1471-2180-9-243
Abstract: wBm protein sequences were aligned using BLAST to the Database of Essential Genes (DEG) version 5.2, a collection of 5,260 experimentally identified essential genes in 15 bacterial strains. A confidence score, the Multiple Hit Score (MHS), was developed to predict each wBm gene's essentiality based on the top alignments to essential genes in each bacterial strain. This method was validated using a jackknife methodology to test the ability to recover known essential genes in a control genome. A second estimation of essentiality, the Gene Conservation Score (GCS), was calculated on the basis of phyletic conservation of genes across Wolbachia's parent order Rickettsiales. Clusters of orthologous genes were predicted within the 27 currently available complete genomes. Druggability of wBm proteins was predicted by alignment to a database of protein targets of known compounds.Ranking wBm genes by either MHS or GCS predicts and prioritizes potentially essential genes. Comparison of the MHS to GCS produces quadrants representing four types of predictions: those with high confidence of essentiality by both methods (245 genes), those highly conserved across Rickettsiales (299 genes), those similar to distant essential genes (8 genes), and those with low confidence of essentiality (253 genes). These data facilitate selection of wBm genes for entry into drug design pipelines.Exponential growth in the amount of available genomic information has produced unprecedented opportunities to computationally predict functional genomics in biologically intractable organisms. One application of these data is facilitation of the rational drug design process. Most high throughput drug discovery techniques screen compounds for biological activity, only determining target and mechanism post hoc. An alternative approach, rational drug design, seeks to utilize genomic information to specifically identify and inhibit targets. Often these methods utilize in silico sequence analysis to choose a tar
Creches com qualidade para a educa o e o desenvolvimento integral da crian a pequena
Katia S. Amorim,Maria Clotilde Rossetti - Ferreira
Psicologia: Ciência e Profiss?o , 1999, DOI: 10.1590/s1414-98931999000200009
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