Translate this page to:
In JoVE (1)
Other Publications (9)
Articles by Bradley I. Coleman in JoVE
A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants
Bradley I. Coleman, Marc-Jan Gubbels
Department of Biology, Boston College
Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.
Other articles by Bradley I. Coleman on PubMed
Genome Research. Nov, 2004 | Pubmed ID: 15479945
Although the cost of generating draft-quality genomic sequence continues to decline, refining that sequence by the process of "sequence finishing" remains expensive. Near-perfect finished sequence is an appropriate goal for the human genome and a small set of reference genomes; however, such a high-quality product cannot be cost-justified for large numbers of additional genomes, at least for the foreseeable future. Here we describe the generation and quality of an intermediate grade of finished genomic sequence (termed comparative-grade finished sequence), which is tailored for use in multispecies sequence comparisons. Our analyses indicate that this sequence is very high quality (with the residual gaps and errors mostly falling within repetitive elements) and reflects 99% of the total sequence. Importantly, comparative-grade sequence finishing requires approximately 40-fold less reagents and approximately 10-fold less personnel effort compared to the generation of near-perfect finished sequence, such as that produced for the human genome. Although applied here to finishing sequence derived from individual bacterial artificial chromosome (BAC) clones, one could envision establishing routines for refining sequences emanating from whole-genome shotgun sequencing projects to a similar quality level. Our experience to date demonstrates that comparative-grade sequence finishing represents a practical and affordable option for sequence refinement en route to comparative analyses.
Cellular Microbiology. Oct, 2008 | Pubmed ID: 18637022
Infection with the apicomplexan parasite Plasmodium falciparum is associated with a high burden of morbidity and mortality across the developing world, yet the mechanisms of transcriptional control in this organism are poorly understood. While P. falciparum possesses many of the characteristics common to eukaryotic transcription, including much of the canonical machinery, it also demonstrates unique patterns of gene expression and possesses unusually AT-rich intergenic sequences. Importantly, several biological processes that are critical to parasite virulence involve highly regulated patterns of gene expression and silencing. The relative scarcity of transcription-associated proteins and specific cis-regulatory motifs recognized in the P. falciparum genome have been thought to reflect a reduced role for transcription factors in transcriptional control in these parasites. New approaches and technologies, however, have led to the discovery of many more of these elements, including an expanded family of DNA-binding proteins, and a re-assessment of this hypothesis is required. We review the current understanding of transcriptional control in P. falciparum, specifically highlighting promoter-driven and epigenetic mechanisms involved in the control of transcription initiation.
Molecular Microbiology. May, 2009 | Pubmed ID: 19400777
Plasmodium falciparum is the most virulent of the Plasmodium species infective to humans. Different P. falciparum strains vary in their dependence on erythrocyte receptors for invasion and their ability to switch in their utilization of different receptor repertoires. Members of the reticulocyte-binding protein-like (RBL) family of invasion ligands are postulated to play a central role in defining ligand-receptor interactions, known as invasion pathways. Here we report the targeted gene disruption of PfRh2b and PfRh2a in W2mef, a parasite strain that is heavily dependent on sialic-acid receptors for invasion, and show that the PfRh2b ligand is functional in this parasite background. Like the parental line, parasites lacking either PfRh2a or PfR2b can switch to a sialic acid-independent invasion pathway. However, both of the switched lines exhibit a reduced efficiency for invasion into sialic acid-depleted cells, suggesting a role for both PfRh2b and PfRh2a in invasion via sialic acid-independent receptors. We also find a strong selective pressure for the reconstitution of PfRh2b expression at the expense of PfRh2a. Our results reveal the importance of genetic background in ligand-receptor usage by P. falciparum parasites, and suggest that the co-ordinate expression of PfRh2a, PfRh2b together mediate efficient sialic acid-independent erythrocyte invasion.
Characterization and Structural Studies of the Plasmodium Falciparum Ubiquitin and Nedd8 Hydrolase UCHL3
The Journal of Biological Chemistry. Feb, 2010 | Pubmed ID: 20042598
Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed the identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected.
"Functional Diversification Between Two Related Plasmodium Falciparum Merozoite Invasion Ligands is Determined by Changes in the Cytoplasmic Domain"
Molecular Microbiology. Jan, 2010 | Pubmed ID: 20059683
Abstract The pathogenesis of Plasmodium falciparum depends on efficient invasion into host erythrocytes. Parasite ligands encoded by multigene families interact with erythrocyte receptors. P. falciparum reticulocyte binding protein homologues (PfRhs) are expressed at the apical surface of invasive merozoites and have divergent ectodomains that are postulated to bind different erythrocyte receptors. Variant expression of these paralogs results in the use of alternative invasion pathways. Two PfRh proteins, PfRh2a and PfRh2b, are identical for 2700 N-terminal amino acids and differ only in a C-terminal 500 amino acid region, which includes a unique ectodomain, transmembrane domain, and cytoplasmic domain. Despite their similarity, PfRh2b is required for a well-defined invasion pathway while PfRh2a is not required or sufficient for this pathway. Mapping the genomic region encoding these proteins revealed a recombinogenic locus with PfRh2a and PfRh2b in a head-to-head orientation. We have generated viable PfRh2a/2b chimeric parasites to identify the regions required for alternative invasion pathway utilization. We find that the differential ability to use these pathways is conferred by the cytoplasmic domains of PfRh2a and PfRh2b, not the ectodomain or transmembrane regions. Our results highlight the importance of the cytoplasmic domain for functional diversification of a major adhesive ligand family in malaria parasites.
Functional Diversification Between Two Related Plasmodium Falciparum Merozoite Invasion Ligands is Determined by Changes in the Cytoplasmic Domain
Molecular Microbiology. Feb, 2010 | Pubmed ID: 20487292
The pathogenesis of Plasmodium falciparum depends on efficient invasion into host erythrocytes. Parasite ligands encoded by multi-gene families interact with erythrocyte receptors. P. falciparum reticulocyte binding protein homologues (PfRhs) are expressed at the apical surface of invasive merozoites and have divergent ectodomains that are postulated to bind different erythrocyte receptors. Variant expression of these paralogues results in the use of alternative invasion pathways. Two PfRh proteins, PfRh2a and PfRh2b, are identical for 2700 N-terminal amino acids and differ only in a C-terminal 500 amino acid region, which includes a unique ectodomain, transmembrane domain and cytoplasmic domain. Despite their similarity, PfRh2b is required for a well-defined invasion pathway while PfRh2a is not required or sufficient for this pathway. Mapping the genomic region encoding these proteins revealed a recombinogenic locus with PfRh2a and PfRh2b in a head-to-head orientation. We have generated viable PfRh2a/2b chimeric parasites to identify the regions required for alternative invasion pathway utilization. We find that the differential ability to use these pathways is conferred by the cytoplasmic domains of PfRh2a and PfRh2b, not the ectodomain or transmembrane regions. Our results highlight the importance of the cytoplasmic domain for functional diversification of a major adhesive ligand family in malaria parasites.
Functional Analysis of Epigenetic Regulation of Tandem RhopH1/clag Genes Reveals a Role in Plasmodium Falciparum Growth
Molecular Microbiology. Apr, 2011 | Pubmed ID: 21320181
The Plasmodium RhopH complex is a high molecular weight antigenic complex consisting of three subunits - RhopH1/clag, RhopH2 and RhopH3 - located in the rhoptry secretory organelles of the invasive merozoite. In Plasmodium falciparum RhopH1/clag is encoded by one of five clag genes. Two highly similar paralogous genes, clag 3.1 and clag 3.2, are mutually exclusively expressed. Here we show clonal switching from the clag 3.2 to the clag 3.1 paralogue in vitro. Chromatin immunoprecitation studies suggest that silencing of either clag 3 paralogue is associated with the enrichment of specific histone modifications associated with heterochromatin. We were able to disrupt the clag 3.2 gene, with a drug cassette inserted into the clag 3.2 locus being readily silenced in a position-dependent and sequence-independent manner. Activation of this drug cassette by drug selection results in parasites with the clag 3.1 locus silenced and lack full-length clag 3.1 or 3.2 transcripts. These clag 3-null parasites demonstrate a significant growth inhibition compared with wild-type parasites, providing the first genetic evidence for a role for these proteins in efficient parasite proliferation. Epigenetic regulation of these chromosomally proximal members of a multigene family provides a mechanism for both immune evasion and functional diversification.
Antimicrobial Agents and Chemotherapy. Jun, 2011 | Pubmed ID: 21422215
This study characterizes aminoindole molecules that are analogs of Genz-644442. Genz-644442 was identified as a hit in a screen of ~70,000 compounds in the Broad Institute's small-molecule library and the ICCB-L compound collection at Harvard Medical School. Genz-644442 is a potent inhibitor of Plasmodium falciparum in vitro (50% inhibitory concentrations [IC₅₀s], 200 to 285 nM) and inhibits P. berghei in vivo with an efficacy of > 99% in an adapted version of Peters' 4-day suppressive test (W. Peters, Ann. Trop. Med. Parasitol. 69:155-171, 1975). Genz-644442 became the focus of medicinal chemistry optimization; 321 analogs were synthesized and were tested for in vitro potency against P. falciparum and for in vitro absorption, distribution, metabolism, and excretion (ADME) properties. This yielded compounds with IC₅₀s of approximately 30 nM. The lead compound, Genz-668764, has been characterized in more detail. It is a single enantiomer with IC₅₀s of 28 to 65 nM against P. falciparum in vitro. In the 4-day P. berghei model, when it was dosed at 100 mg/kg of body weight/day, no parasites were detected on day 4 postinfection. However, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in cures of 3/5 animals. The compound had comparable activity against P. falciparum blood stages in a human-engrafted NOD-scid mouse model. Genz-668764 had a terminal half-life of 2.8 h and plasma trough levels of 41 ng/ml when it was dosed twice a day orally at 55 mg/kg/day. Seven-day rat safety studies showed a no-observable-adverse-effect level (NOAEL) at 200 mg/kg/day; the compound was not mutagenic in Ames tests, did not inhibit the hERG channel, and did not have potent activity against a broad panel of receptors and enzymes. Employing allometric scaling and using in vitro ADME data, the predicted human minimum efficacious dose of Genz-668764 in a 3-day once-daily dosing regimen was 421 mg/day/70 kg, which would maintain plasma trough levels above the IC₉₀ against P. falciparum for at least 96 h after the last dose. The predicted human therapeutic index was approximately 3, on the basis of the exposure in rats at the NOAEL. We were unable to select for parasites with >2-fold decreased sensitivity to the parent compound, Genz-644442, over 270 days of in vitro culture under drug pressure. These characteristics make Genz-668764 a good candidate for preclinical development.
Identification and Validation of Tetracyclic Benzothiazepines As Plasmodium Falciparum Cytochrome Bc1 Inhibitors
Chemistry & Biology. Dec, 2011 | Pubmed ID: 22195562
Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.