Mark Carrington

TriTrypDB: a functional genomic resource for the Trypanosomatidae

TriTrypDB (http://tritrypdb.org) is an integrated database providing access to genome-scale datasets for kinetoplastid parasites, and supporting a variety of complex queries driven by research and development needs. TriTrypDB is a collaborative project, utilizing the GUS/WDK computational infrastructure developed by the Eukaryotic Pathogen Bioinformatics Resource Center (EuPathDB.org) to integrate genome annotation and analyses from GeneDB and elsewhere with a wide variety of functional genomics datasets made available by members of the global research community, often pre-publication. Currently, TriTrypDB integrates datasets from Leishmania braziliensis, L. infantum, L. major, L. tarentolae, Trypanosoma brucei and T. cruzi. Users may examine individual genes or chromosomal spans in their genomic context, including syntenic alignments with other kinetoplastid organisms. Data within TriTrypDB can be interrogated utilizing a sophisticated search strategy system that enables a user to construct complex queries combining multiple data types. All search strategies are stored, allowing future access and integrated searches. ‘User Comments’ may be added to any gene page, enhancing available annotation; such comments become immediately searchable via the text search, and are forwarded to curators for incorporation into the reference annotation when appropriate.

Identification of human-infective trypanosomes in animal reservoir of sleeping sickness in Uganda by means of serum-resistance-associated (SRA) gene

BACKGROUND The expansion of sleeping sickness caused by Trypanosoma brucei rhodesiense beyond its traditional focus in southeast Uganda has been linked with large-scale livestock restocking. To assess the risk presented to the human population by domestic livestock, human-infective T b rhodesiense must be distinguished from non-human-infective T brucei brucei, since both parasites can be present in cattle. We investigated the use of a simple genetic marker to characterise parasites collected from cattle in villages within the new sleeping sickness focus in Soroti District, Uganda. METHODS 70 T brucei sl samples of known human infectivity status collected from human beings and cattle in Tororo District, Uganda, from 1989 to 1991 were screened for the presence of the human-serum-resistance-associated (SRA) gene by conventional PCR. In 2000-01, blood samples from 200 randomly selected cattle in six villages and two markets in Soroti District were screened for T brucei sl parasites by PCR; positive samples were screened for the presence of the SRA gene. FINDINGS The SRA gene was present in all 29 samples from patients with sleeping sickness in Tororo District. Of the 41 samples collected from cattle at the same time, the SRA gene was present in the eight samples that tested resistant to human serum in vitro, whereas it was absent from all 33 isolates that were sensitive to human serum in vitro. Of the 200 cattle sampled in Soroti District, we estimated that up to 18% (95% CI 12-23) were infected with T b rhodesiense. INTERPRETATION Detection of the SRA gene could provide the basis for a simple diagnostic test to enable targeted control of T b rhodesiense in the domestic livestock reservoir, thereby reducing the public-health burden of sleeping sickness in east Africa.

The trypanosome flagellar pocket

Trypanosomes are important disease agents and excellent models for the study of evolutionary cell biology. The trypanosome flagellar pocket is a small invagination of the plasma membrane where the flagellum exits the cytoplasm and participates in many cellular processes. It is the only site of exocytosis and endocytosis and part of a multiorganelle complex that is involved in cell polarity and cell division. Several flagellar pocket-associated proteins have been identified and found to contribute to trafficking and virulence. In this Review we discuss the contribution of the flagellar pocket to protein trafficking, immune evasion and other processes.

Sleeping sickness in Uganda: a thin line between two fatal diseases

Abstract Objective To determine, through the use of molecular diagnostic tools, whether the two species of parasite that cause human African trypanosomiasis have become sympatric. Design Blood sampling of all available patients between June 2001 and June 2005 in central Uganda and between July and September 2003 in northwest Uganda and analysis of subcounty sleeping sickness records in Uganda between 1985 and 2005. Setting Sleeping sickness treatment centres in central and northwest Uganda and in south Sudan. Participants Patients presenting at the treatment centres and diagnosed as having sleeping sickness. Main outcome measure Classification of parasites from patients from each disease focus as either Trypanosoma brucei rhodesiense (acute form) or T b gambiense (chronic form). Results Blood from 231 patients with sleeping sickness in central Uganda and from 91 patients with sleeping sickness in northwest Uganda and south Sudan were screened for T b rhodesiense (detection of SRA gene) and T b gambiense (detection of TgsGP gene). All samples from central Uganda were classified as T b rhodesiense, and all samples from northwest Uganda and south Sudan were identified as T b gambiense. Conclusions The two focuses of human African trypanosomiasis remain discrete, but the area of Uganda affected by the acute form of human sleeping sickness has increased 2.5-fold since 1985, spreading to three new districts within the past five years through movement of infected livestock. Without preventive action targeted at the livestock reservoir of this zoonotic disease, it is likely that the two disease focuses will converge. This will have a major impact on diagnosis and treatment of this neglected disease. Real time monitoring is recommended, using molecular diagnostic tools (at a regional surveillance centre, for example) targeted at both livestock and human patients.

GeneDB—an annotation database for pathogens

GeneDB (http://www.genedb.org) is a genome database for prokaryotic and eukaryotic pathogens and closely related organisms. The resource provides a portal to genome sequence and annotation data, which is primarily generated by the Pathogen Genomics group at the Wellcome Trust Sanger Institute. It combines data from completed and ongoing genome projects with curated annotation, which is readily accessible from a web based resource. The development of the database in recent years has focused on providing database-driven annotation tools and pipelines, as well as catering for increasingly frequent assembly updates. The website has been significantly redesigned to take advantage of current web technologies, and improve usability. The current release stores 41 data sets, of which 17 are manually curated and maintained by biologists, who review and incorporate data from the scientific literature, as well as other sources. GeneDB is primarily a production and annotation database for the genomes of predominantly pathogenic organisms.

Heat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169

In trypanosomes there is an almost total reliance on post-transcriptional mechanisms to alter gene expression; here, heat shock was used to investigate the response to an environmental signal. Heat shock rapidly and reversibly induced a decrease in polysome abundance, and the consequent changes in mRNA metabolism were studied. Both heat shock and polysome dissociation were necessary for (1) a reduction in mRNA levels that was more rapid than normal turnover, (2) an increased number of P-body-like granules that contained DHH1, SCD6 and XRNA, (3) the formation of stress granules that remained largely separate from the P-body-like granules and localise to the periphery of the cell and, (4) an increase in the size of a novel focus located at the posterior pole of the cell that contain XRNA, but neither DHH1 nor SCD6. The response differed from mammalian cells in that neither the decrease in polysomes nor stress-granule formation required phosphorylation of eIF2α at the position homologous to that of serine 51 in mammalian eIF2α and in the occurrence of a novel XRNA-focus.

Variant specific glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues

The complete amino acid sequences for nine variant specific glycoproteins (VSGs) of Trypanosoma brucei are presented. These have more than doubled the size of the VSG sequence data base and have enabled a new and more rigorous comparison to be made between amino acid sequences of different VSGs. Each VSG can be defined as a combination of an N-terminal domain type and a C-terminal domain type, based on the distribution of cysteine residues within the molecule. This identifies three N-terminal domain types and at least four C-terminal domain types. Different combinations of N and C-terminal domains can be formed; for example, in the sequences presented here, two different N-terminal domains are found in association with each of three different C-terminal domains. The biological context of the domain structure of VSGs is discussed.

A role for Caf1 in mRNA deadenylation and decay in trypanosomes and human cells

The eukaryotic Ccr4/Caf1/Not complex is involved in deadenylation of mRNAs. The Caf1 and Ccr4 subunits both potentially have deadenylating enzyme activity. We investigate here the roles of Ccr4 and Caf1 in deadenylation in two organisms that separated early in eukaryotic evolution: humans and trypanosomes. In Trypanosoma brucei, we found a complex containing CAF1, NOT1, NOT2 and NOT5, DHH1 and a possible homologue of Caf130; no homologue of Ccr4 was found. Trypanosome CAF1 has deadenylation activity, and is essential for cell survival. Depletion of trypanosome CAF1 delayed deadenylation and degradation of constitutively expressed mRNAs. Human cells have two isozymes of Caf1: simultaneous depletion of both inhibited degradation of an unstable reporter mRNA. In both species, depletion of Caf1 homologues inhibited deadenylation of bulk RNA and resulted in an increase in average poly(A) tail length.

Evolution of the primate trypanolytic factor APOL1

Significance African trypanosomes are parasites that can cause African sleeping sickness in humans. Humans and some primates, but not other mammals, have a gene called APOL1 that protects against certain trypanosomes. Genetic variants in APOL1 that arose in Africa are strongly associated with kidney disease in African Americans. These kidney disease-associated variants may have risen to high frequency in Africa because they can defend humans against a particularly pathogenic trypanosome. In this paper, we show how APOL1 has evolved by analyzing the distribution of these variants in Africa and then elucidating the molecular mechanisms that enhance their trypanosome killing capacity. We also show that these antitrypanosomal APOL1 variants may have adverse consequences for the host. ApolipoproteinL1 (APOL1) protects humans and some primates against several African trypanosomes. APOL1 genetic variants strongly associated with kidney disease in African Americans have additional trypanolytic activity against Trypanosoma brucei rhodesiense, the cause of acute African sleeping sickness. We combined genetic, physiological, and biochemical studies to explore coevolution between the APOL1 gene and trypanosomes. We analyzed the APOL1 sequence in modern and archaic humans and baboons along with geographic distribution in present day Africa to understand how the kidney risk variants evolved. Then, we tested Old World monkey, human, and engineered APOL1 variants for their ability to kill human infective trypanosomes in vivo to identify the molecular mechanism whereby human trypanolytic APOL1 variants evade T. brucei rhodesiense virulence factor serum resistance-associated protein (SRA). For one APOL1 kidney risk variant, a two-residue deletion of amino acids 388 and 389 causes a shift in a single lysine residue that mimics the Old World monkey sequence, which augments trypanolytic activity by preventing SRA binding. A second human APOL1 kidney risk allele, with an amino acid substitution that also restores sequence alignment with Old World monkeys, protected against T. brucei rhodesiense due in part to reduced SRA binding. Both APOL1 risk variants induced tissue injury in murine livers, the site of transgenic gene expression. Our study shows that both genetic variants of human APOL1 that protect against T. brucei rhodesiense have recapitulated molecular signatures found in Old World monkeys and raises the possibility that APOL1 variants have broader innate immune activity that extends beyond trypanosomes.

The cyclin A1-CDK2 complex regulates DNA double-strand break repair.

ABSTRACT Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and γ-irradiation which was mediated by p53. cyclin A1−/− cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1−/− cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.