Translin facilitates RNA polymerase II dissociation and suppresses genome instability during RNase H2- and Dicer-deficiency.

The conserved nucleic acid binding protein Translin contributes to numerous facets of mammalian biology and genetic diseases. It was first identified as a binder of cancer-associated chromosomal translocation breakpoint junctions leading to the suggestion that it was involved in genetic recombination. With a paralogous partner protein, Trax, Translin has subsequently been found to form a hetero-octomeric RNase complex that drives some of its functions, including passenger strand removal in RNA interference (RNAi). The Translin-Trax complex also degrades the precursors to tumour suppressing microRNAs in cancers deficient for the RNase III Dicer. This oncogenic activity has resulted in the Translin-Trax complex being explored as a therapeutic target. Additionally, Translin and Trax have been implicated in a wider range of biological functions ranging from sleep regulation to telomere transcript control. Here we reveal a Trax- and RNAi-independent function for Translin in dissociating RNA polymerase II from its genomic template, with loss of Translin function resulting in increased transcription-associated recombination and elevated genome instability. This provides genetic insight into the longstanding question of how Translin might influence chromosomal rearrangements in human genetic diseases and provides important functional understanding of an oncological therapeutic target.

Population genomic scan for candidate signatures of balancing selection to guide antigen characterization in malaria parasites.

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10_0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10_0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now prioritized for functional study.

Epigenetic dysregulation of virulence gene expression in severe Plasmodium falciparum malaria.

Chronic infections with the human malaria parasite Plasmodium falciparum depend on antigenic variation. P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major erythrocyte surface antigen mediating parasite sequestration in the microvasculature, is encoded in parasites by a highly diverse family of var genes. Antigenic switching is mediated by clonal variation in var expression, and recent in vitro studies have demonstrated a role for epigenetic processes in var regulation. Expression of particular PfEMP1 variants may result in parasite enrichment in different tissues, a factor in the development of severe disease. Here, we study in vivo human infections and provide evidence that infection-induced stress responses in the host can modify PfEMP1 expression via the perturbation of epigenetic mechanisms. Our work suggests that severe disease may not be the direct result of an adaptive virulence strategy to maximize parasite survival but that it may indicate a loss of control of the carefully regulated process of antigenic switching that maintains chronic infections.

Statistical estimation of cell-cycle progression and lineage commitment in Plasmodium falciparum reveals a homogeneous pattern of transcription in ex vivo culture.

We have cultured Plasmodium falciparum directly from the blood of infected individuals to examine patterns of mature-stage gene expression in patient isolates. Analysis of the transcriptome of P. falciparum is complicated by the highly periodic nature of gene expression because small variations in the stage of parasite development between samples can lead to an apparent difference in gene expression values. To address this issue, we have developed statistical likelihood-based methods to estimate cell cycle progression and commitment to asexual or sexual development lineages in our samples based on microscopy and gene expression patterns. In cases subsequently matched for temporal development, we find that transcriptional patterns in ex vivo culture display little variation across patients with diverse clinical profiles and closely resemble transcriptional profiles that occur in vitro. These statistical methods, available to the research community, assist in the design and interpretation of P. falciparum expression profiling experiments where it is difficult to separate true differential expression from cell-cycle dependent expression. We reanalyze an existing dataset of in vivo patient expression profiles and conclude that previously observed discrete variation is consistent with the commitment of a varying proportion of the parasite population to the sexual development lineage.

Distinct roles for FOXP3 and FOXP3 CD4 T cells in regulating cellular immunity to uncomplicated and severe Plasmodium falciparum malaria.

Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to pathogenesis of severe malaria. To determine whether this balance is maintained by classical regulatory T cells (CD4(+) FOXP3(+) CD127(-/low); Tregs) we compared cellular responses between Gambian children (n = 124) with severe Plasmodium falciparum malaria or uncomplicated malaria infections. Although no significant differences in Treg numbers or function were observed between the groups, Treg activity during acute disease was inversely correlated with malaria-specific memory responses detectable 28 days later. Thus, while Tregs may not regulate acute malarial inflammation, they may limit memory responses to levels that subsequently facilitate parasite clearance without causing immunopathology. Importantly, we identified a population of FOXP3(-), CD45RO(+) CD4(+) T cells which coproduce IL-10 and IFN-gamma. These cells are more prevalent in children with uncomplicated malaria than in those with severe disease, suggesting that they may be the regulators of acute malarial inflammation.

Erythrocyte invasion and merozoite ligand gene expression in severe and mild Plasmodium falciparum malaria.

Erythrocyte invasion is central to malaria parasite replication and virulence. Plasmodium falciparum parasites use different alternative erythrocyte receptors and vary in expression of erythrocyte-binding antigenic (EBA) proteins and reticulocyte-binding protein homologues (Rh). Parasite invasion phenotypes and schizont-stage transcript expression profiles of the 8 eba and Rh protein-coding genes without internal stop codons were determined for 163 clinical isolates cultured ex vivo in The Gambia. There was extensive diversity in ability to invade erythrocytes treated with neuraminidase, trypsin, or chymotrypsin, and severe malaria isolates were less restricted by trypsin treatment than were mild malaria isolates (P = .015). Expression profiles of the eba and Rh genes showed distinct clusters indicating coordinated alternative transcription. The most divergent of 5 major clusters was dominated by Rh2b, with virtually no expression of eba175 or eba140 genes (which were dominant in the other 4 clusters). Particular transcripts were significantly correlated with parasitemia (Rh5 was positively correlated and eba140 negatively correlated; P < .01 for both) and age of patients (eba181 was positively correlated and eba175 negatively correlated; P < .001 for both) but not with invasion phenotypes or severity of malaria. Severe and mild malaria isolates were also evenly represented across the different expression clusters.

Gene copy number variation throughout the Plasmodium falciparum genome.

BACKGROUND: Gene copy number variation (CNV) is responsible for several important phenotypes of the malaria parasite Plasmodium falciparum, including drug resistance, loss of infected erythrocyte cytoadherence and alteration of receptor usage for erythrocyte invasion. Despite the known effects of CNV, little is known about its extent throughout the genome. RESULTS: We performed a whole-genome survey of CNV genes in P. falciparum using comparative genome hybridisation of a diverse set of 16 laboratory culture-adapted isolates to a custom designed high density Affymetrix GeneChip array. Overall, 186 genes showed hybridisation signals consistent with deletion or amplification in one or more isolate. There is a strong association of CNV with gene length, genomic location, and low orthology to genes in other Plasmodium species. Sub-telomeric regions of all chromosomes are strongly associated with CNV genes independent from members of previously described multigene families. However, approximately 40% of CNV genes were located in more central regions of the chromosomes. Among the previously undescribed CNV genes, several that are of potential phenotypic relevance are identified. CONCLUSION: CNV represents a major form of genetic variation within the P. falciparum genome; the distribution of gene features indicates the involvement of highly non-random mutational and selective processes. Additional studies should be directed at examining CNV in natural parasite populations to extend conclusions to clinical settings.

Immuno-epidemiology of human Schistosoma haematobium infection: preferential IgG3 antibody responsiveness to a recombinant antigen dependent on age and parasite burden.

BACKGROUND: Schistosomiasis is a major parasitic disease affecting over 200 million people in the developing world with a further 400 million people at risk of infection. The aim of this study was to identify a single antigen from adult Schistosoma haematobium worms and subsequently use this antigen to study the development of schistosome-acquired immunity in a human population. METHODS: The full-length cDNA sequence of a S. haematobium protein, a putative orthologue of the S. mansoni tegumental antigen Sm13, was obtained from a cDNA library of adult S. haematobium worms and named Sh13 following a small-scale expressed sequence tags (EST) project. The recombinant Sh13 protein expressed in E. coli, was used to investigate immuno-epidemiological patterns in 147 Zimbabweans (7-18 years old) exposed to S. haematobium. RESULTS: Sequence analysis of the full-length cDNA sequence of the S. haematobium protein Sh13, indicated that the protein has an N-terminal signal peptide and encodes an 85-amino acid mature protein with a highly conserved predicted transmembrane domain (86 % identity with the S. mansoni tegumental antigen Sm13). The recombinant Sh13 protein was used in ELISA assays to determine the reactivity of sera from the study participants. Antibody responses against Sh13 were predominantly IgG3 isotype compared to responses against crude worm antigens which were predominantly IgG1 and IgG4. The relationship between anti-Sh13 IgG3 levels and infection intensity varied significantly with host age. The youngest children (7-10 years old) had relatively low levels of both infection and anti-Sh13 IgG3. In older children (11-12 years old) rising infection levels were accompanied by a significant increase in anti-Sh13 IgG3 levels. Subsequently, infection intensity declined significantly in 13-18 year olds but levels of the antibody continued to rise. The changing relationship between infection intensity and anti-Sh13 IgG3 levels with host age is consistent with the profile of a protective immune response predicted from theoretical work. CONCLUSION: We have identified and characterised a novel S. haematobium antigen Sh13, a putative tegumental protein, and shown that it is recognised predominantly by IgG3 antibodies from people infected with/exposed to S. haematobium parasites. We have also shown that, the anti-Sh13 IgG3 response is maximal in older individuals with the lowest infection intensity, and that the age profile of the relationship between anti-Sh13 IgG3 and infection intensity is consistent with that predicted by theoretical work for a protective response stimulated by and directed against adult worms.

Heterologous expression of the filarial nematode alt gene products reveals their potential to inhibit immune function.

BACKGROUND: Parasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Brugia malayi termed abundant larval transcript (alt), expression of which reaches ~5% of total transcript at the time parasites enter the human host. RESULTS: To test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania mexicana. We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro, reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo, and fewer mice were able to clear infection of organisms expressing ALT. alt-transfected parasites were more resistant to IFN-gamma-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection. CONCLUSION: Leishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells.

Translin and Trax differentially regulate telomere-associated transcript homeostasis.

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.

Abundant larval transcript-1 and -2 genes from Brugia malayi: diversity of genomic environments but conservation of 5' promoter sequences functional in Caenorhabditis elegans.

The genomic organisation of two abundant larval transcript (alt) genes from the filarial nematode Brugia malayi has been defined. The products of these genes are 78% identical in amino acid sequence, and are highly expressed in a stage-specific manner by mosquito-borne infective larvae. alt-1 is present as two near-identical copies organised in an inverted repeat of approximately 7.6 kb, occupying a total of 16 kb of the genome. alt-2 is a single-copy gene at a different locus to alt-1. The two alt-1 genes (alt-1.1 and -1.2) are 99.7% identical in coding sequence and 99.5% in intronic sequences. Both alt-1 and -2 contain 3 introns, and the third intron of alt-2 exhibits a size polymorphism evident in different individual parasites from the laboratory-maintained strain. Genomic sequence up- and down-stream from alt-1.1/1.2 (26 and 6 kb, respectively) and alt-2 (6 and 4 kb, respectively) show that neither gene is in a multiple array or an operon. Most notably, the neighbouring genes of alt-1 and -2 show no similarity to each other, or to the genes flanking the distant alt homologue in Caenorhabditis elegans. Despite this diversity in flanking genes, the 5' UTR tracts extending some 800 bp upstream of each B. malayi alt gene show a high degree of similarity (overall 59% identity with tracts of 77-86% identity). Surmising that this region may contain conserved promoter elements, constructs containing the B. malayi alt 5' UTR with or without coding sequence were made fused to beta-galactosidase reporter protein. These constructs were injected into the syncytical gonad of C. elegans and progeny stained for beta-gal expression. Our results show relatively strong expression in the gut cells of C. elegans for both alt-1 and -2 constructs, commencing in larval worms and continuing into adulthood. Moreover, expression was enhanced when constructs contained segments of alt-1 coding and intronic sequence in addition to the 5' UTR. We conclude that the high level of alt transcription in filarial L3s is not due to expression from a multi-copy gene family but to a set of strong promoter elements shared between the two alt genes.

Continued decline of malaria in The Gambia with implications for elimination.

BACKGROUND: A substantial decline in malaria was reported to have occurred over several years until 2007 in the western part of The Gambia, encouraging consideration of future elimination in this previously highly endemic region. Scale up of interventions has since increased with support from the Global Fund and other donors. METHODOLOGY/PRINCIPAL FINDINGS: We continued to examine laboratory records at four health facilities previously studied and investigated six additional facilities for a 7 year period, adding data from 243,707 slide examinations, to determine trends throughout the country until the end of 2009. We actively detected infections in a community cohort of 800 children living in rural villages throughout the 2008 malaria season, and assayed serological changes in another rural population between 2006 and 2009. Proportions of malaria positive slides declined significantly at all of the 10 health facilities between 2003 (annual mean across all sites, 38.7%) and 2009 (annual mean, 7.9%). Statistical modelling of trends confirmed significant seasonality and decline over time at each facility. Slide positivity was lowest in 2009 at all sites, except two where lowest levels were observed in 2006. Mapping households of cases presenting at the latter sites in 2007-2009 indicated that these were not restricted to a few residual foci. Only 2.8% (22/800) of a rural cohort of children had a malaria episode in the 2008 season, and there was substantial serological decline between 2006 and 2009 in a separate rural area. CONCLUSIONS: Malaria has continued to decline in The Gambia, as indicated by a downward trend in slide positivity at health facilities, and unprecedented low incidence and seroprevalence in community surveys. We recommend intensification of control interventions for several years to further reduce incidence, prior to considering an elimination programme.

Quantitative detection of Plasmodium falciparum DNA in saliva, blood, and urine.

BACKGROUND: Current methods for detecting malaria parasites are invasive and associated with poor compliance when repeated sampling is required. New methods to detect and quantify parasites in a less-invasive manner would greatly enhance the potential for longitudinal surveillance in clinical trials. METHODS: Saliva, urine, and blood samples from 386 Gambian outpatients with suspected malaria infections were analyzed by nested polymerase chain reaction (nPCR) to detect infection and to evaluate diagnostic accuracy in comparison to expert microscopy. The amount of parasite DNA in malaria-positive samples was estimated using real-time quantitative PCR (qPCR). RESULTS: Blood parasite density as estimated by qPCR correlated well with parasite counts established by microscopy (p = 0.94; P < .001). qPCR results for saliva had a significant correlation with microscopy counts (p = 0.58; P < .001), whereas qPCR results for urine had a positive but poor correlation with microscopy counts (p = 0.20; P = .117). The mean amounts of parasite DNA quantified in blood were greater than the mean amounts quantified in saliva and urine samples obtained concurrently from the same individual, by approximately 600-fold and approximately 2500-fold, respectively. When nPCR results were compared with microscopy results, nPCR of saliva had a sensitivity of 73% and a specificity of 97%; its sensitivity increased to 82% in samples with a parasite density of > or = 1000 parasites/microL. nPCR of urine had a sensitivity of 32% and a specificity of 98%. CONCLUSION: Saliva sampling is a promising less-invasive approach for detecting malaria infection.

C-type lectins from the nematode parasites Heligmosomoides polygyrus and Nippostrongylus brasiliensis.

The C-type lectin superfamily is highly represented in all metazoan phyla so far studied. Many members of this superfamily are important in innate immune defences against infection, while others serve key developmental and structural roles. Within the superfamily, many proteins contain multiple canonical carbohydrate-recognition domains (CRDs), together with additional non-lectin domains. In this report, we have studied two gastrointestinal nematode parasites which are widely used in experimental rodent systems, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. From cDNA libraries, we have isolated 3 new C-type lectins from these species; all are single-CRD proteins with short additional N-terminal domains. The predicted Hp-CTL-1 protein contains 156 aa, Nb-CTL-1 191 aa and Nb-CTL-2 183 aa; all encode predicted signal peptides, as well as key conserved sequence motifs characteristic of the CTL superfamily. These lectins are most similar to C. elegans CLEC-48, 49 and 50, as well as to the lectin domains of mammalian immune system proteins CD23 and CD206. RT-PCR showed that these H. polygyrus and N. brasiliensis genes are primarily expressed in the gut-dwelling adult stages, although Nb-CTL-2 transcripts are also prominent in the free-living infective larval (L3) stage. Polyclonal antibodies raised to Hp-CTL-1 and Nb-CTL-1 reacted to both proteins by ELISA, and in Western blot analysis recognised a 15-kDa band in secreted proteins of adult N. brasiliensis (NES) and a 19-kDa band in H. polygyrus ES (HES). Anti-CTL-1 antibody also bound strongly to the cuticle of adult H. polygyrus. Hence, live parasites release C-type lectins homologous to some key receptors of the mammalian host immune system, raising the possibility that these products interfere in some manner with immunological recognition or effector function.

Helminth parasites--masters of regulation.

Immune regulation by parasites is a global concept that includes suppression, diversion, and conversion of the host immune response to the benefit of the pathogen. While many microparasites escape immune attack by antigenic variation or sequestration in specialized niches, helminths appear to thrive in exposed extracellular locations, such as the lymphatics, bloodstream, or gastrointestinal tract. We review here the multiple layers of immunoregulation that have now been discovered in helminth infection and discuss both the cellular and the molecular interactions involved. Key events among the host cell population are dominance of the T-helper 2 cell (Th2) phenotype and the selective loss of effector activity, against a background of regulatory T cells, alternatively activated macrophages, and Th2-inducing dendritic cells. Increasingly, there is evidence of important effects on other innate cell types, particularly mast cells and eosinophils. The sum effect of these changes to host reactivity is to create an anti-inflammatory environment, which is most favorable to parasite survival. We hypothesize therefore that parasites have evolved specific molecular strategies to induce this conducive landscape, and we review the foremost candidate immunomodulators released by helminths, including cytokine homologs, protease inhibitors, and an intriguing set of novel products implicated in immune suppression.