Detecting signatures of balancing selection to identify targets of anti-parasite immunity.

Parasite antigen genes might evolve under frequency-dependent immune selection. The distinctive patterns of polymorphism that result can be detected using population genetic methods that test for signatures of balancing selection, allowing genes encoding important targets of immunity to be identified. Analyses can be complicated by population structures, histories and features of a parasite's genome. However, new sequencing technologies facilitate scans of polymorphism throughout parasite genomes to identify the most exceptional gene specific signatures. We focus on malaria parasites to illustrate challenges and opportunities for detecting targets of frequency-dependent immune selection to discover new potential vaccine candidates

The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation

BACKGROUND: Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion. RESULTS: We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains. CONCLUSIONS: Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens

Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters

Background: The filamentous fungus Aspergillus nidulans has been a tractable model organism for cell biology and genetics for over 60 years. It is among a large number of Aspergilli whose genomes have been sequenced since 2005, including medically and industrially important species. In order to advance our knowledge of its biology and increase its utility as a genetic model by improving gene annotation we sequenced the transcriptome of A. nidulans with a focus on 5' end analysis. Results: Strand-specific whole transcriptome sequencing showed that 80-95% of annotated genes appear to be expressed across the conditions tested. We estimate that the total gene number should be increased by approximately 1000, to 11,800. With respect to splicing 8.3% of genes had multiple alternative transcripts, but alternative splicing by exon-skipping was very rare. 75% of annotated genes showed some level of antisense transcription and for one gene, meaB, we demonstrated the antisense transcript has a regulatory role. Specific sequencing of the 5' ends of transcripts was used for genome wide mapping of transcription start sites, allowing us to interrogate over 7000 promoters and 5' untranslated regions. Conclusions: Our data has revealed the complexity of the A. nidulans transcriptome and contributed to improved genome annotation. The data can be viewed on the AspGD genome browser

Old World cutaneous leishmaniasis treatment response varies depending on parasite species, geographical location and development of secondary infection

Background: In the Kingdom of Saudi Arabia (KSA), Leishmania major and L. tropica are the main causative agents of Old World cutaneous leishmaniasis (CL). The national CL treatment regimen consists of topical 1% clotrimazole/2% fusidic acid cream followed by 1–2 courses of intralesional sodium stibogluconate (SSG); however, treatment efficacy is highly variable and the reasons for this are not well understood. In this study, we present a complete epidemiological map of CL and determined the efficacy of the standard CL treatment regime in several endemic regions of KSA.Results: Overall, three quarters of patients in all CL-endemic areas studied responded satisfactorily to the current treatment regime, with the remaining requiring only an extra course of SSG. The majority of unresponsive cases were infected with L. tropica. Furthermore, the development of secondary infections (SI) around or within the CL lesion significantly favoured the treatment response of L. major patients but had no effect on L. tropica cases.Conclusions: The response of CL patients to a national treatment protocol appears to depend on several factors, including Leishmania parasite species, geographical location and occurrences of SI. Our findings suggest there is a need to implement alternative CL treatment protocols based on these parameters

Evidence of Gene Conversion in Genes Encoding the Gal/GalNac Lectin Complex of Entamoeba

The human gut parasite Entamoeba histolytica, uses a lectin complex on its cell surface to bind to mucin and to ligands on the intestinal epithelia. Binding to mucin is necessary for colonisation and binding to intestinal epithelia for invasion, therefore blocking this binding may protect against amoebiasis. Acquired protective immunity raised against the lectin complex should create a selection pressure to change the amino acid sequence of lectin genes in order to avoid future detection. We present evidence that gene conversion has occurred in lineages leading to E. histolytica strain HM1:IMSS and E. dispar strain SAW760. This evolutionary mechanism generates diversity and could contribute to immune evasion by the parasites

The Entamoeba lysine and glutamic acid rich protein (KERP1) virulence factor gene is present in the genomes of Entamoeba nuttalli, Entamoeba dispar and Entamoeba moshkovskii

The lysine and glutamic acid rich protein KERP1 is a cell surface-expressed virulence factor in the human pathogen Entamoeba histolytica. It was originally suggested that the gene was absent from the related, avirulent human commensal Entamoeba dispar, an absence which would be relevant to the differential virulence of these species. Here, the gene is shown to be present in E. dispar, and its sequence is presented, as well as in a virulent parasite of macaques, Entamoeba nuttalli, and the primarily free living, opportunistically parasitic Entamoeba moshkovskii

DNA barcoding of nematodes using the MinION

Many nematode species are parasitic and threaten the health of plants and animals, including humans, on a global scale. Advances in DNA sequencing techniques have allowed for the rapid and accurate identification of many organisms including nematodes. However, the steps taken from sample collection in the field to molecular analysis and identification can take many days and depend on access to both immovable equipment and a specialized laboratory. Here, we present a protocol to genetically identify nematodes using 18S SSU rRNA sequencing using the MinION, a portable third generation sequencer, and proof that it is possible to perform all the molecular preparations on a fully portable molecular biology lab – the Bentolab. We show that both parasitic and free-living nematode species (Anisakis simplex, Panagrellus redivivus, Turbatrix aceti, and Caenorhabditis elegans) can be identified with a 96–100% accuracy compared to Sanger sequencing, requiring only 10–15 min of sequencing. This protocol is an essential first step toward genetically identifying nematodes in the field from complex natural environments (such as feces, soil, or marine sediments). This increased accessibility could in turn improve global information of nematode presence and distribution, aiding near-real-time global biomonitoring

Characterization of the glutathione S-transferase genes in the sand flies Phlebotomus papatasi and Lutzomyia longipalpis shows expansion of the novel glutathione S-transferase xi (X) class

Leishmaniasis control often relies upon insecticidal control of phlebotomine sandfly vector populations. Such methods are vulnerable to the evolution of insecticide resistance via a range of molecular mechanisms. There is evidence that two major resistance mechanisms, target site insensitivity and metabolic resistance, have evolved in some sandfly populations and further genetic characterization of resistance would be useful to understand and combat it. To facilitate the study of the mechanisms of metabolic resistance, here we improved the annotation and characterized a major detoxification gene family, the glutathione-s-transferases (GST), in the genomes of two sand fly species: Phlebotomus papatasi and Lutzomyia longipalpis. The compositions of the GST gene family differ markedly from those of Aedes and Anopheles mosquitoes. Most strikingly, the xi (X) class of GSTs appears to have expanded in both sand fly genomes. Our results provide a basis for further studies of metabolic resistance mechanisms in these important disease vector species

Morphotypes of the common beadlet anemone Actinia equina (L.) are genetically distinct

Anemones of the genus Actinia are ecologically important and familiar organisms on many rocky shores. However, this genus is taxonomically problematical and prior evidence suggests that the North Atlantic beadlet anemone, Actinia equina, may actually consist of a number of cryptic species. Previous genetic work has been largely limited to allozyme electrophoresis and there remains a dearth of genetic resources with which to study this genus. Mitochondrial DNA sequencing may help to clarify the taxonomy of Actinia. Here, the complete mitochondrial genome of the beadlet anemone Actinia equina (Cnidaria: Anthozoa: Actinaria: Actiniidae) is shown to be 20,690 bp in length and to contain the standard complement of Cnidarian features including 13 protein coding genes, two rRNA genes, two tRNAs and two Group I introns, one with an in-frame truncated homing endonuclease gene open reading frame. However, amplification and sequencing of the standard mtDNA barcoding region of the cytochrome oxidase I gene revealed only two haplotypes, differing by a single base pair, in widely geographically separated A. equina and its congener A. prasina. COI barcoding shows that whilst A. equina and A. prasina share the common mtDNA haplotype, haplotype frequency differed significantly between A. equina with red/orange pedal discs and those with green pedal discs, consistent with the hypothesis that these morphotypes represent incipient species