Functional genomics of parasitic worms: the dawn of a new era

The study of gene function in parasitic worms is technically demanding due to difficulties associated with life-cycle propagation and, hence, molecular genetics. Exploitation of the free-living nematode, Caenorhabditis elegans, coupled with recent major advances in molecular studies of parasitic nematodes, have opened up new avenues for understanding the biology of these parasites and present opportunities for novel strategies of therapeutic intervention and control.     

Toward next-generation sequencing of mitochondrial genomes — Focus on parasitic worms of animals and biotechnological implications

Helminths (worms) include parasitic nematodes (roundworms) and platyhelminths (flatworms). These worms are abundant, and many of them are of agricultural, aquacultural, veterinary and medical importance and cause substantial socioeconomic losses worldwide. The genetic characterization of parasitic nematodes using advanced molecular tools is central to the diagnosis of infections and the control of parasitism. The accurate analysis of genetic variation also underpins studies of their taxonomy, epidemiology and evolutionary history. Although the nuclear genome contains suitable genetic markers (e.g., in ribosomal DNA) for the identification of many species, the large size and high variability of the mt genome consistently provides a rich source of such markers for informative systematic and epidemiological studies both within and among species. There is significant value in establishing a practical platform for the rapid sequencing, annotation and analysis of mt genomic datasets to underpin such fundamental and applied studies of parasitic worms (= helminths). In the last decade, there have been some important advances in the mt genomics of helminths, but next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation. In this article, we provide a background on mt genomics, cover technological challenges and recent advances, and provide a perspective on future mt genome research of parasitic helminths and its fundamental scientific and biotechnological implications.     

Approaches to functional genomics in filamentous fungi

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.     

Taxonomic and geographic bias in the genetic study of helminth parasites

The use of genetic information is now fundamental in parasite taxonomy and systematics, for resolving parasite phylogenies, discovering cryptic species, and elucidating patterns of gene flow among parasite populations. The accumulation of available gene sequences per geographical area or per parasite taxonomic group is likely proportional to species richness, but not without some biases. Certain areas and certain taxonomic groups receive more research effort than others, possibly causing a deficit in the relative number of parasite species being characterized genetically in some areas or taxonomic groups. Here, we use data on the number of parasite records per country or helminth family from the London Natural History Museum host-parasite database, and matching data on the number of gene sequences available from the National Center for Biotechnology Information (NCBI) GenBank database, to determine how available gene sequences scale with species richness across countries or parasitic helminth families. Our quantitative analysis identified countries/regions of the world and helminth families that have received the most effort in genetic research. More importantly, it allowed us to generate lists (based on residuals from the statistical model) of the 20 countries/regions and the 20 helminth families with the largest deficit in available gene sequences relative to their helminth species richness. We propose these lists as useful guides toward future allocation of effort to maximise advances in parasite biodiscovery, systematics and population structure.     

CRISPR/Cas9: A new tool for the study and control of helminth parasites

Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9‐targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9‐mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.     

Gene manipulation in parasitic helminths

In light of recent growth in available DNA sequence information for a number of parasitic helminths, it is crucial that suitable gene manipulation technologies are developed to facilitate functional genomic studies in these organisms. In this review we discuss recent progress in the development of these technologies in nematode and platyhelminth parasites of medical and veterinary importance. Specifically, the current status of transient transfection, double-stranded RNA interference and antisense RNA as viable techniques for the manipulation of parasitic helminth gene expression is presented. In addition, the potential for the development of stable, or germ-line, transformation methods in these organisms is also discussed.     

Adenylyl and guanylyl cyclases from the malaria parasite Plasmodium falciparum

Completion of several malaria parasite genome sequences and advances in Plasmodium gene manipulation technology, will lead to significant advances in our knowledge of the biology of these organisms. Biochemical analysis of the cyclic nucleotide signalling pathways of P. falciparum has provided important information on malaria parasite development. The Plasmodium purine nucleotide cyclase enzymes have extremely unusual structures and the regulatory mechanisms controlling parasite enzyme activity are distinct from those operating on the analogous host molecules. Study of these enzymes could therefore lead to novel strategies for anti-malarial intervention in addition to providing unique insights into the intriguing biology of the parasite.     

Trypanosomatid comparative genomics: Contributions to the study of parasite biology and different parasitic diseases

In 2005, draft sequences of the genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, also known as the Tri-Tryp genomes, were published. These protozoan parasites are the causative agents of three distinct insect-borne diseases, namely sleeping sickness, Chagas disease and leishmaniasis, all with a worldwide distribution. Despite the large estimated evolutionary distance among them, a conserved core of ~6,200 trypanosomatid genes was found among the Tri-Tryp genomes. Extensive analysis of these genomic sequences has greatly increased our understanding of the biology of these parasites and their host-parasite interactions. In this article, we review the recent advances in the comparative genomics of these three species. This analysis also includes data on additional sequences derived from other trypanosmatid species, as well as recent data on gene expression and functional genomics. In addition to facilitating the identification of key parasite molecules that may provide a better understanding of these complex diseases, genome studies offer a rich source of new information that can be used to define potential new drug targets and vaccine candidates for controlling these parasitic infections.     

Parelaphostrongylus odocoilei in Columbian black-tailed deer from Oregon

Documenting the occurrence of Parelaphostrongylus odocoilei has historically relied on the morphological examination of adult worms collected from the skeletal muscle of definitive hosts, including deer. Recent advances in the knowledge of protostrongylid genetic sequences now permit larvae to be identified. Dorsal-spined larvae (DSLs) collected in 2003-2004 from the lung and feces of six Columbian black-tailed deer (Odocoileus hemionus columbianus) from Oregon were characterized genetically. The sequences from unknown DSLs were compared to those from morphologically validated adults and larvae of P. odocoilei at both the second internal transcribed spacer (ITS-2) of ribosomal DNA and the mitochondrial cytochrome oxidase II gene. We provide the first unequivocal identification of P. odocoilei in Columbian black-tailed deer from Oregon. The broader geographic distribution, prevalence, and pathology of P. odocoilei are not known in populations of Oregon deer.     

Synthetic biology of secondary metabolite biosynthesis in actinomycetes: Engineering precursor supply as a way to optimize antibiotic production

Actinomycetes are a rich source for the synthesis of medically and technically useful natural products. The genes encoding the enzymes for their biosynthesis are normally organized in gene clusters, which include also the information for resistance (in the case of antibacterial compounds), regulation, and transport. This facilitates the manipulation of such pathways by molecular genetic techniques. Recent advances in DNA sequencing and analytical chemistry revealed that not only new strains isolated from yet unexplored habitats, but also already known strains possess a large potential for the synthesis of novel compounds. Synthetic Biology now offers a new perspective to exploit this potential further by generating novel pathways, and thereby novel products, by combining different biosynthetic steps originating from different bacteria. The supply of precursors, which are subsequently incorporated into the final product, is often already organized in a modular manner in nature and may directly be exploited for Synthetic Biology. Here we report examples for the synthesis of building blocks and possibilities to modify and optimize antibiotic biosynthesis, exemplary for the synthesis of the manipulation of the synthesis of the glycopeptide antibiotic balhimycin.     

The immunobiology of schistosomiasis

Schistosomes are parasitic worms that are a prime example of a complex multicellular pathogen that flourishes in the human host despite the development of a pronounced immune response. Understanding how the immune system deals with such pathogens is a daunting challenge. The past decade has seen the use of a wide range of new approaches to determine the nature and function of the immune response to schistosomes. Here, we attempt to summarize advances in our understanding of the immunology of schistosomiasis, with the bulk of the review reflecting the experimental focus on Schistosoma mansoni infection in mice.     

Rapidly evolving lineages impede the resolution of phylogenetic relationships among Clitellata (Annelida)

The phylogenetic relationships of the Clitellata were investigated using a data set with published and new complete or partial 18S rRNA and mtCOI gene sequences of 13 and 49 taxa representing 8 and 14 families, respectively. Three different alignments were considered for 18S, and the possible influence of departures from rate constancy among sites was evaluated by analyses using a Gamma model of rate heterogeneity. Maximum-likelihood estimates of the shape parameter alpha of the Gamma distribution were very low, whatever the alignment or the gene considered, suggesting that phylogenetic reconstructions taking into account the rate heterogeneity among sites are likely to be the most reliable. Analyzed separately, the two genes did not resolve the relationships among the Clitellata, but the consensus tree was congruent with the morphology-based relationships. Our data suggest the inclusion of the Euhirudinea, Acanthobdellida, and Branchiobdellida in the Oligochaeta and suggest the Lumbriculidae as the link between both assemblages. Although separate analyses of both genes, as well as different alignments for the 18S rRNA sequences, yielded conflicting results concerning the phylogenetic position of leeches and leech-like worms vis-à-vis the Oligochaeta, subsequent analyses using the Gamma model greatly reduced the observed inconsistencies. Our analyses show that among the Clitellata, the leeches and the leech-like and gutless worms represent significantly faster evolving lineages. It is suggested that the observed higher mutation rates may be explained by the fact that these lineages contain almost exclusively commensal and/or parasitic taxa.     

Pattern recognition molecules and innate immunity to parasites

Recent pioneering advances in understanding how plants, insects and worms eliminate pathogens has led to the realization that innate immunity plays a vital role in protecting humans from infection. This comprehensive review examines the molecules involved in innate immune responses, how they act to control parasites and if their engagement can explain many immune features characteristic of parasitic infections.     

Chemistry,manufacturing and controls for gene modified hematopoietic stem cells

Gene modification of hematopoietic stem cells is increasingly becoming popular as a therapeutic approach, given the recent approvals and the number of new applications for clinical trials targeting monogenetic and immunodeficiency disorders. Technological advances in stem cell selection, culture, transduction and gene editing now allow for efficient ex vivo genetic manipulation of stem cells. Gene-addition techniques using viral vectors (mainly retrovirus- and lentivirus-based) and gene editing using various targeted nuclease platforms (e.g., Zinc finger, TALEN and Crispr/Cas9) are being applied to the treatment of multiple genetic and immunodeficiency disorders. Herein, the current state of the art in manufacturing and critical assays that are required for ex vivo manipulation of stem cells are addressed. Important quality control and safety assays that need to be planned early in the process development phase of these products for regulatory approval are also highlighted.     

Advances in QTL mapping in pigs

Over the past 15 years advances in the porcine genetic linkage map and discovery of useful candidate genes have led to valuable gene and trait information being discovered. Early use of exotic breed crosses and now commercial breed crosses for quantitative trait loci (QTL) scans and candidate gene analyses have led to 110 publications which have identified 1,675 QTL. Additionally, these studies continue to identify genes associated with economically important traits such as growth rate, leanness, feed intake, meat quality, litter size, and disease resistance. A well developed QTL database called PigQTLdb is now as a valuable tool for summarizing and pinpointing in silico regions of interest to researchers. The commercial pig industry is actively incorporating these markers in marker-assisted selection along with traditional performance information to improve traits of economic performance. The long awaited sequencing efforts are also now beginning to provide sequence available for both comparative genomics and large scale single nucleotide polymorphism (SNP) association studies. While these advances are all positive, development of useful new trait families and measurement of new or underlying traits still limits future discoveries. A review of these developments is presented.     

Advances in genetic engineering of the avian genome: “Realising the promise”

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express “humanised” antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.     

Genome-wide high-throughput screens in functional genomics

The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function. Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals. Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function. Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells. Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms. Integration of such data with other genomic approaches will extend our understanding of cellular networks.