Molecular Parasitology Research Articles

Molecular confirmation of Hymenolepis hibernia in field mice (Apodemus sylvaticus) from St Kilda has potential to resolve a host-parasite relationship.

Insular wildlife populations provide opportunities to examine biological questions in systems that are relatively closed and potentially tractable, striking examples being the long-term studies of ecology and evolution in the red deer and feral sheep populations on the Hebridean islands of Rum and St Kilda. In the case of parasitology, Understanding of parasitic infections insular wildlife populations in conjunction with knowledge of their origins has the potential to add a fresh perspective to disease control in humans and domestic animals. In the case of parasitology, understanding infections of insular wildlife populations, in conjunction with knowledge of their origins, has the potential to add a fresh perspective to disease control in humans and domestic animals. With this in mind, gross and molecular examination for the presence of cyclophyllidean tapeworms was performed on the viscera and rectal contents of 17 preserved specimens of Apodemus sylvaticus field mice and on the naturally voided faeces of a further four mice on the remote archipelago of St Kilda. Molecular speciation of hexacanth embryos extracted from the faeces of two mice, using nucleotide sequence analysis of the ribosomal cytochrome c-oxidase subunit-1, confirmed infection with Hymenolepis hibernia. Phylogenetic analysis showed that these were genetically distinct from Hymenolepis diminuta, previously reported in the insular A. sylvaticus mice, and from other published H. hibernia haplotypes. There was insufficient hymenolepidid tapeworm phylogeographic variation to resolve the origins of the co-evolved St Kilda mice, primarily due to a lack of published H. hibernia Cox-1 sequence data across the parasite's geographical range. Nevertheless, the Maximum Likelihood haplotype tree shows the potential for molecular parasitology to resolve a host-parasite relationship once more data become available. Morphological diagnostic features of zoonotic H. hibernia eggs are also described.

Computing the Diameter of the Space of Maximum Parsimony Reconciliations in the Duplication-Transfer-Loss Model

Phylogenetic tree reconciliation is widely used in the fields of molecular evolution, cophylogenetics, parasitology, and biogeography to study the evolutionary histories of pairs of entities. In these contexts, reconciliation is often performed using maximum parsimony under the Duplication-Transfer-Loss (DTL) event model. In general, the number of maximum parsimony reconciliations (MPRs) can grow exponentially with the size of the trees. While a number of previous efforts have been made to count the number of MPRs, find representative MPRs, and compute the frequencies of events across the space of MPRs, little is known about the structure of MPR space. In particular, how different are MPRs in terms of the events that they comprise? One way to address this question is to compute the diameter of MPR space, defined to be the maximum number of DTL events that distinguish any two MPRs in the solution space. We show how to compute the diameter of MPR space in polynomial time and then apply this algorithm to a large biological dataset to study the variability of events.

Parasites! Graphic Exploration of Tropical Disease Drug Development.

Parasites!, a 2010 comic sponsored by the Wellcome Trust Centre for Molecular Parasitology, demonstrates that a graphic narrative can play a role in energizing public debate. Part of the genre known as graphic medicine-comics about illness, treatment, disability, and caregiving-Parasites! is intended to educate readers of all ages about illnesses less known in the developed world. Two visual strategies in particular enable the comic to offer an alternative and aesthetic response to questions about developing drugs to treat tropical diseases for profit. By including visuals and text, and not just one of these formats, viewers must reorient themselves aesthetically and epistemologically to ethical, social, cultural, and political structures that adversely affect human health.

Corrigendum.

Parasite ImmunologyVolume 40, Issue 1 e12504 CORRIGENDUMFree Access Corrigendum This article corrects the following: Evaluating the potential of a new isotope-labelled glyco-ligand for estimating the remnant liver function of schistosoma-infected mice P.-C. Cheng, P.-F. Chiang, K.-M. Lee, C.-H. Yeh, K.-L. Hsu, S.-W. Liu, L.-H. Shen, C.-L. Peng, C.-K. Fan, T.-Y. Luo, Volume 35Issue 3-4Parasite Immunology pages: 129-139 First Published online: February 5, 2013 First published: 21 December 2017 https://doi.org/10.1111/pim.12504AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL In the article “Evaluating the potential of a new isotope-labelled glyco-ligand for estimating the remnant liver function of schistosoma-infected mice,” it has been noted that the first affiliation which appeared at the beginning of the article was incorrect. The correct first affiliation for the article is shown below. 1Department of Molecular Parasitology and Tropical Diseases, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan We apologize for any inconvenience caused. REFERENCE 1Cheng P-C, Chiang P-F, Lee K-M, et al. Evaluating the potential of a new isotope-labelled glyco-ligand for estimating the remnant liver function of schistosoma-infected mice. Parasite Immunol. 2013; 35: 129- 139. https://doi.org/10.1111/pim.12022. Volume40, Issue1January 2018e12504 ReferencesRelatedInformation

Transporters of Trypanosoma brucei-phylogeny, physiology, pharmacology.

Trypanosoma brucei comprise the causative agents of sleeping sickness, T. b. gambiense and T. b. rhodesiense, as well as the livestock-pathogenic T. b. brucei. The parasites are transmitted by the tsetse fly and occur exclusively in sub-Saharan Africa. T. brucei are not only lethal pathogens but have also become model organisms for molecular parasitology. We focus here on membrane transport proteins of T. brucei, their contribution to homeostasis and metabolism in the context of a parasitic lifestyle, and their pharmacological role as potential drug targets or routes of drug entry. Transporters and channels in the plasma membrane are attractive drug targets as they are accessible from the outside. Alternatively, they can be exploited to selectively deliver harmful substances into the trypanosome's interior. Both approaches require the targeted transporter to be essential: in the first case to kill the trypanosome, in the second case to prevent drug resistance due to loss of the transporter. By combining functional and phylogenetic analyses, we were mining the T. brucei predicted proteome for transporters of pharmacological significance. Here, we review recent progress in the identification of transporters of lipid precursors, amino acid permeases and ion channels in T. brucei.

Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.

Taming Parasites by Tailoring Them

The next-generation gene editing based on CRISPR (clustered regularly interspaced short palindromic repeats) has been successfully implemented in a wide range of organisms including some protozoan parasites. However, application of such a versatile game-changing technology in molecular parasitology remains fairly underexplored. Here, we briefly introduce state-of-the-art in human and mouse research and usher new directions to drive the parasitology research in the years to come. In precise, we outline contemporary ways to embolden existing apicomplexan and kinetoplastid parasite models by commissioning front-line gene-tailoring methods, and illustrate how we can break the enduring gridlock of gene manipulation in non-model parasitic protists to tackle intriguing questions that remain long unresolved otherwise. We show how a judicious solicitation of the CRISPR technology can eventually balance out the two facets of pathogen-host interplay.

How Antiparasitic Drugs Work-And Sometimes Stop Working!

How Antiparasitic Drugs Work-And Sometimes Stop Working!

Update on pathology of ocular parasitic disease.

Parasites are a group of eukaryotic organisms that may be free-living or form a symbiotic or parasitic relationship with the hosts. Consisting of over 800,000 recognized species, parasites may be unicellular (Protozoa) or multicellular (helminths and arthropods). The association of parasites with human population started long before the emergence of civilization. Parasitic zoonotic diseases are prevalent worldwide including India. Appropriate epidemiological data are lacking on existing zoonotic parasitic diseases, and newer diseases are emerging in our scenario. Systemic diseases such as cysticercosis, paragonimiasis, hydatidosis, and toxoplasmosis are fairly common. Acquired Toxoplasma infections are rising in immune-deficient individuals. Amongst the ocular parasitic diseases, various protozoas such as Cystoidea, trematodes, tissue flagellates, sporozoas etc. affect humans in general and eyes in particular, in different parts of the world. These zoonoses seem to be a real health related problem globally. Recent intensification of research throughout the world has led to specialization in biological fields, creating a conducive situation for researchers interested in this subject. The basics of parasitology lie in morphology, pathology, and with recent updates in molecular parasitology, the scope has extended further. The current review is to address the recent update in ophthalmic parasites with special reference to pathology and give a glimpse of further research in this field.

Macromolecular crystallography and what it can contribute to antiparasite drug discovery.

This issue of Acta Crystallographica Section F is dedicated towards structural investigations on proteins associated with molecular parasitology, specifically research linked to protozoan pathogens. It is in line with the previous special sections or issues contributed by the RIKEN-UK structural genomics consortium (December 2009), the Joint Center for Structural Genomics in October 2010 (Weiss & Einspahr, 2010 ▶) and the Seattle Structural Genomics Center for Infectious Diseases in September 2011 (Weiss & Einspahr, 2011 ▶). In contrast to the previous issues however, it is thematically much more focused on protozoan pathogens. Interest in these primitive eukaryotic organisms is often to exploit them as model systems and many significant advances have resulted from such research. As examples there are the fundamentally important discoveries relating to RNA editing, antigenic variation and the occurrence of glycosylphosphatidylinositol tags that anchor proteins on the cell surface, all accrued from research with the African trypanosome. However, a major impetus to study protists is the drastic toll they take on human life. The World Health Organization provide documentation and sobering statistics on the diseases and their impact on human health. For example http://www.who.int/mediacentre/factsheets/fs375/en/ is their recently updated information sheet on Leishmaniasis. Researchers have sought to understand the processes that allow pathogens to exist, to invade a host, to evade the immune response, and to cause debilitating and often fatal diseases. Extending from such investigations and alongside drug discovery efforts enter structural biology, in particular crystallography and the high quality chemical information it can provide. Remember that most drugs are smallish organic molecules that exert their beneficial effect by interacting with certain macromolecules. The discovery of such molecules has transformed human existence. Indeed the emergence of the modern pharmaceutical industry, founded as it was on the basis of antimicrobial and analgesic agents, might be considered one of the major developments of the last 100 years. Different disciplines contribute to drug discovery, and increasingly the search is on for new targets, better technologies and model systems to assist the process. Structural studies contribute a key role by providing accurate chemical information to guide assessment of targets, the development of reliable structure–activity relationships or templates for computational design projects in the search for lead compounds. There are numerous excellent studies published in which specific parasite systems have been targeted and dissected. In addition structural genomics efforts have involved cohorts of protist proteins and the Protein Data Bank has been greatly enriched in this area with both NMR and crystal structures. We now present an issue of fundamental studies in molecular parasitology and also research directed toward protozoan drug target characterization. To set the scene there is an overview and perspective from Professor Wim Hol. In this he also details a few of his favourite examples where structural information contributes to the search for improved therapies. We commissioned Wim for an overview because of his outstanding contributions and infectious enthusiasm for the area; he continues to be an inspirational champion for the role of crystallography in drug discovery. There is also a short review from Professor Inari Kursula on cytoskeletal proteins related to the infection process. We thought it relevant to address this aspect and would highlight this as one in which there may be opportunities in future drug discovery work. We are pleased to also have a number of communications from structural genomics efforts which sit comfortably alongside more focused projects. Section F’s mandate is to allow the structural biology community to efficiently publish their data and we hope this special issue will serve to inform our community and perhaps others in the life sciences or medicinal chemistry areas, generate interest and provide encouragement to consider the journal as a natural home for results in molecular parasitology. We look forward to seeing more structural information on parasite proteins, more advances in basic biology and results to support future efforts in early stage drug discovery.

Department of Molecular and Cellular Parasitology

Department of Molecular and Cellular Parasitology

False‐colored scanning electron microscopy images of Trypanosoma brucei gambiense (length: 15 micrometers) laying on the peritoneal epithelium and awaiting entrance into the bloodstream of an infected mouse. The parasite is responsible for human sleeping sickness in Western and Central Africa. Image is from the Laboratory of Molecular Parasitology and Center for Microscopy and Molecular Imaging, Université Libre de Bruxelles, Gosselies,

False‐colored scanning electron microscopy images of <i>Trypanosoma brucei gambiense</i> (length: 15 micrometers) laying on the peritoneal epithelium and awaiting entrance into the bloodstream of an infected mouse. The parasite is responsible for human sleeping sickness in Western and Central Africa. Image is from the Laboratory of Molecular Parasitology and Center for Microscopy and Molecular Imaging, Université Libre de Bruxelles, Gosselies,

David James Kemp 1945–2013

David Kemp's seminal contributions to molecular parasitology of malaria and scabies have placed Australian science at the forefront of research on these important human pathogens. Immunoscreening of expression clones led to the identification of several vaccine candidates against malaria. His contributions to scabies research are pivotal to our understanding of bacteria–parasite–human interactions. Other notable achievements are: the discovery of one of the earliest known multi-gene families; the first cloning of linked variable-region genes in the immunoglobulin heavy-chain locus; the invention of highly cited molecular biology methods, namely Northern blotting and inverted-PCR; and contributions to ‘molecular public health' by his work on various bacterial infections relevant to the health of Indigenous Australians. Kemp's manifest enthusiasm for science was highly infectious. He mentored many high-achieving scientists. In addition to his exemplary career as a scientist, he was a musician at heart and a passionate rock fossicker.

Cell density-dependent ectopic expression in bloodstream form Trypanosoma brucei.

Ectopic expression of either wild type or mutant proteins is a standard method in cell biology, and a vital part of the tool kit of molecular parasitology. During study of protein expression levels mediating intracellular trafficking, we became aware of highly variable expression between experiments. When investigated systematically it became apparent that ectopic expression of proteins from a ribosomal promoter diminished at high cell culture density in bloodstream form Trypanosoma brucei. This phenomenon was not restricted to expression of a specific protein or cell line or the vector backbone. While procyclic form cells did not exhibit detectable density-related expression changes, bloodstream form cells manifest significant reduction in expression at high density, confirmed by qRT PCR, Western blotting and fluorescence microscopy. Culturing in conditioned media unveiled a similar reduction in expression at lower cell densities. Taken together we concluded that this effect is likely related to the influence of a diffusible factor present in conditioned media and has implications for accurate quantification of ectopic expression using transgenic expression systems.

Assessing the validity of an ELISA test for the serological diagnosis of human fascioliasis in different epidemiological situations

To improve the diagnosis of human fascioliasis caused by Fasciola hepatica and Fasciola gigantica, we evaluated the diagnostic accuracy of an enzyme-linked immunosorbent assay (ELISA), with Fasciola antigen from the adult liver fluke, for the detection of IgG against fascioliasis in human sera. The sera of 54 fascioliasis cases, originating from three endemic areas, were used in this evaluation: (i) a hyperendemic F. hepatica area where humans usually shed a great number of parasite eggs in faeces (11 sera); (ii) an epidemic F. hepatica area where humans usually shed small amounts of parasite eggs (24 sera) and (iii) an overlap area of both Fasciola species and where human shedding of parasite eggs in faeces is usually scarce or non-existent (19 sera). One hundred and sixty-eight patients with other parasitic infections and 89 healthy controls were also analysed. The respective sensitivity and specificity of this assay were 95.3% (95% confidence intervals, 82.9-99.2%) and 95.7% (95% confidence intervals, 92.3-97.5%). No correlation between egg output and the OD450 values of the F. hepatica IgG ELISA test was observed. This test could be used both as an individual serodiagnostic test for human fascioliasis when backed up by a compatible clinical history together with a second diagnostic technique for other cross-reactive helminth infections, and in large-scale epidemiological studies of human fascioliasis worldwide.

Molecular evolution and phylogenetics of rodent malaria parasites

BackgroundOver the last 6 decades, rodent Plasmodium species have become key model systems for understanding the basic biology of malaria parasites. Cell and molecular parasitology have made much progress in identifying genes underpinning interactions between malaria parasites, hosts, and vectors. However, little attention has been paid to the evolutionary genetics of parasites, which provides context for identifying potential therapeutic targets and for understanding the selective forces shaping parasites in natural populations. Additionally, understanding the relationships between species, subspecies, and strains, is necessary to maximize the utility of rodent malaria parasites as medically important infectious disease models, and for investigating the evolution of host-parasite interactions.ResultsHere, we collected multi-locus sequence data from 58 rodent malaria genotypes distributed throughout 13 subspecies belonging to P. berghei, P. chabaudi, P. vinckei, and P. yoelii. We employ multi-locus methods to infer the subspecies phylogeny, and use population-genetic approaches to elucidate the selective patterns shaping the evolution of these organisms. Our results reveal a time-line for the evolution of rodent Plasmodium and suggest that all the subspecies are independently evolving lineages (i.e. species). We show that estimates of species-level polymorphism are inflated if subspecies are not explicitly recognized, and detect purifying selection at most loci.ConclusionsOur work resolves previous inconsistencies in the phylogeny of rodent malaria parasites, provides estimates of important parameters that relate to the parasite’s natural history and provides a much-needed evolutionary context for understanding diverse biological aspects from the cross-reactivity of immune responses to parasite mating patterns.

Programmed cell death in Leishmania: biochemical evidence and role in parasite infectivity

Demonstration of features of a programmed cell death (PCD) pathway in protozoan parasites initiated a great deal of interest and debate in the field of molecular parasitology. Several of the markers typical of mammalian apoptosis have been shown in Leishmania which suggested the existence of an apoptosis like death in these organisms. However, studies to elucidate the downstream events associated with phosphotidyl serine exposure, loss of mitochondrial membrane potential, cytochrome c release, and caspase-like activities in cells undergoing such cell death remain an ongoing challenge. Recent advances in genome sequencing, chemical biology should help to solve some of these challenges. Leishmania genetic mutants that lack putative regulators/effectors of PCD pathway should not only help to demonstrate the mechanisms of PCD but also provide tools to better understand the putative role for this pathway in population control and in the establishment of a successful infection of the host.

Inhibition of unwinding and ATPase activities of Plasmodium falciparum Dbp5/DDX19 homologue

We have recently reported the isolation and characterization of Plasmodium falciparum Dbp5/DDX19 homologue PfD66 and the results indicate that it contains ATP-dependent bipolar DNA and RNA unwinding activity, intrinsic nucleic acid-dependent ATPase and RNA-binding activities (Molecular and Biochemical Parasitology, (In press) 10.1016/j.molbiopara.2010.12.003). In the present study we report the effect of a number of compounds such as actinomycin D, aphidicolin, camptothecin, cyclophosphamide, 4’,6’-di-amidino-2-phenylindole (DAPI), daunorubicin, distamycin, ethidium bromide, ellipticine, genistein, mitoxantrone, nalidixic acid, netropsin, nogalamycin, novobiocin and VP-16 on the DNA unwinding and ATPase activities of PfD66. The results indicate that DAPI, ethidium bromide, netropsin and nogalamycin efficiently inhibited the helicase and ATPase activities of PfD66. These studies will make an important contribution in understanding the mechanism of DNA unwinding by Plasmodium falciparum helicase PfD66.

Profile of Paul Roepe

Profile of Paul Roepe

Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System

The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed.