Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers.

This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)-immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.

Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi.

Ivermectin (IVM) is a broad-spectrum anthelmintic used in filariasis control programs. By binding to nematode glutamate-gated chloride channels (GluCls), IVM disrupts neurotransmission processes regulated by GluCl activity. IVM treatment of filarial infections is characterized by an initial dramatic drop in the levels of circulating microfilariae, followed by long-term suppression of their production, but the drug has little direct effect on microfilariae in culture at pharmacologically relevant concentrations. We localized Brugia malayi GluCl expression solely in a muscle structure that surrounds the microfilarial excretory-secretory (ES) vesicle, which suggests that protein release from the ES vesicle is regulated by GluCl activity. Consistent with this hypothesis, exposure to IVM in vitro decreased the amount of protein released from microfilariae. To better understand the scope of IVM effects on protein release by the parasite, three different expression patterns were identified from immunolocalization assays on a representative group of five microfilarial ES products. Patterns of expression suggest that the ES apparatus is the main source of regulated ES product release from microfilariae, as it is the only compartment that appears to be under neuromuscular control. Our results show that IVM treatment of microfilariae results in a marked reduction of protein release from the ES apparatus. Under in vivo conditions, the rapid microfilarial clearance induced by IVM treatment is proposed to result from suppression of the ability of the parasite to secrete proteins that enable evasion of the host immune system.

When Secretomes Meet Anthelmintics: Lessons for Therapeutic Interventions.

Helminth secretomes comprise many potential immunomodulators. The molecular and functional diversity of these entities and their importance at the host-parasite interface have been increasingly recognized. It is now common to hypothesize that parasite-derived molecules (PDMs) are essential mediators used by parasites to establish and remain in their hosts. Suppression of PDM release has been reported for two anthelmintic drug classes, the benzimidazoles and macrocyclic lactones, the mechanisms of action of which remain incompletely resolved. We propose that bringing together recent insights from different streams of parasitology research, for example, immunoparasitology and pharmacology, will stimulate the development of new ways to alter the host-parasite interface in the search for novel anthelmintic strategies.

Mining nematode protein secretomes to explain lifestyle and host specificity.

Parasitic nematodes are highly successful pathogens, inflicting disease on humans, animals and plants. Despite great differences in their life cycles, host preference and transmission modes, these parasites share a common capacity to manipulate their host's immune system. This is at least partly achieved through the release of excretory/secretory proteins, the most well-characterized component of nematode secretomes, that are comprised of functionally diverse molecules. In this work, we analyzed published protein secretomes of parasitic nematodes to identify common patterns as well as species-specific traits. The 20 selected organisms span 4 nematode clades, including plant pathogens, animal parasites, and the free-living species Caenorhabditis elegans. Transthyretin-like proteins were the only component common to all adult secretomes; many other protein classes overlapped across multiple datasets. The glycolytic enzymes aldolase and enolase were present in all parasitic species, but missing from C. elegans. Secretomes from larval stages showed less overlap between species. Although comparison of secretome composition across species and life-cycle stages is challenged by the use of different methods and depths of sequencing among studies, our workflow enabled the identification of conserved protein families and pinpointed elements that may have evolved as to enable parasitism. This strategy, extended to more secretomes, may be exploited to prioritize therapeutic targets in the future.

Identification of anti-horn fly vaccine antigen candidates using a reverse vaccinology approach.

BACKGROUND: The horn fly, Haematobia irritans irritans, causes significant production losses to the cattle industry. Horn fly control relies on insecticides; however, alternative control methods such as vaccines are needed due to the fly's capacity to quickly develop resistance to insecticides, and the pressure for eco-friendly options. METHODS: We used a reverse vaccinology approach comprising three vaccine prediction and 11 annotation tools to evaluate and rank 79,542 translated open reading frames (ORFs) from the horn fly's transcriptome, and selected 10 transcript ORFs as vaccine candidates for expression in Pichia pastoris. The expression of the 10 selected transcripts and the proteins that they encoded were investigated in adult flies by reverse transcription polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Then, we evaluated the immunogenicity of a vaccine candidate in an immunization trial and the antigen's effects on horn fly mortality and fecundity in an in vitro feeding assay. RESULTS: Six of the ten vaccine candidate antigens were successfully expressed in P. pastoris. RT-PCR confirmed the expression of all six ORFs in adult fly RNA. One of the vaccine candidate antigens, BI-HS009, was expressed in sufficient quantity for immunogenicity and efficacy trials. The IgG titers of animals vaccinated with BI-HS009 plus adjuvant were significantly higher than those of animals vaccinated with buffer plus adjuvant only from days 42 to 112, with a peak on day 56. Progeny of horn flies feeding upon blood from animals vaccinated with BI-HS009 plus adjuvant collected on day 56 had 63% lower pupariation rate and 57% lower adult emergence than the control group (ANOVA: F (1, 6) = 8.221, P = 0.028 and F (1, 6) = 8.299, P = 0.028, respectively). CONCLUSIONS: The reverse vaccinology approach streamlined the discovery process by prioritizing possible vaccine antigen candidates. Through a thoughtful process of selection and in vivo and in vitro evaluations, we were able to identify a promising antigen for an anti-horn fly vaccine.

Dirofilaria immitis JYD-34 isolate: whole genome analysis.

BACKGROUND: Macrocyclic lactone (ML) anthelmintics are used for chemoprophylaxis for heartworm infection in dogs and cats. Cases of dogs becoming infected with heartworms, despite apparent compliance to recommended chemoprophylaxis with approved preventives, has led to such cases being considered as suspected lack of efficacy (LOE). Recently, microfilariae collected from a small number of LOE isolates were used as a source of infection of new host dogs and confirmed to have reduced susceptibility to ML in controlled efficacy studies using L3 challenge in dogs. A specific Dirofilaria immitis laboratory isolate named JYD-34 has also been confirmed to have less than 100% susceptibility to ML-based preventives. For preventive claims against heartworm disease, evidence of 100% efficacy is required by FDA-CVM. It was therefore of interest to determine whether JYD-34 has a genetic profile similar to other documented LOE and confirmed reduced susceptibility isolates or has a genetic profile similar to known ML-susceptible isolates. METHODS: In this study, the 90Mbp whole genome of the JYD-34 strain was sequenced. This genome was compared using bioinformatics tools to pooled whole genomes of four well-characterized susceptible D. immitis populations, one susceptible Missouri laboratory isolate, as well as the pooled whole genomes of four LOE D. immitis populations. Fixation indexes (FST), which allow the genetic structure of each population (isolate) to be compared at the level of single nucleotide polymorphisms (SNP) across the genome, have been calculated. Forty-one previously reported SNP, that appeared to differentiate between susceptible and LOE and confirmed reduced susceptibility isolates, were also investigated in the JYD-34 isolate. RESULTS: The FST analysis, and the analysis of the 41 SNP that appeared to differentiate reduced susceptibility from fully susceptible isolates, confirmed that the JYD-34 isolate has a genome similar to previously investigated LOE isolates, and isolates confirmed to have reduced susceptibility, and to be dissimilar to the susceptible isolates. CONCLUSIONS: These results provide additional evidence for the link between genotype and the reduced susceptibility phenotype observed in such isolates as JYD-34. Further work on other isolates showing reduced susceptibility to ML is required to demonstrate the value of genetic analysis in predicting the response to ML chemoprophylaxis. The authors suggest that genetic analysis may be useful in helping to interpret the results of in vivo efficacy testing of ML heartworm preventives against D. immitis isolates.

Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite.

Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPß and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways.

Expansion of host cellular niche can drive adaptation of a zoonotic malaria parasite to humans.

The macaque malaria parasite Plasmodium knowlesi has recently emerged as an important zoonosis in Southeast Asia. Infections are typically mild but can cause severe disease, achieving parasite densities similar to fatal Plasmodium falciparum infections. Here we show that a primate-adapted P. knowlesi parasite proliferates poorly in human blood due to a strong preference for young red blood cells (RBCs). We establish a continuous in vitro culture system by using human blood enriched for young cells. Mathematical modelling predicts that parasite adaptation for invasion of older RBCs is a likely mechanism leading to high parasite densities in clinical infections. Consistent with this model, we find that P. knowlesi can adapt to invade a wider age range of RBCs, resulting in proliferation in normal human blood. Such cellular niche expansion may increase pathogenesis in humans and will be a key feature to monitor as P. knowlesi emerges in human populations.

Macrocyclic lactone anthelmintics: spectrum of activity and mechanism of action.

Macrocyclic lactones (MLs), exemplified by the prototype of the class, ivermectin (IVM), are mainstays of programs for the control of nematode and arthropod parasites and pests. Since their introduction 30 years ago, research has revealed that they act on a family of ligand-gated chloride channels gated by glutamate, which is largely restricted to animals in the phyla Nematoda and Arthropoda. Studies on IVM in model organisms have contributed greatly to our understanding of ML pharmacology, but our understanding of the basis for differences among species and among MLs in potency and spectrum remains far from complete.

First analysis of the secretome of the canine heartworm, Dirofilaria immitis.

BACKGROUND: The characterization of proteins released from filariae is an important step in addressing many of the needs in the diagnosis and treatment of these clinically important parasites, as well as contributing to a clearer understanding of their biology. This report describes findings on the proteins released during in vitro cultivation of adult Dirofilaria immitis , the causative agent of canine and feline heartworm disease. Differences in protein secretion among nematodes in vivo may relate to the ecological niche of each parasite and the pathological changes that they induce. METHODS: The proteins in the secretions of cultured adult worms were run on Tris-Glycine gels, bands separated and peptides from each band analysed by ultra mass spectrometry and compared with a FastA dataset of predicted tryptic peptides derived from a genome sequence of D. immitis. RESULTS: This study identified 110 proteins. Of these proteins, 52 were unique to D. immitis. A total of 23 (44%) were recognized as proteins likely to be secreted. Although these proteins were unique, the motifs were conserved compared with proteins secreted by other nematodes. CONCLUSION: The present data indicate that D. immitis secretes proteins that are unique to this species, when compared with Brugia malayi. The two major functional groups of molecules represented were those representing cellular and of metabolic processes. Unique proteins might be important for maintaining an infection in the host environment, intimately involved in the pathogenesis of disease and may also provide new tools for the diagnosis of heartworm infection.

Proteomic profiles of soluble proteins from the esophageal gland in female Meloidogyne incognita.

Meloidogyne incognita can infect multiple plant species. Proteins synthesized in the esophageal glands and secreted through the stylet of plant parasitic nematodes play critical roles in the plant-nematode interactions. Female M. incognita live for approximately 15days, embedded in a host plant, but their esophageal gland proteins have not yet been comprehensively analyzed. In this study, a new bacterium-contamination-resistant method for collecting soluble proteins from esophageal gland cells (SPEGC) of female M. incognita was established. Approximately 5µg of freeze-dried proteins could be extracted from 150 female M. incognita. Bands of a one-dimensional SDS-polyacrylamide gel were excised after electrophoresis of 20µg of protein and were analyzed. Two hundred and forty-six proteins from SPEGC of female M. incognita were identified by LC-MS/MS. Gene Ontology analysis suggests that many of the secreted proteins are involved in protein or carbohydrate metabolism and proteolysis. Some of the SPEGC (46.3%) were predicted to be secreted through classical or non-classical secretory pathways. The described method presents a new approach for the identification of proteins stored in SPEGC of an important plant parasitic nematode. This global proteomic profile of SPEGC provides a basis for future studies to elucidate the functions of proteins secreted from female M. incognita on plant responses.

Proteomic analysis of excretory-secretory products of Heligmosomoides polygyrus assessed with next-generation sequencing transcriptomic information.

The murine parasite Heligmosomoides polygyrus is a convenient experimental model to study immune responses and pathology associated with gastrointestinal nematode infections. The excretory-secretory products (ESP) produced by this parasite have potent immunomodulatory activity, but the protein(s) responsible has not been defined. Identification of the protein composition of ESP derived from H. polygyrus and other relevant nematode species has been hampered by the lack of genomic sequence information required for proteomic analysis based on database searches. To overcome this, a transcriptome next generation sequencing (RNA-seq) de novo assembly containing 33,641 transcripts was generated, annotated, and used to interrogate mass spectrometry (MS) data derived from 1D-SDS PAGE and LC-MS/MS analysis of ESP. Using the database generated from the 6 open reading frames deduced from the RNA-seq assembly and conventional identification programs, 209 proteins were identified in ESP including homologues of vitellogenins, retinol- and fatty acid-binding proteins, globins, and the allergen V5/Tpx-1-related family of proteins. Several potential immunomodulators, such as macrophage migration inhibitory factor, cysteine protease inhibitors, galectins, C-type lectins, peroxiredoxin, and glutathione S-transferase, were also identified. Comparative analysis of protein annotations based on the RNA-seq assembly and proteomics revealed processes and proteins that may contribute to the functional specialization of ESP, including proteins involved in signalling pathways and in nutrient transport and/or uptake. Together, these findings provide important information that will help to illuminate molecular, biochemical, and in particular immunomodulatory aspects of host-H. polygyrus biology. In addition, the methods and analyses presented here are applicable to study biochemical and molecular aspects of the host-parasite relationship in species for which sequence information is not available.

Stage- and gender-specific proteomic analysis of Brugia malayi excretory-secretory products.

INTRODUCTION: While we lack a complete understanding of the molecular mechanisms by which parasites establish and achieve protection from host immune responses, it is accepted that many of these processes are mediated by products, primarily proteins, released from the parasite. Parasitic nematodes occur in different life stages and anatomical compartments within the host. Little is known about the composition and variability of products released at different developmental stages and their contribution to parasite survival and progression of the infection. METHODOLOGY/PRINCIPAL FINDINGS: To gain a deeper understanding on these aspects, we collected and analyzed through 1D-SDS PAGE and LC-MS/MS the Excretory-Secretory Products (ESP) of adult female, adult male and microfilariae of the filarial nematode Brugia malayi, one of the etiological agents of human lymphatic filariasis. This proteomic analysis led to the identification of 228 proteins. The list includes 76 proteins with unknown function as well as also proteins with potential immunoregulatory properties, such as protease inhibitors, cytokine homologues and carbohydrate-binding proteins. Larval and adult ESP differed in composition. Only 32 proteins were shared between all three stages/genders. Consistent with this observation, different gene ontology profiles were associated with the different ESP. CONCLUSIONS/SIGNIFICANCE: A comparative analysis of the proteins released in vitro by different forms of a parasitic nematode dwelling in the same host is presented. The catalog of secreted proteins reflects different stage- and gender-specific related processes and different strategies of immune evasion, providing valuable insights on the contribution of each form of the parasite for establishing the host-parasite interaction.