Developmental Stages Of Parasite Research Articles

A nuclear protein, PfMORC confers melatonin dependent synchrony of the human malaria parasite P. falciparum in the asexual stage

The host hormone melatonin is known to modulate the asexual cell-cycle of the human malaria parasite Plasmodium falciparum and the kinase PfPK7 is fundamental in the downstream signaling pathways. The nuclear protein PfMORC displays a histidine kinase domain and is involved in parasite cell cycle control. By using a real-time assay, we show a 24 h (h) rhythmic expression of PfMORC at the parasite asexual cycle and the expression is dramatically changed when parasites were treated with 100 nM melatonin for 17 h. Moreover, PfMORC expression was severely affected in PfPK7 knockout (PfPK7−) parasites following melatonin treatment. Parasites expressing 3D7morc-GFP shows nuclear localization of the protein during the asexual stage of parasite development. Although the PfMORC knockdown had no significant impact on the parasite proliferation in vitro it significantly changed the ratio of the different asexual intraerythrocytic stages of the parasites upon the addition of melatonin. Our data reveal that in addition to the upstream melatonin signaling pathways such as IP3 generation, calcium, and cAMP rise, a nuclear protein, PfMORC is essential for the hormone response in parasite synchronization.

Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation–reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.

Lysercell M enhances the detection of stage-specific Plasmodium-infected red blood cells in the automated hematology analyzer XN-31 prototype

The automated hematology analyzers XN-30 (for research) and XN-31 prototype (for diagnosis support) can easily and rapidly detect Plasmodium-infected red blood cells (iRBCs) and distinguish the developmental stages of the parasite in approximately 1min. Two dedicated reagents, Lysercell M and Fluorocell M, are available with the analyzers. Lysercell M plays an indispensable role in enhancing the fluorescence intensity of the nucleic acid staining dye in Fluorocell M and altering cell morphology. These effects of Lysercell M have been empirically determined but insufficiently analyzed. In this study, the properties of Lysercell M were analyzed using two flow cytometers and a fluorescence microscope. First, the fluorescence intensity emitted by iRBCs treated with Lysercell M or phosphate-buffered saline (PBS) was evaluated. Second, the size of RBCs treated with Lysercell M or PBS was measured. Finally, the morphology of individual parasites was observed after reconstruction of an M scattergram, a cytogram of the XN-31 prototype system, using an imaging flow cytometer. These analyses showed that treatment of iRBCs with Lysercell M increased the fluorescence intensity of stained parasite nucleic acids by approximately 10-fold and reduced the size of iRBCs in a stage-specific manner, facilitating the identification and quantification of ring form, trophozoite, and schizont stage iRBCs. These properties suggest that Lysercell M is useful for rapidly detecting iRBCs and accurately distinguishing the parasite developmental stages, thereby contributing to the usability of the XN-30 and XN-31 prototype analyzers.

Erythrocyte phospho-signalling is dynamically altered during infection with Plasmodium falciparum.

It is well established that intracellular pathogens mobilise signalling pathways to manipulate gene expression of their host cell to promote their own survival. Surprisingly, there is evidence that specific host signalling molecules are likewise activated in a-nucleated erythrocytes in response to infection with malaria parasites. In this paper (Adderley et al., Nature Communications 2020), we report the system-wide assessment of host erythrocyte signalling during the course of infection with Plasmodium falciparum. This was achieved through the use of antibody microarrays containing >800 antibodies directed against human signalling proteins, which enabled us to interrogate the status of host erythrocyte signalling pathways at the ring, trophozoite and schizont stages of parasite development. This not only confirmed the pre-existing fragmentary data on the activation of a host erythrocyte PAK-MEK pathway, but also identified dynamic changes to many additional signalling elements, with trophozoite-infected erythrocytes displaying the largest mobilisation of host cell signalling. This study generated a comprehensive dataset on the modulation of host erythrocyte signalling during infection with P. falciparum, and provides the proof of principle that human protein kinases activated by Plasmodium infection represent attractive targets for antimalarial intervention.

Retargeting azithromycin analogues to have dual-modality antimalarial activity

BackgroundResistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium spp. malaria parasites is urgently needed. Azithromycin is a clinically used macrolide antibiotic proposed as a partner drug for combination therapy in malaria, which has also been tested as monotherapy. However, its slow-killing ‘delayed-death’ activity against the parasite’s apicoplast organelle and suboptimal activity as monotherapy limit its application as a potential malaria treatment. Here, we explore a panel of azithromycin analogues and demonstrate that chemical modifications can be used to greatly improve the speed and potency of antimalarial action.ResultsInvestigation of 84 azithromycin analogues revealed nanomolar quick-killing potency directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin with less than 48 hrs treatment in vitro. Analogues were effective against zoonotic Plasmodium knowlesi malaria parasites and against both multi-drug and artemisinin-resistant Plasmodium falciparum lines. Metabolomic profiles of azithromycin analogue-treated parasites suggested activity in the parasite food vacuole and mitochondria were disrupted. Moreover, unlike the food vacuole-targeting drug chloroquine, azithromycin and analogues were active across blood-stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on ‘quick-killing’ activity. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity.ConclusionWe show that azithromycin and analogues can rapidly kill malaria parasite asexual blood stages via a fast action mechanism. Development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed-death mechanism of action in a single, multifactorial chemotype.

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as β-actin, ef-1β, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

Host and Parasite Transcriptomic Changes upon Successive Plasmodium falciparum Infections in Early Childhood.

Children are highly susceptible to clinical malaria, and in regions where malaria is endemic, their immune systems must face successive encounters with Plasmodium falciparum parasites before they develop immunity, first against severe disease and later against uncomplicated malaria. Understanding cellular and molecular interactions between host and parasites during an infection could provide insights into the processes underlying this gradual acquisition of immunity, as well as to how parasites adapt to infect hosts that are successively more malaria experienced. Here, we describe methods to analyze the host and parasite gene expression profiles generated simultaneously from blood samples collected from five consecutive symptomatic P. falciparum infections in three Malian children. We show that the data generated enable statistical assessment of the proportions of (i) each white blood cell subset and (ii) the parasite developmental stages, as well as investigations of host-parasite gene coexpression. We also use the sequences generated to analyze allelic variations in transcribed regions and determine the complexity of each infection. While limited by the modest sample size, our analyses suggest that host gene expression profiles primarily clustered by individual, while the parasite gene expression profiles seemed to differentiate early from late infections. Overall, this study provides a solid framework to examine the mechanisms underlying acquisition of immunity to malaria infections using whole-blood transcriptome sequencing (RNA-seq).IMPORTANCE We show that dual RNA-seq from patient blood samples allows characterization of host/parasite interactions during malaria infections and can provide a solid framework to study the acquisition of antimalarial immunity, as well as the adaptations of P. falciparum to malaria-experienced hosts.

Habronematidosis in Equids: Current Status, Advances, Future Challenges.

Over the past few decades, among equine parasitoses caused by gastrointestinal nematodes, habronematidosis has been discontinuously studied worldwide. Habronematidosis is a parasitic disease distributed all over the world. It is caused by Habronema microstoma, Habronema muscae, and Draschia megastoma (Spirurida, Habronematidae), and it is maintained in the environment by muscid flies which act as intermediate hosts. At larval and adult stages these species live in the stomach of domestic and wild equids. However, the larvae can also be found on the skin, causing lesions known as “summer sores”, and occasionally on other body areas, such as ocular and genital mucosa (muco-cutaneous habronematidosis) and lung, liver, brain parenchyma. Depending on the parasite's developmental stage and localization site, clinical signs vary from mild to severe. Habronematidosis is responsible for significant economic losses, mostly when sport horses are affected, because their performances are impaired and the infection can be unaesthetic. We used three on-line databases for searching the articles on habronematidosis according to the selected inclusion criteria; a total of 250 contributions, published between 1911 and 2020 were analyzed. This review summarizes the key features of pathogenesis, epizootiology, diagnosis, treatment, and control of habronematidosis, and highlights the current knownledge about its geographical distribution and spread. Anthelmintic drugs are the most widely-used tools against habronematidosis; given the known risk of anthelmintic resistance in some nematodes affecting horses, this aspect should also be explored for habronematidosis. Dedicated research is essential to fill gaps of knowledge and increase the understanding of habronematidosis to maximize equine health, reduce economic losses and sanitary impact associated with this parasitic infection.

The first analysis of Trichinella spiralis and Trichinella britovi adult worm excretory-secretory proteins by two-dimensional electrophoresis coupled with LC-MS/MS

The two most common Trichinella species present in European countries are Trichinella spiralis and Trichinella britovi. The consumption of raw or undercooked meat with invasive larvae results in trichinellosis. Currently, the most commonly used sources for detecting specific anti-Trichinella antibodies is ELISA with the muscle larvae (ML) excretory-secretory (E-S) proteins. However, these serological methods cannot be efficiently applied at early stage of infection. The aim of the current study was to identify the common and species-specific E-S proteins of T. spiralis and T. britovi adult worms which could have potential for accurate diagnosis of Trichinella infection at an early stage of invasion. Different sets of immunoreactive proteins were identified in T. spiralis and T. britovi proteomes by a combination of two-dimensional electrophoresis (2-DE), immunoblot and LC-MS/MS analysis. Polyubiquitin-B, a possible enoyl-CoA hydratase/isomerase YngF or polyubiquitin-like protein was found to be common for both species; gene ontology analysis confirmed its involvement in proteolysis, oxidation-reduction and translation processes, as well as in molecular transport. These molecules, being secreted or excreted at an early stage of parasite development, may play a critical role in the processes occurring during the initial steps of the host invasion and hence be suitable for diagnostic test development.

The influence of feeding behaviour and temperature on the capacity of mosquitoes to transmit malaria.

Insecticide-treated bed nets reduce malaria transmission by limiting contact between mosquito vectors and human hosts when mosquitoes feed during the night. However, malaria vectors can also feed in the early evening and in the morning when people are not protected. Here, we explored how timing of blood feeding interacts with environmental temperature to influence the capacity of Anopheles mosquitoes to transmit the human malaria parasite, Plasmodium falciparum. In laboratory experiments, we found no effect of biting time itself on the proportion of mosquitoes that became infectious (vector competence) at constant temperature. However, when mosquitoes were maintained under more realistic fluctuating temperatures there was a significant increase in competence for mosquitoes feeding in the evening (18:00h), and a significant reduction in competence for those feeding in the morning (06:00h), relative to those feeding at midnight (00:00h). These effects appear to be due to thermal sensitivity of malaria parasites during the initial stages of parasite development within the mosquito, and the fact that mosquitoes feeding in the evening experience cooling temperatures during the night, whereas mosquitoes feeding in the morning quickly experience warming temperatures that are inhibitory to parasite establishment. A transmission dynamics model illustrates that such differences in competence could have important implications for malaria prevalence, the extent of transmission that persists in the presence of bed nets, and the epidemiological impact of behavioural resistance. These results indicate the interaction of temperature and feeding behaviour could be a major ecological determinant of the vectorial capacity of malaria mosquitoes.

Magnitude and direction of parasite-induced phenotypic alterations: a meta-analysis in acanthocephalans.

Several parasite species have the ability to modify their host's phenotype to their own advantage thereby increasing the probability of transmission from one host to another. This phenomenon of host manipulation is interpreted as the expression of a parasite extended phenotype. Manipulative parasites generally affect multiple phenotypic traits in their hosts, although both the extent and adaptive significance of such multidimensionality in host manipulation is still poorly documented. To review the multidimensionality and magnitude of host manipulation, and to understand the causes of variation in trait value alteration, we performed a phylogenetically corrected meta-analysis, focusing on a model taxon: acanthocephalan parasites. Acanthocephala is a phylum of helminth parasites that use vertebrates as final hosts and invertebrates as intermediate hosts, and is one of the few parasite groups for which manipulation is predicted to be ancestral. We compiled 279 estimates of parasite-induced alterations in phenotypic trait value, from 81 studies and 13 acanthocephalan species, allocating a sign to effect size estimates according to the direction of alteration favouring parasite transmission, and grouped traits by category. Phylogenetic inertia accounted for a low proportion of variation in effect sizes. The overall average alteration of trait value was moderate and positive when considering the expected effect of alterations on trophic transmission success (signed effect sizes, after the onset of parasite infectivity to the final host). Variation in the alteration of trait value was affected by the category of phenotypic trait, with the largest alterations being reversed taxis/phobia and responses to stimuli, and increased vulnerability to predation, changes to reproductive traits (behavioural or physiological castration) and immunosuppression. Parasite transmission would thereby be facilitated mainly by changing mainly the choice of micro-habitat and the anti-predation behaviour of infected hosts, and by promoting energy-saving strategies in the host. In addition, infection with larval stages not yet infective to definitive hosts (acanthella) tends to induce opposite effects of comparable magnitude to infection with the infective stage (cystacanth), although this result should be considered with caution due to the low number of estimates with acanthella. This analysis raises important issues that should be considered in future studies investigating the adaptive significance of host manipulation, not only in acanthocephalans but also in other taxa. Specifically, the contribution of phenotypic traits to parasite transmission and the range of taxonomic diversity covered deserve thorough attention. In addition, the relationship between behaviour and immunity across parasite developmental stages and host-parasite systems (the neuropsychoimmune hypothesis of host manipulation), still awaits experimental evidence. Most of these issues apply more broadly to reported cases of host manipulation by other groups of parasites.

Transcriptome analysis of Anopheles dirus and Plasmodium vivax at ookinete and oocyst stages

Malaria is transmitted by Plasmodium parasites through the bite of female Anopheles mosquitoes. One of the most important mosquito vectors in the Greater Mekong Subregion is Anopheles dirus. This study reports RNA sequencing (RNA-Seq) transcriptome analysis of An. dirus at 18 hours and 7 days after a P. vivax-infected blood meal, which represent infection at the ookinete and oocyst parasite developmental stages, respectively. Following infection, 582 An. dirus transcripts were modulated. The 2,408 P. vivax transcripts could be classified into ookinete-specific, two-stage, and oocyst-specific groups. Results were validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analysis of the vector and parasite revealed several biological pathways for both, providing a better understanding of Anopheles–Plasmodium interactions at the ookinete and oocyst stages.

Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence

The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria.

A simple monochromatic flow cytometric assay for assessment of intraerythrocytic development of Plasmodium falciparum

BackgroundGold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner.MethodsIn this study, ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, was used for assessing in vitro development of P. falciparum using flow cytometry.ResultsFluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSGhigh population, and growing trophozoites and ring-shaped forms were observed in the VSGintermediate and VSGlow populations. The efficacy of VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitaemia estimated by Giemsa staining. Moreover, when applying VSG for anti-malarial drug test, it was able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy.ConclusionsTaken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.

Plasmepsin Inhibitors in Antimalarial Drug Discovery: Medicinal Chemistry and Target Validation (2000 to Present).

Plasmepsins represent novel antimalarial drug targets. However, plasmepsin-based antimalarial drug discovery efforts in the past 2 decades have generally suffered some drawbacks including lack of translatability of target inhibition to potent parasite inhibition in vitro and in vivo as well as poor selectivity over the related human aspartic proteases. Most studies reported in this period have over-relied on the use of hemoglobinase plasmepsins I-IV (particularly I and II) as targets for the new inhibitors even though these are known to be nonessential at the asexual stage of parasite development. Therefore, future antimalarial drug discovery efforts seeking to identify plasmepsin inhibitors should focus on incorporating non-hemoglobinase plasmepsins such as V, IX, and X in their screening in order to maximize chances of success. Additionally, there is need to go beyond just target enzymatic activity profiling to establishing cellular activity, physicochemical as well as drug metabolism and pharmacokinetics properties and finally in vivo proof-of-concept while ensuring selectivity over related human host proteases.

Analysis of the Physiological and Metabolic State of Leishmania Using Heavy Water Labeling.

This protocol describes the use of heavy water (2H2O) labeling to determine the growth rate and metabolic state of Leishmania parasites in culture and in infected animals. In vitro labeling studies are undertaken by cultivating defined parasite developmental stages in standard medium supplemented with 5% 2H2O, resulting in the incorporation of deuterium (2H) into a range of metabolic precursors used in macromolecule (DNA, RNA, protein, lipid, and glycan) synthesis. The rate of turnover of different parasite macromolecules can subsequently be determined by analysis of deuterium enrichment in the different constituents of these macromolecules by gas chromatography-mass spectrometry (GC-MS). To measure the growth rate and physiological state of parasite stages in lesion tissue, infected mice were provided with 9% 2H2O in their drinking water for various periods of time and 2H-enrichment in the macromolecular constituents of isolated lesion-derived parasite stages determined by GC-MS. This protocol provides quantitative information on key cellular processes, such as replication (DNA turnover), transcription (RNA turnover), translation (protein turnover), membrane biogenesis (lipid turnover), and central carbon metabolism (glycan turnover) that define the growth state and phenome of different parasite stages in vitro and in vivo. This approach can be used to assess the impact of host immune responses on parasite growth and physiology (using different Leishmania strains/species, mouse lines), characterize different parasite populations during chronic and acute infections, and assess parasite responses to drug treatments. It is also broadly applicable to other microbial pathogens.

Liver echinococcosis: which treatment method to prefer?

The aim — to determine the role and effectiveness of various surgical treatment methods for liver echinococcosis, depending on the type of echinococcal cyst, its location and size.Materials and methods. A treatment results analysis of 56 patients with liver echinococcosis who were treated in the clinic from 2008 to 2018 was performed. There were 42.9 % of men and 57.1 % of women. The mean age of the patients was 48 ± 2.3 years. The standard examination algorithm included mandatory contrast enhanced CT and the detection of antibodies to echinococcus in blood serum in addition to routine laboratory and instrumental tests. CL type of cysts were detected in 3 patients (5.4 %), type CE1 in 27 patients (48.2 %), type CE2 in 10 cases (17.9 %), type CE3 in 12 cases (21.4 %), type CE4 — 5 — in 4 patients (7.2 %). Patients underwent various surgical interventions, depending on the type of echinococcal cyst. PAIR was used in 3 patients (5.4 %), PAIRD was used in 7 patients (12.5 %). Echinococcectomy was performed in 17 patients (30.4 %) The open approach was performed in 8 patients (14.3 %), and laparoscopic approach — in 9 cases (16.1 %). Pericystectomy was performed in 19 patients (33.8 %)/The open approach was used in 16 cases (28.4 %), and laparoscopic approach — in 3 cases (5.4 %). In another 10 cases (17.9 %), liver resections varying from segmentectomy to hemihepatectomy were performed.Results and discussion.Bile leakage via the drainages of the abdominal cavity was observed in 7 cases (12.5 %). Allergic reactions of varying severity were observed in 3 cases (5.6 %). The recurrence of decease was noted in 2 patients (4 %). There was no lethality in this group of patients.Conclusions. The absence of a unified surgical tactics for liver echinococcosis requires the use of an individualized approach. The choice of treatment method depends on the location and size of the cyst, the stage of parasite development. Postoperative therapy with albendazole (mebendazole) in the presence of a live parasite is required. In the presence of a non‑living parasite (cysts of type CE4 — CE5) it is advisable to use observant tactics.

Identification of Plasmodiumdipeptidyl aminopeptidase allosteric inhibitors by high throughput screening.

Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas’s disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

Experimental characterization of the complete life cycle of Haemoproteus columbae, with a description of a natural host-parasite system used to study this infection

Characterization of complete life cycles of haemoparasites requires the maintenance of suitable susceptible vertebrate hosts and vectors for long periods in captivity, in order to follow the complete parasitic cycle in definitive and intermediate hosts. Currently, there are few host-parasite models established in avian haemosporidian research, and those have been developed mainly for species of Passeriformes and their parasites. This study aimed to develop an experimental methodology to access the complete life cycle of Haemoproteus columbae (cytb lineage HAECOL1), which parasitizes the Rock Pigeon (Columba livia) and louse fly (Pseudolynchia canariensis). A colony of louse flies, which are the natural vectors of this parasite, was established. Thirty newly emerged insects were exposed to H. columbae infection and used to infect naïve Rock Pigeons. The peak of parasitaemia (acute stage) was seen between 27 and 32 days p.i. when up to 70.8% of red blood cells were infected. The crisis occurred approximately 1 week after the peak, and the long-lasting chronic parasitaemia stage followed. Exo-erythrocytic meronts were seen mainly in the lungs where extensive tissue damage was reported, but also in the kidneys and spleen. In the vector, the sporogonic cycle of H. columbae was completed between 13 and 16 days p.i., at an average temperature ranging between 12 and 15 °C. This host-parasite model is tractable for maintenance in captivity. It is recommended for use in studies aiming for detailed characterization of host-parasite relationships in areas such as physiology, pathology, immunobiology, genetics, as well as for evaluative treatments and to follow the infection in any stage of parasite development both in the vertebrate or invertebrate host.

Combination of quercetin and ivermectin: In vitro and in vivo effects against Haemonchus contortus

The aim of the present study was to evaluate the in vitro effect of quercetin combined with ivermectin (IVM) on Haemonchus contortus larvae and adults with different resistance profiles and demonstrate the in vivo anthelmintic action of this combination when used in sheep naturally infected. The effect of combination was evaluated based on the analysis of the mean effective concentration (EC50) obtained for larvae using the larval migration inhibition test and for adults using the motility test on females. The tests with larvae and adults were conducted using isolates with different degrees of susceptibility to IVM (sensitive, intermediate and highly resistant). The in vivo effect was evaluated based on the reduction in the egg count (FEC) and reduction in the count of adult helminths recovered after parasitological necropsy. Using the combination of quercetin with IVM, it was observed that in larvae, quercetin did not significantly reduce the EC50 for IVM in the sensitive and highly resistant isolates, but led to a significant reduction in the EC50 for IVM in the intermediate isolate. In adults, quercetin did not significantly reduce the EC50 for IVM in any of the isolates. No significant effect of the combination was found regarding the reduction in FEC or total count of parasites. The results of the in vitro and in vivo tests performed in the present study on quercetin activity underscore the importance of evaluating resistance-reversing agents among different stages of parasite development as well as among isolates with different resistance profiles. The action of quercetin combined with IVM on the motility of H. contortus larvae and adults was influenced by the degree of resistance and development stage of the parasite. The combination was effective only on intermediate resistant larvae. No action of the combination against adults was found. Moreover, this combination, when administered through the intra-abomasal route, was not effective at reducing the FEC and parasite load of naturally infected sheep.