Epidrugs: alternative chemotherapy targeting Theileria annulata schizont stage parasites.

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally.

Antibodies against a Plasmodium falciparum RON12 inhibit merozoite invasion into erythrocytes.

Proteins coating Plasmodium merozoite surface and secreted from its apical organelles are considered as promising vaccine candidates for blood-stage malaria. The rhoptry neck protein 12 of Plasmodium falciparum (PfRON12) was recently reported as a protein specifically expressed in schizonts and localized to the rhoptry neck of merozoites. Here, we assessed its potential as a vaccine candidate. We expressed a recombinant PfRON12 protein by a wheat germ cell-free system to obtain anti-PfRON12 antibody. Immunoblot analysis of schizont lysates detected a single band at approximately 40 kDa under reducing conditions, consistent with the predicted molecular weight. Additionally, anti-PfRON12 antibody recognized a single band around 80 kDa under non-reducing conditions, suggesting native PfRON12 forms a disulfide-bond-mediated multimer. Immunofluorescence assay and immunoelectron microscopy revealed that PfRON12 localized to the rhoptry neck of merozoites in schizonts and to the surface of free merozoites. The biological activity of anti-PfRON12 antibody was tested by in vitro growth inhibition assay (GIA), and the rabbit antibodies significantly inhibited merozoite invasion of erythrocytes. We then investigated whether PfRON12 is immunogenic in P. falciparum-infected individuals. The sera from P. falciparum infected individuals in Thailand and Mali reacted with the recombinant PfRON12. Furthermore, human anti-PfRON12 antibodies affinity-purified from Malian serum samples inhibited merozoite invasion of erythrocytes in vitro. Moreover, pfron12 is highly conserved with only 4 non-synonymous mutations in the coding sequence from approximately 200 isolates deposited in PlasmoDB. These results suggest that PfRON12 might be a potential blood-stage vaccine candidate antigen against P. falciparum.

Characterization of Plasmodium falciparum Atypical Kinase PfPK7- Dependent Phosphoproteome.

PfPK7 is an "orphan" kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7 - parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the pfpk7 - cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways.

Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals.

BACKGROUND: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane. RESULTS: In this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum. CONCLUSIONS: The results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

Plasmodium falciparum merozoite surface protein 3: oligomerization, self-assembly, and heme complex formation.

Merozoite surface protein 3 of Plasmodium falciparum, a 40-kDa protein that also binds heme, has been biophysically characterized for its tendency to form highly elongated oligomers. This study aims to systematically analyze the regions in MSP3 sequence involved in oligomerization and correlate its aggregation tendency with its high affinity for binding with heme. Through size exclusion chromatography, dynamic light scattering, and transmission electron microscopy, we have found that MSP3, previously known to form elongated oligomers, actually forms self-assembled filamentous structures that possess amyloid-like characteristics. By expressing different regions of MSP3, we observed that the previously described leucine zipper region at the C terminus of MSP3 may not be the only structural element responsible for oligomerization and that other peptide segments like MSP3(192-196) (YILGW) may also be required. MSP3 aggregates on incubation were transformed to long unbranched amyloid fibrils. Using immunostaining methods, we found that 5-15-µm-long fibrillar structures stained by anti-MSP3 antibodies were attached to the merozoite surface and also associated with erythrocyte membrane. We also found MSP3 to bind several molecules of heme by UV spectrophotometry, HPLC, and electrophoresis. This study suggested that its ability to bind heme is somehow related to its inherent characteristics to form oligomers. Moreover, heme interaction with a surface protein like MSP3, which does not participate in hemozoin formation, may suggest a protective role against the heme released from unprocessed hemoglobin released after schizont egress. These studies point to the other roles that MSP3 may play during the blood stages of the parasite, in addition to be an important vaccine candidate.

Structural and biochemical characterization of Plasmodium falciparum 12 (Pf12) reveals a unique interdomain organization and the potential for an antiparallel arrangement with Pf41.

Plasmodium falciparum is the most devastating agent of human malaria. A major contributor to its virulence is a complex lifecycle with multiple parasite forms, each presenting a different repertoire of surface antigens. Importantly, members of the 6-Cys s48/45 family of proteins are found on the surface of P. falciparum in every stage, and several of these antigens have been investigated as vaccine targets. Pf12 is the archetypal member of the 6-Cys protein family, containing just two s48/45 domains, whereas other members have up to 14 of these domains. Pf12 is strongly recognized by immune sera from naturally infected patients. Here we show that Pf12 is highly conserved and under purifying selection. Immunofluorescence data reveals a punctate staining pattern with an apical organization in late schizonts. Together, these data are consistent with an important functional role for Pf12 in parasite-host cell attachment or invasion. To infer the structural and functional diversity between Pf12 and the other 11 6-Cys domain proteins, we solved the 1.90 Å resolution crystal structure of the Pf12 ectodomain. Structural analysis reveals a unique organization between the membrane proximal and membrane distal domains and clear homology with the SRS-domain containing proteins of Toxoplasma gondii. Cross-linking and mass spectrometry confirm the previously identified Pf12-Pf41 heterodimeric complex, and analysis of individual cross-links supports an unexpected antiparallel organization. Collectively, the localization and structure of Pf12 and details of its interaction with Pf41 reveal important insight into the structural and functional properties of this archetypal member of the 6-Cys protein family.

The GPI-anchored 6-Cys protein Pv12 is present in detergent-resistant microdomains of Plasmodium vivax blood stage schizonts.

Plasmodium vivax malaria remains one of the tropical diseases causing an enormous burden on global public health. Several proteins located on this parasite species' merozoite surface have been considered the most suitable antigens for being included in an anti-malarial vaccine, given the functional role they play during the parasite's interaction with red blood cells. The present study identifies and characterizes the P. vivax Pv12 surface protein which was evaluated by using molecular biology and immunochemistry assays; its antigenic potential was also examined in natural and experimental P. vivax malaria infections. The P. vivax VCG-1 strain Pv12 gene encodes a 362 amino acid-long protein exhibiting a signal peptide, a glycosylphosphatidylinositol (GPI) anchor sequence and two 6-Cys domains. The presence of the Pv12 protein on the parasite's surface and its association with detergent-resistant membrane complexes, together with its antigenic potential, supports the notion that this antigen could play an important role as a red blood cell binding ligand. Further studies aimed at establishing the immunogenicity and protection-inducing ability of the Pv12 protein or its products in the Aotus experimental model are thus suggested.

Proteomic and genetic analyses demonstrate that Plasmodium berghei blood stages export a large and diverse repertoire of proteins.

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.

Proteomic analysis of the Theileria annulata schizont.

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.

Isolation of erythrocytes infected with viable early stages of Plasmodium falciparum by flow cytometry.

The erythrocytic life cycle of Plasmodium falciparum is highly associated with severe clinical symptoms of malaria that causes hundreds of thousands of death each year. The parasite develops within human erythrocytes leading to the disruption of the infected red blood cell (iRBC) prior to the start of a new cycle of erythrocyte infection. Emerging mechanisms of resistance against antimalarial drugs require improved knowledge about parasite's blood stages to facilitate new alternative antimalarial strategies. For the analysis of young blood stages of Plasmodium at the molecular level, the isolation of ring stages is essential. However, early stages can hardly be separated from both, late stages and non-infected red blood cells using conventional methods. Here, iRBCs were stained with the DNA-binding dyes Vybrant® DyeCycle™ Violet and SYBR® Green I. Subsequently, cells were subjected to flow-cytometric analysis. This enabled the discrimination of early stage iRBCs as well as late-stage iRBCs from non-infected erythrocytes and the properties of the used dyes were evaluated. Moreover, early stage iRBCs were isolated with high purity (>98%) by FACS. Subsequently, development of sorted early stages of the parasite was monitored over time and compared with control cultures. The described flow cytometry method, based on staining with Vybrant DyeCycle Violet, allows the isolation of viable ring stages of the malarial agent P. falciparum, and thereby provides the basis for new, broad-range molecular investigations of the parasite.

Insights into the Plasmodium falciparum schizont phospho-proteome.

It is becoming clear that, as is the case with many human diseases, targeting protein phosphorylation in strategies aimed at developing the next generation of anti-malarials is likely to bear considerable fruit. A major barrier to this development, however, is the paucity of information regarding the role of protein phosphorylation in malaria. A major step has recently been taken in this area with the publication of the first analyses of the phospho-proteome of the most virulent species of human malaria Plasmodium falciparum. Here, we discuss these studies.

Plasmodium falciparum proteome changes in response to doxycycline treatment.

BACKGROUND: The emergence of Plasmodium falciparum resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in P. falciparum have not yet been clearly defined, particularly at the protein level. METHODS: A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ). RESULTS: After doxycycline treatment, 32 and 40 P. falciparum proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis. CONCLUSIONS: In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts.

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins. Proteins from seven known complexes were identified including MSP-1/7, the low (RAP1/2 and RAP1/3), and high (RhopH1/H2/H3) molecular weight rhoptry complexes, and the invasion motor complex (GAP45/GAP50/myosinA). Remarkably, a large proportion of identified spectra were derived from only 4 proteins: the GPI-anchored proteins MSP-1 and Pf92, the putative GPI-anchored protein Pf113 and RAP-1, the core component of the two RAP complexes. Each of these proteins predominated in high molecular weight species suggesting their aggregation in much larger complexes than anticipated. To demonstrate that the procedure had isolated novel complexes we focussed on MSP-1, which predominated as a distinct species at approximately 500 kDa by BN-PAGE, approximately twice its expected size. Chemical cross-linking supports the existence of a stable MSP-1 oligomer of approximately 500 kDa, probably comprising a highly stable homodimeric species. Our observations also suggests that oligomerization of MSP-1 is likely to occur outside the C-terminal epidermal growth factor (EGF)-like domains. Confirmation of MSP-1 oligomerization, together with the isolation of a number of known complexes by BN-PAGE, makes it highly likely that novel interactions occur amongst members of this proteome.