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BMC Microbiology Sep 2023Plasmodium berghei has been used as a preferred model for studying human malaria, but only a limited number of disease-associated genes of P. berghei have been reported...
BACKGROUND
Plasmodium berghei has been used as a preferred model for studying human malaria, but only a limited number of disease-associated genes of P. berghei have been reported to date. Identification of new disease-related genes as many as possible will provide a landscape for better understanding the pathogenesis of P. berghei.
METHODS
Network module analysis method was developed and applied to identify disease-related genes in P. berghei genome. Sequence feature identification, gene ontology annotation, and T-cell epitope analysis were performed on these genes to illustrate their functions in the pathogenesis of P. berghei.
RESULTS
33,314 genes were classified into 4,693 clusters. 4,127 genes shared by six malaria parasites were identified and are involved in many aspects of biological processes. Most of the known essential genes belong to shared genes. A total of 63 clusters consisting of 405 P. berghei genes were enriched in rodent malaria parasites. These genes participate in various stages of parasites such as liver stage development and immune evasion. Combination of these genes might be responsible for P. berghei infecting mice. Comparing with P. chabaudi, none of the clusters were specific to P. berghei. P. berghei lacks some proteins belonging to P. chabaudi and possesses some specific T-cell epitopes binding by class-I MHC, which might together contribute to the occurrence of experimental cerebral malaria (ECM).
CONCLUSIONS
We successfully identified disease-associated P. berghei genes by network module analysis. These results will deepen understanding of the pathogenesis of P. berghei and provide candidate parasite genes for further ECM investigation.
Topics: Humans; Animals; Mice; Plasmodium berghei; Gene Ontology; Genes, Essential; Immune Evasion; Molecular Sequence Annotation
PubMed: 37735351
DOI: 10.1186/s12866-023-03019-0 -
MSphere Aug 2023During invasion, parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A...
During invasion, parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A sporozoite stage-specific gene silencing system revealed that rhoptry neck protein 2 (RON2), RON4, and RON5 are important for sporozoite invasion of mosquito salivary glands. Here, we further investigated the roles of RON4 during sporozoite infection of the liver . Following intravenous inoculation of RON4-knockdown sporozoites into mice, we demonstrated that sporozoite RON4 has multiple functions during sporozoite traversal of sinusoidal cells and infection of hepatocytes. infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.IMPORTANCEMalarial parasite transmission to mammals is established when sporozoites are inoculated by mosquitoes and migrate through the bloodstream to infect hepatocytes. Many aspects of the molecular mechanisms underpinning migration and cellular invasion remain largely unelucidated. By applying a sporozoite stage-specific gene silencing system in the rodent malarial parasite, , we demonstrated that rhoptry neck protein 4 (RON4) is crucial for sporozoite infection of the liver . Combined with investigations, it was revealed that RON4 functions during a crossing of the sinusoidal cell layer and invading hepatocytes, at an early stage of liver infection, by mediating the sporozoite capacity for adhesion and the onset of motility. Since RON4 is also expressed in merozoites and tachyzoites, our findings contribute to understanding the conserved invasion mechanisms of parasites.
Topics: Animals; Mice; Plasmodium berghei; Liver; Malaria; Sporozoites; Protozoan Proteins; Hepatocytes
PubMed: 37272704
DOI: 10.1128/msphere.00587-22 -
Molecular Microbiology Mar 2024Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade...
Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.
Topics: Animals; Culicidae; Parasites; Protozoan Proteins; Oocysts; Plasmodium berghei; Sporozoites
PubMed: 37314965
DOI: 10.1111/mmi.15078 -
Nature Communications Dec 2023Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian...
Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian hosts. To better understand the 3D organisation of male gametogenesis, we used serial block face scanning electron microscopy (SBF-SEM) and serial-section cellular electron tomography (ssET) of P. berghei microgametocytes to examine key structures during male gamete formation. Our data reveals an elaborate organisation of axonemes coiling around the nucleus in opposite directions forming a central axonemal band in microgametocytes. Furthermore, we discover the nucleus of microgametes to be tightly coiled around the axoneme in a complex structure whose formation starts before microgamete emergence during exflagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlight some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite.
Topics: Male; Animals; Plasmodium berghei; Haploidy; Germ Cells; Anopheles; Flagella; Mammals
PubMed: 38092766
DOI: 10.1038/s41467-023-43877-w -
Malaria Journal Nov 2019Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a...
BACKGROUND
Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice.
METHODS
To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria.
RESULTS
Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice.
CONCLUSIONS
Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.
Topics: Animals; Antimalarials; Malaria, Cerebral; Malaria, Falciparum; Mice; Mice, Inbred C57BL; Plasmodium berghei; Plasmodium falciparum; Tamoxifen
PubMed: 31775753
DOI: 10.1186/s12936-019-3012-7 -
Experimental Parasitology Feb 2018Plasmodium berghei (Pb) XAT, a rodent malaria parasite, is an irradiation-attenuated variant derived from the lethal strain Pb NK65. Differences in genome sequence,... (Comparative Study)
Comparative Study
Plasmodium berghei (Pb) XAT, a rodent malaria parasite, is an irradiation-attenuated variant derived from the lethal strain Pb NK65. Differences in genome sequence, protein structure and function between Pb XAT and Pb NK65 are currently unknown. In this study, to investigate genetic alterations in Pb XAT, we performed comparative genomics and proteomics analyses of nonlethal and lethal strains of Pb. We found mutations, such as a deletion mutation in rhoptry-associated protein (rap) 1, and deletion of rap2/3 and skeleton-binding protein 1 (sbp1), in Pb XAT. RAP1 is required for targeting of RAP2 to the rhoptries. However, the contribution of RAP2/3 to the lethality of Plasmodium is unclear. Therefore, we generated RAP1- and RAP2/3-deficient mutants of Pb ANKA, a reference strain of P. berghei. Furthermore, we investigated the effect of RAP1 and RAP2/3 deficiency on the outcome of infection. The parasitemia in mice infected with RAP1-deficient parasites was increased compared to that in control parasite-infected mice during the early phase of infection. However, mice infected with RAP1-deficient parasites survived longer than did control parasite-infected mice. Moreover, mice infected with RAP2/3-deficient parasites showed low levels of parasitemia and ultimately recovered from the infection The aim of this study was to investigate the effect of RAP2/3 expression on the outcome of infection with Pb XAT using a RAP2/3-expressing Pb XAT. Results showed that complementation of RAP2/3 expression in Pb XAT partially restored virulence. Our findings suggest that RAP1 and RAP2/3 contribute to virulence and a decrease in their expression explains the loss of virulence of the Pb XAT strain.
Topics: Animals; Chromatography, Liquid; DNA, Protozoan; Erythrocytes; Female; Genomics; Malaria; Mice; Mice, Inbred C57BL; Parasitemia; Plasmodium berghei; Polymerase Chain Reaction; Proteomics; Protozoan Proteins; Reverse Transcription; Sequence Deletion; Specific Pathogen-Free Organisms; Tandem Mass Spectrometry; Virulence
PubMed: 29307564
DOI: 10.1016/j.exppara.2018.01.001 -
Free Radical Biology & Medicine Jun 2016Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use...
Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions.
Topics: Animals; Antioxidants; Cell Nucleus; DNA Damage; DNA, Mitochondrial; Gene Knockout Techniques; Glutamate-Cysteine Ligase; Glutathione; Glutathione Reductase; Life Cycle Stages; Malaria; Oxidative Stress; Plasmodium berghei; Thioredoxins
PubMed: 26952808
DOI: 10.1016/j.freeradbiomed.2016.02.032 -
Experimental Parasitology Sep 2015Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes...
Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies.
Topics: Animals; Dose-Response Relationship, Drug; Electrophoresis, Gel, Two-Dimensional; Gametogenesis; Hydroxyurea; Male; Mice; Mice, Inbred BALB C; Phosphoproteins; Phosphorylation; Plasmodium berghei; Protozoan Proteins; Tandem Mass Spectrometry; Titanium
PubMed: 26008612
DOI: 10.1016/j.exppara.2015.05.010 -
Journal of Leukocyte Biology May 2016
Topics: Animals; Humans; Malaria; Parasites; Plasmodium berghei
PubMed: 27130887
DOI: 10.1189/jlb.4CE1115-494R -
Open Biology Aug 2022Protein phosphatase 1 (PP1) is a key enzyme for development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the...
Protein phosphatase 1 (PP1) is a key enzyme for development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1-LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of oocyst development through its interaction with PbLRR1.
Topics: Animals; Leucine-Rich Repeat Proteins; Oocysts; Phosphorylation; Plasmodium berghei; Protein Phosphatase 1
PubMed: 35920043
DOI: 10.1098/rsob.220015