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Frontiers in Cellular and Infection... 2021Colonization of the mosquito host by parasites is achieved by sexually differentiated gametocytes. Gametocytogenesis, gamete formation and fertilization are tightly...
Colonization of the mosquito host by parasites is achieved by sexually differentiated gametocytes. Gametocytogenesis, gamete formation and fertilization are tightly regulated processes, and translational repression is a major regulatory mechanism for stage conversion. Here, we present a characterization of a RNA binding protein, UIS12, that contains two conserved eukaryotic RNA recognition motifs (RRM). Targeted gene deletion resulted in viable parasites that replicate normally during blood infection, but form fewer gametocytes. Upon transmission to mosquitoes, both numbers and size of midgut-associated oocysts were reduced and their development stopped at an early time point. As a consequence, no salivary gland sporozoites were formed indicative of a complete life cycle arrest in the mosquito vector. Comparative transcript profiling in mutant and wild-type infected red blood cells revealed a decrease in transcript abundance of mRNAs coding for signature gamete-, ookinete-, and oocyst-specific proteins in parasites. Together, our findings indicate multiple roles for UIS12 in regulation of gene expression after blood infection in good agreement with the pleiotropic defects that terminate successful sporogony and onward transmission to a new vertebrate host.
Topics: Animals; Anopheles; Oocysts; Plasmodium berghei; RNA-Binding Proteins; Sporozoites
PubMed: 33747980
DOI: 10.3389/fcimb.2021.624945 -
Emerging Microbes & Infections Dec 2022The family of apicomplexan specific proteins contains caspases-like proteins called "metacaspases". These enzymes are present in the malaria parasite but absent in...
The family of apicomplexan specific proteins contains caspases-like proteins called "metacaspases". These enzymes are present in the malaria parasite but absent in human; therefore, these can be explored as potential drug targets. We deleted the MCA-2 gene from genome using a gene knockout strategy to decipher its precise function. This study has identified that MCA-2 plays an important role in parasite transmission since it is critical for the formation of gametocytes and for maintaining an appropriate number of infectious sporozoites required for sporogony. It is noticeable that a significant reduction in gametocyte, oocysts, ookinete and sporozoites load along with a delay in hepatocytes invasion were observed in the MCA-2 knockout parasite. Furthermore, a study found the two MCA-2 inhibitory molecules known as C-532 and C-533, which remarkably inhibited the MCA-2 activity, abolished the parasite growth, and also impaired the transmission cycle of and in . Our findings indicate that the deletion of MCA-2 hampers the development during erythrocytic and exo-erythrocytic stages, and its inhibition by C-532 and C-533 critically affects the malaria transmission biology.
Topics: Animals; Gametogenesis; Humans; Malaria; Plasmodium berghei; Protozoan Proteins; Sporozoites
PubMed: 35264080
DOI: 10.1080/22221751.2022.2052357 -
BMC Genomics Sep 2017The clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the... (Comparative Study)
Comparative Study
BACKGROUND
The clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the malaria parasite must differentiate into sexual forms, termed gametocytes, which are ingested by a mosquito vector. Sexual differentiation produces either female or male gametocytes, and involves significant morphological and biochemical changes. These transformations prepare gametocytes for the rapid progression to gamete formation and fertilisation, which occur within 20 min of ingestion. Here we present the transcriptomes of asexual, female, and male gametocytes in P. berghei, and a comprehensive statistically-based differential-expression analysis of the transcriptional changes that underpin this sexual differentiation.
RESULTS
RNA-seq analysis revealed numerous differences in the transcriptomes of female and male gametocytes compared to asexual stages. Overall, there is net downregulation of transcripts in gametocytes compared to asexual stages, with this trend more marked in female gametocytes. Our analysis identified transcriptional changes in previously-characterised gametocyte-specific pathways, which validated our approach. We also detected many previously-unreported female- and male-specific pathways and genes. Transcriptional biases in stage and gender were then used to investigate sex-specificity and sexual dimorphism of Plasmodium in an evolutionary context. Sex-related gene expression is well conserved between Plasmodium species, but relatively poorly conserved in related organisms outside this genus. This pattern of conservation is most evident in genes necessary for both male and female gametocyte formation. However, this trend is less pronounced for male-specific genes, which are more highly conserved outside the genus than genes specific to female development.
CONCLUSIONS
We characterised the transcriptional changes that are integral to the development of the female and male sexual forms of Plasmodium. These differential-expression patterns provide a vital insight into understanding the gender-specific characteristics of this essential stage that is the primary target for treatments that block parasite transmission. Our results also offer insight into the evolution of sex genes through Alveolata, and suggest that many Plasmodium sex genes evolved within the genus. We further hypothesise that male gametocytes co-opted pre-existing cellular machinery in their evolutionary history, whereas female gametocytes evolved more through the development of novel, parasite-specific pathways.
Topics: Gene Expression Profiling; Nucleotide Motifs; Phylogeny; Plasmodium berghei; RNA, Messenger; Sequence Homology, Nucleic Acid
PubMed: 28923023
DOI: 10.1186/s12864-017-4100-0 -
Acta Parasitologica Sep 2019Piperaquine (PQ) is one of the major components of artemisinin-based combination therapy for malaria. However, the mechanism of PQ resistance has remained unclear. (Comparative Study)
Comparative Study
BACKGROUND
Piperaquine (PQ) is one of the major components of artemisinin-based combination therapy for malaria. However, the mechanism of PQ resistance has remained unclear.
METHODS
In this study, we infected mice with PQ-resistant Plasmodium berghei ANKA strain line (PbPQR) or PQ-sensitive P. berghei ANKA strain line (PbPQS) and their survival rates, parasitemia, and spleen sizes were compared. In addition, we constructed genomic DNA subtractive library of spleens from the infected mice, and screened the potential PQ-resistant related genes from genomic DNA of PbPQR line using the representational difference analysis (RDA) method. Clones of the subtractive library were screened by PCR, and related genes were sequenced and analyzed using BLAST software of NCBI.
RESULTS
Compared to PbPQS-infected mice, PbPQR-infected mice survived significantly longer, and had significantly lowered parasitemia rate and significantly increased splenomegaly. Among the total of 502 clones picked, 494 were sequenced and 96 unique PCR fragments were obtained; in which 24 DNA fragments were homologous to chromosomes related to immune function of mice. ORF Finder blasting showed that at the protein level, 26 encoded proteins were homologous to 18 hypothetical PbANKA proteins and 13 encoded proteins were homologous to "ferlin-like protein" family of PbANKA. In addition, there were more immune-related DNA molecules, ubiquitous PbANKA homology at the ORF fragment level, and enriched ferlin-like protein families identified from PbPQR-infected mice than those from PbPQS-infected mice.
CONCLUSION
These findings suggest that PbPQR may induce stronger protective immune response than that of PbPQS in infected mice.
Topics: Animals; Antimalarials; Disease Models, Animal; Humans; Malaria; Male; Mice; Plasmodium berghei; Protozoan Proteins; Quinolines; Virulence
PubMed: 31321598
DOI: 10.2478/s11686-019-00100-5 -
PloS One 2015Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the...
Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ) and artemisinin (ART) are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ) and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT) and the multidrug resistant gene (pfmdr), CQ resistance has also been associated with increased parasite glutathione (GSH) levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs) encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko) or higher (pbggcs-oe) levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ). Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.
Topics: Animals; Artemisinins; Chloroquine; Drug Resistance; Glutamate-Cysteine Ligase; Glutathione; Malaria; Mice; Plasmodium berghei; Protozoan Proteins
PubMed: 26010448
DOI: 10.1371/journal.pone.0128212 -
Cellular Microbiology Jan 2017Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an...
Characterization of the Plasmodium falciparum and P. berghei glycerol 3-phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast.
Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti-malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3-phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N-terminal targeting sequence to GFP and 3' tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site-directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3-phosphate acyltransferase in malaria parasites.
Topics: Apicoplasts; Bacteria; DNA Mutational Analysis; Fatty Acids; Gene Knockout Techniques; Genetic Complementation Test; Glycerol-3-Phosphate O-Acyltransferase; Plasmodium berghei; Plasmodium falciparum; Protein Transport
PubMed: 27324409
DOI: 10.1111/cmi.12633 -
BMC Research Notes Feb 2022The current culture system for P. berghei still requires modifications in consistency and long-term maintenance of parasites considering their pathogenicity in culture...
OBJECTIVES
The current culture system for P. berghei still requires modifications in consistency and long-term maintenance of parasites considering their pathogenicity in culture media. Therefore, this study designed to further improvement of culture conditions and designing a cost-effective culture medium with minimum changes in pathogenicity for in vitro culture of P. berghei.
RESULTS
Results indicated that the rate of parasitaemia in our modified method remained statistically stable between days one to seven (P = 0.07). The current modified cultivation method was more efficient in maintaining of parasites for further days. Furthermore, in current method the stability of parasitaemia rate during day1 to day7 was in better rate compared to that in Ronan Jambou et al. and the differences between two methods were statistically significant (P = 0.001). The virulence of cultivated parasites in our modified method remained similar to frozen stock parasites as positive control group. No significant differences were seen in survival time between two groups of mice those were infected with either cultivated parasites or stock freeze parasites (P = 0.39) with the mean survival time of 20.83 ± 3.84 and 19.66 ± 1.21 days, respectively. Herein, we achieved a simple, cost-effective and applicable technique for culture of P. berghei.
Topics: Animals; Cost-Benefit Analysis; Culture Media; Mice; Mice, Inbred BALB C; Parasitemia; Plasmodium berghei
PubMed: 35168649
DOI: 10.1186/s13104-022-05946-z -
Pathogens and Global Health Mar 2015Plasmodium berghei was identified as a parasite of thicket rats (Grammomys dolichurus) and Anopheles dureni mosquitoes in African highland forests. Successful adaptation... (Review)
Review
Plasmodium berghei was identified as a parasite of thicket rats (Grammomys dolichurus) and Anopheles dureni mosquitoes in African highland forests. Successful adaptation to a range of rodent and mosquito species established P. berghei as a malaria model parasite. The introduction of stable transfection technology, permitted classical reverse genetics strategies and thus systematic functional profiling of the gene repertoire. In the past 10 years following the publication of the P. berghei genome sequence, many new tools for experimental genetics approaches have been developed and existing ones have been improved. The infection of mice is the principal limitation towards a genome-wide repository of mutant parasite lines. In the past few years, there have been some promising and most welcome developments that allow rapid selection and isolation of recombinant parasites while simultaneously minimising animal usage. Here, we provide an overview of all the currently available tools and methods.
Topics: Animals; Anopheles; Disease Models, Animal; Genetic Vectors; Genome-Wide Association Study; Host-Parasite Interactions; Life Cycle Stages; Malaria; Mice; Molecular Sequence Data; Organisms, Genetically Modified; Plasmodium berghei; Rats; Transfection
PubMed: 25789828
DOI: 10.1179/2047773215Y.0000000006 -
Malaria Journal Jun 2021Malaria is a fatal disease that presents clinically as a continuum of symptoms and severity, which are determined by complex host-parasite interactions. Clearance of...
BACKGROUND
Malaria is a fatal disease that presents clinically as a continuum of symptoms and severity, which are determined by complex host-parasite interactions. Clearance of infection is believed to be accomplished by the spleen and mononuclear phagocytic system (MPS), independent of artemisinin treatment. The spleen filters infected red blood cells (RBCs) from circulation through immune-mediated recognition of the infected RBCs followed by phagocytosis. This study evaluated the tolerance of four different strains of mice to Plasmodium berghei strain K173 (P. berghei K173), and the differences in the role of the spleen in controlling P. berghei K173 infection.
METHODS
Using different strains of mice (C57BL/6, BALB/C, ICR, and KM mice) infected with P. berghei K173, the mechanisms leading to splenomegaly, histopathology, splenocyte activation and proliferation, and their relationship to the control of parasitaemia and host mortality were examined and evaluated.
RESULTS
Survival time of mice infected with P. berghei K173 varied, although the infection was uniformly lethal. Mice of the C57BL/6 strain were the most resistant, while mice of the strain ICR were the most susceptible. BALB/c and KM mice were intermediate. In the course of P. berghei K173 infection, all infected mice experienced significant splenomegaly. Parasites were observed in the red pulp at 3 days post infection (dpi) in all animals. All spleens retained late trophozoite stages as well as a fraction of earlier ring-stage parasites. The percentages of macrophages in infected C57BL/6 and KM mice were higher than uninfected mice on 8 dpi. Spleens of infected ICR and KM mice exhibited structural disorganization and remodelling. Furthermore, parasitaemia was significantly higher in KM versus C57BL/6 mice at 8 dpi. The percentages of macrophages in ICR infected mice were lower than uninfected mice, and the parasitaemia was higher than other strains.
CONCLUSIONS
The results presented here demonstrate the rate of splenic mechanical filtration and that splenic macrophages are the predominant roles in controlling an individual's total parasite burden. This can influence the pathogenesis of malaria. Finally, different genetic backgrounds of mice have different splenic mechanisms for controlling malaria infection.
Topics: Animals; Hematologic Tests; Malaria; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Plasmodium berghei; Spleen; Mice
PubMed: 34090420
DOI: 10.1186/s12936-021-03786-z -
Nucleic Acids Research Jan 2020The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered...
The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.
Topics: Animals; Anopheles; Argonaute Proteins; Female; Genes, Reporter; Genetic Engineering; Green Fluorescent Proteins; Life Cycle Stages; Mice; Mice, Inbred C57BL; Mosquito Vectors; Organisms, Genetically Modified; Perforin; Plasmodium berghei; Protozoan Proteins; RNA Interference; RNA, Small Interfering; Transgenes
PubMed: 31680162
DOI: 10.1093/nar/gkz927