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Experimental Parasitology Apr 1989In this study the DNA content and the karyotype of clones of Plasmodium berghei, which differed in the capability to produce gametocytes, were determined. The DNA...
In this study the DNA content and the karyotype of clones of Plasmodium berghei, which differed in the capability to produce gametocytes, were determined. The DNA content per haploid genome was established by cytofluorometric methods after staining of the haploid merozoites with DNA-specific fluorescent dyes. Field inversion gel electrophoresis was used to establish the number and size of the chromosomes. Parasites of a high gametocyte producer clone (original HP) and a low producer clone (original LP) contained 13 to 14 chromosomes in the size range of 0.5-3.8 megabase. In four independent experiments parasites of the original HP clone were maintained in mice and were mechanically transmitted for prolonged periods of time (up to 90 weeks). During the transmission period the capability to produce gametocytes decreased in all four lines. After mosquito transmission of parasites from these low producer lines, the gametocyte production returned to the level of the original HP clone. The total DNA content per haploid genome of low producer parasites was not significantly different from that of HP parasites. During prolonged periods of asexual multiplication of the HP clone in vivo, changes in the relative size of several chromosomes were detected. Mosquito transmission of the original HP clone did not result in a change of the karyotype. However, novel karyotypes were readily observed after mosquito transmission of parasites of the low producer lines. The decrease of the capability to produce gametocytes did not necessarily involve detectable changes in DNA content or in karyotype.
Topics: Animals; Anopheles; DNA; Electrophoresis, Agar Gel; Flow Cytometry; Karyotyping; Mice; Plasmodium berghei; Polymorphism, Genetic; Rats; Rats, Inbred Strains
PubMed: 2649389
DOI: 10.1016/0014-4894(89)90109-4 -
International Journal For Parasitology Feb 2007Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a natural malaria infection. The molecular mechanisms underlying sporozoite...
Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a natural malaria infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that infection by Plasmodium falciparum and Plasmodium yoelii sporozoites depends on CD81 and cholesterol-dependent tetraspanin-enriched microdomains (TEMs) on the hepatocyte surface. Here we have analyzed the role of CD81 and TEMs during infection by sporozoites from the rodent parasite Plasmodium berghei. We found that depending on the host cell type, P. berghei sporozoites can use several distinct pathways for invasion. Infection of human HepG2, HuH7 and HeLa cells by P. berghei does not depend on CD81 or host membrane cholesterol, whereas both CD81 and cholesterol are required for infection of mouse hepatoma Hepa1-6 cells. In primary mouse hepatocytes, both CD81-dependent and -independent mechanisms participate in P. berghei infection and the relative contribution of the different pathways varies, depending on mouse genetic background. The existence of distinct invasion pathways may explain why P. berghei sporozoites are capable of infecting a wide range of host cell types in vitro. It could also provide a means for human parasites to escape immune responses and face polymorphisms of host receptors. This may have implications for the development of an anti-malarial vaccine targeting sporozoites.
Topics: Animals; Antigens, CD; Cell Line; Cholesterol; Hepatocytes; Host-Parasite Interactions; Humans; Membrane Proteins; Mice; Plasmodium berghei; Rats; Sporozoites; Tetraspanin 28; Tetraspanins
PubMed: 17112526
DOI: 10.1016/j.ijpara.2006.10.005 -
PloS One 2018Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for...
Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs.
Topics: Animals; Anti-Retroviral Agents; Antimalarials; Aspartic Acid Endopeptidases; Aspartic Acid Proteases; Lopinavir; Mice; Models, Molecular; Molecular Docking Simulation; Plasmodium berghei; Protease Inhibitors; Protozoan Proteins; Reverse Genetics; Saquinavir
PubMed: 30067811
DOI: 10.1371/journal.pone.0201556 -
Experimental Parasitology Oct 1974
Topics: Animals; Anopheles; Blood; Malaria; Mice; Microscopy, Electron; Plasmodium berghei; Salivary Glands
PubMed: 4606913
DOI: 10.1016/0014-4894(74)90058-7 -
Experimental Parasitology Feb 1992We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied...
We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events.
Topics: Animals; Gametogenesis; Genetic Variation; Karyotyping; Malaria; Mice; Mitosis; Mutation; Plasmodium berghei; Reproduction, Asexual
PubMed: 1730264
DOI: 10.1016/0014-4894(92)90133-u -
Frontiers in Immunology 2021Reticulon and the REEP family of proteins stabilize the high curvature of endoplasmic reticulum tubules. The REEP5 homolog in , YOP1 (YOP1), plays an important role in...
Reticulon and the REEP family of proteins stabilize the high curvature of endoplasmic reticulum tubules. The REEP5 homolog in , YOP1 (YOP1), plays an important role in the erythrocytic cycle of the ANKA and the pathogenesis of experimental cerebral malaria (ECM), but the mechanisms are largely unknown. Here, we show that protection from ECM in yop1Δ-infected mice is associated with reduced intracerebral Th1 accumulation, decreased expression of pro-inflammatory cytokines and chemokines, and attenuated pathologies in the brainstem, though the total number of CD4 and CD8 T cells sequestered in the brain are not reduced. Expression of adhesive molecules on brain endothelial cells, including ICAM-1, VCAM-1, and CD36, are decreased, particularly in the brainstem, where fatal pathology is always induced during ECM. Subsequently, CD8 T cell-mediated cell apoptosis in the brain is compromised. These findings suggest that yop1Δ parasites can be a useful tool for mechanistic investigation of cerebral malaria pathogenesis.
Topics: Animals; Disease Models, Animal; Female; Malaria, Cerebral; Mice; Mice, Inbred C57BL; Plasmodium berghei; Protozoan Proteins; T-Lymphocytes
PubMed: 34025654
DOI: 10.3389/fimmu.2021.642585 -
Molecular and Biochemical Parasitology Nov 2005Plasmodium falciparum contains two genes encoding different isotypes of alpha-tubulin, alpha-tubulin I and alpha-tubulin II. alpha-Tubulin II is highly expressed in male...
Plasmodium falciparum contains two genes encoding different isotypes of alpha-tubulin, alpha-tubulin I and alpha-tubulin II. alpha-Tubulin II is highly expressed in male gametocytes and forms part of the microtubules of the axoneme of male gametes. Here we present the characterization of Plasmodium berghei alpha-tubulin I and alpha-tubulin II that encode proteins of 453 and 450 amino acids, respectively. alpha-Tubulin II lacks the well-conserved three amino acid C-terminal extension including a terminal tyrosine residue present in alpha-tubulin I. Investigation of transcription by Northern analysis and RT-PCR and analysis of promoter activity by GFP tagging showed that alpha-tubulin I is expressed in all blood and mosquito stages. As expected, alpha-tubulin II was highly expressed in the male gametocytes, but transcription was also observed in the asexual blood stages, female gametocytes, ookinetes and oocysts. Gene disruption experiments using standard transfection technologies did not produce viable parasites indicating that both alpha-tubulin isotypes are essential for the asexual blood stages. Targeted modification of alpha-tubulin II by the addition of the three C-terminal amino acids of alpha-tubulin I did not affect either blood stage development nor male gamete formation. Attempts to modify the C-terminal region by adding a TAP tag to the endogenous alpha-tubulin II gene were not successful. Introduction of a transgene, expressing TAP-tagged alpha-tubulin II, next to the endogenous alpha-tubulin II gene, had no effect on the asexual blood stages but strongly impaired formation of male gametes. These results show that alpha-tubulin II not only plays an important role in the male gamete but is also expressed in and essential for asexual blood stage development.
Topics: Animals; Gene Expression; Germ Cells; Life Cycle Stages; Male; Plasmodium berghei; Promoter Regions, Genetic; Protozoan Proteins; Tubulin
PubMed: 16115694
DOI: 10.1016/j.molbiopara.2005.07.003 -
Molecular Microbiology Mar 2012The importance of pathogen-induced host cell remodelling has been well established for red blood cell infection by the human malaria parasite Plasmodium falciparum....
The importance of pathogen-induced host cell remodelling has been well established for red blood cell infection by the human malaria parasite Plasmodium falciparum. Exported parasite-encoded proteins, which often possess a signature motif, termed Plasmodium export element (PEXEL) or host-targeting (HT) signal, are critical for the extensive red blood cell modifications. To what extent remodelling of erythrocyte membranes also occurs in non-primate hosts and whether it is in fact a hallmark of all mammalian Plasmodium parasites remains elusive. Here we characterize a novel Plasmodium berghei PEXEL/HT-containing protein, which we term IBIS1. Temporal expression and spatial localization determined by fluorescent tagging revealed the presence of IBIS1 at the parasite/host interface during both liver and blood stages of infection. Targeted deletion of the IBIS1 protein revealed a mild impairment of intra-erythrocytic growth indicating a role for these structures in the rapid expansion of the parasite population in the blood in vivo. In red blood cells, the protein localizes to dynamic, punctate structures external to the parasite. Biochemical and microscopic data revealed that these intra-erythrocytic P. berghei-induced structures (IBIS) are membranous indicating that P. berghei, like P. falciparum, creates an intracellular membranous network in infected red blood cells.
Topics: Amino Acid Motifs; Animals; Cell Line; Cell Membrane; Erythrocytes; Female; Humans; Malaria; Mice; Mice, Inbred C57BL; Plasmodium berghei; Protein Transport; Protozoan Proteins
PubMed: 22329949
DOI: 10.1111/j.1365-2958.2012.08004.x -
Experimental Parasitology Jun 1976
Topics: Animals; Anopheles; Antigens; Blood; Female; Immunization; Malaria; Mice; Plasmodium berghei; Radiation Effects
PubMed: 773654
DOI: 10.1016/0014-4894(76)90048-5 -
MBio Jun 2016Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei...
UNLABELLED
Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity.
IMPORTANCE
Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite's successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite's autophagic pathway may represent a novel approach to control malarial infections.
Topics: Acyl Carrier Protein; Animals; Apicoplasts; Autophagy; Autophagy-Related Protein 8 Family; Hepatocytes; Humans; Liver; Malaria; Membrane Proteins; Merozoites; Mice, Transgenic; Mutation; Organelles; Plasmodium berghei; Protozoan Proteins
PubMed: 27353755
DOI: 10.1128/mBio.00682-16