Ability of Plasmodium falciparum gametocytes to become extracellular during gametogenesis in the mosquito midgut is a key step of the parasite life cycle. Reliable and quantitative measurement of the ...efficiency of gamete egress is currently constrained by the fact that this phenomenon is usually observed and quantified in vitro either by live microscopy, by statistically limited ultrastructural analysis or by surface antibody-based protocols which can interfere with this fast and complex cellular process.
A protocol was developed based on fluorescent wheat germ agglutinin (WGA) surface staining of erythrocytes containing mature P. falciparum gametocytes. After a single centrifugation step and within minutes from the induction of gametogenesis, the activated gametes can be inspected for presence or absence of the fluorescent WGA staining of the host erythrocyte membrane and scored respectively as intracellular or emerged from the erythrocyte.
Gametogenesis and gamete egress from WGA surface stained, infected erythrocytes occur with normal kinetics and efficiencies. Quantitative measurements of gamete egress can be obtained in live and in paraformaldehyde-fixed cells, which validates this protocol as a suitable tool both for live imaging studies and for higher throughput applications. The protocol was used here to provide functional information on the ability of gametes to egress through a single exit point induced in the host red blood cell membrane, and to re-analyse the phenotype of Pfg377- and osmiophilic body-defective gametes, suggesting that such parasite components are not directly involved in disruption and shedding of the erythrocyte membrane in female gamete egress.
The development of a reliable, fast, non-invasive and quantitative protocol to finely describe and to measure efficiency of P. falciparum gamete egress is a significant improvement in the tools for functional studies on this key process of the parasite life cycle. This protocol can be used to investigate the molecular mechanisms underlying gamete egress and its adaptation to high throughput applications will enable identification of transmission blocking inhibitors.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles ...lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infection.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Subtelomeric RIFIN genes constitute the most abundant multigene family in Plasmodium falciparum. RIFIN products are targets for the human immune response and contribute to the antigenic variability ...of the parasite. They are transmembrane proteins grouped into two sub-families (RIF_A and RIF_B). Although recent data show that RIF_A and RIF_B have different sub-cellular localisations and possibly different functions, the same structural organisation has been proposed for members of the two sub-families. Despite recent advances, our knowledge of the regulation of RIFIN gene expression is still poor and the biological role of the protein products remain obscure.
Comparative studies on RIFINs in three clones of P. falciparum (3D7, HB3 and Dd2) by Multidimensional scaling (MDS) showed that gene sequences evolve differently in the 5'upstream, coding, and 3'downstream regions, and suggested a possible role of highly conserved 3' downstream sequences. Despite the expected polymorphism, we found that the overall structure of RIFIN repertoires is conserved among clones suggesting a balance between genetic drift and homogenisation mechanisms which guarantees emergence of novel variants but preserves the functionality of genes. Protein sequences from a bona fide set of 3D7 RIFINs were submitted to predictors of secondary structure elements. In contrast with the previously proposed structural organisation, no signal peptide and only one transmembrane helix were predicted for the majority of RIF_As. Finally, we developed a strategy to obtain a reliable 3D-model for RIF_As. We generated 265 possible structures from 53 non-redundant sequences, from which clustering and quality assessments selected two models as the most representative for putative RIFIN protein structures.
First, comparative analyses of RIFIN repertoires in different clones of P. falciparum provide insights on evolutionary mechanisms shaping the multigene family. Secondly, we found that members of the two sub-families RIF_As and RIF_Bs have different structural organization in accordance with recent experimental results. Finally, representative models for RIF_As have an "Armadillo-like" fold which is known to promote protein-protein interactions in diverse contexts.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We ...developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility.
Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA).
A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector.
The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.
Summary
Obligate intracellular pathogens actively remodel their host cells to boost propagation, survival, and persistence. Plasmodium falciparum, the causative agent of the most severe form of ...malaria, assembles a complex secretory system in erythrocytes. Export of parasite factors to the erythrocyte membrane is essential for parasite sequestration from the blood circulation and a major factor for clinical complications in falciparum malaria. Historic and recent molecular reports show that host cell remodelling is not exclusive to P. falciparum and that parasite‐induced intra‐erythrocytic membrane structures and protein export occur in several Plasmodia. Comparative analyses of P. falciparum asexual and sexual blood stages and imaging of liver stages from transgenic murine Plasmodium species show that protein export occurs in all intracellular phases from liver infection to sexual differentiation, indicating that mammalian Plasmodium species evolved efficient strategies to renovate erythrocytes and hepatocytes according to the specific needs of each life cycle phase. While the repertoireof identified exported proteins is remarkably expanded in asexual P. falciparum blood stages, the putative export machinery and known targeting signatures are shared across life cycle stages. A better understanding of the molecular mechanisms underlying Plasmodium protein export could assist in designing novel strategies to interrupt transmission between Anopheles mosquitoes and humans.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Summary
In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties ...rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite‐induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive ‘knob’ structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface bythese parasites, and the expression of the var gene family, which encodes 50–60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Phallosome morphology (DV/D ratio) and allozyme variation were used to reexamine the transition from Culex pipiens pipiens L. to Cx. p. quinquefasciatus Say, detected in California from the northern ...Central Valley to the Mexican border of the United States of America. Significant deficiency of heterozygotes was observed at the diagnostic locus Mdhp-2 in populations from the central part of the hybrid zone. Long tails of introgression were detected: populations from both north and south ends of the transect were not genetically pure Cx. p. pipiens or Cx. p. quinquefasciatus, respectively, as previously considered, but included approximately 5% introgressed individuals. A narrow reversed cline from the Delta area into the Sacramento Valley, characterized by increasing frequencies of Cx. p. quinquefasciatus alleles proceeding to the north, was confirmed. Both these clines appear to be related mainly to temperature gradients. Over the last 50 yr, an increase in the proportion of Cx. p. pipiens DV/D phenotypes was detected proceeding north to south along the main latitudinal cline, as well as in the narrow reversed cline. Accordingly, the center of the main latitudinal hybrid zone has apparently moved approximately 100 km to the south. This phenomenon is only partially paralleled by the differentiated locus Pgm of the 3 for which comparison was possible. Similarities to and differences from previous studies are discussed, also in relation with comparable data from another hybrid zone between Cx. p. pipiens and Cx. p. quinquefasciatus recently detected in Madagascar. Hybrid index scores based on differentiated allozymes and the diagnostic locus Mdhp-2 prove to be better descriptors than the DV/D ratio of hybridization and introgression occurring between Cx. p. pipiens and Cx. p. quinquefasciatus. This seems to be caused mainly by the influence of temperature on male genitalia development
A genome-wide expression analysis was undertaken to identify novel genes specifically activated from early stages of gametocytogenesis in
Plasmodium falciparum. A comparative analysis was conducted ...on sexually induced cultures of reference parasite clone 3D7 and its gametocyteless derivative clone F12. Competitive hybridisations on long-oligomer microarrays representing 4488
P. falciparum genes identified a remarkably small number of transcripts differentially produced in the two clones. Upregulation of the mRNAs for the early gametocyte markers Pfs16 and Pfg27 was however readily detected in 3D7, and such genes were used as reference transcripts in a comparative time course analysis of 3D7 and F12 parasites between 30 and 40
h post-invasion in cultures induced to enter gametocytogenesis. One hundred and seventeen genes had expression profiles which correlated to those of
pfs16 and
pfg27, and Northern blot analysis and published proteomic data identified those whose expression was gametocyte-specific. Immunofluorescence analysis with antibodies against two of these gene products identified two novel parasite membrane associated, sexual stage-specific proteins. One was produced from stage I gametocytes and the second showed peak production in stage II gametocytes. The two proteins were named Pfpeg-3 and Pfpeg-4, for
P. falciparum proteins of
early
gametocytes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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