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Female inheritance of malarial lap genes is essential for mosquito transmission.

Raine JD, Ecker A, Mendoza J, Tewari R, Stanway RR, Sinden RE - PLoS Pathog. (2007)

Bottom Line: Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Deltapblap sporozoites, but not if the individual Deltapblap parasite lines were crossed amongst each other.We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation.This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, London, United Kingdom.

ABSTRACT
Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite-mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Deltapblap sporozoites, but not if the individual Deltapblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Deltapbs47) and male-deficient (Deltapbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Deltapblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages.

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Transmission Electron Micrographs of wt and Δpblap OocystsAll images taken on day 13 p.i. unless otherwise indicated. Scale bar = 1 μm (A, B, F) or 5 μm (C–E). ep, midgut epithelium.(A) wt oocyst showing normal morphology of the endoplasmic reticulum (er).(B) Δpblap2 oocyst showing extensive expansion of the endoplasmic reticulum (er).(C) Δpblap1 oocyst (day 27 p.i.) showing extensive expansion of the endoplasmic reticulum (er) and some budding sporozoites (s).(D) wt oocyst showing normal morphology following cytokinesis to produce hundreds of daughter sporozoites (s).(E) Δpblap2 oocyst showing extensive degeneration and few nuclei (some of which are labelled n).(F) Degenerate Δpblap4 oocyst showing prominent melanization (m) of the extracellular oocyst wall (cw) which appears to spread into the mosquito basal lamina (bl).
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ppat-0030030-g003: Transmission Electron Micrographs of wt and Δpblap OocystsAll images taken on day 13 p.i. unless otherwise indicated. Scale bar = 1 μm (A, B, F) or 5 μm (C–E). ep, midgut epithelium.(A) wt oocyst showing normal morphology of the endoplasmic reticulum (er).(B) Δpblap2 oocyst showing extensive expansion of the endoplasmic reticulum (er).(C) Δpblap1 oocyst (day 27 p.i.) showing extensive expansion of the endoplasmic reticulum (er) and some budding sporozoites (s).(D) wt oocyst showing normal morphology following cytokinesis to produce hundreds of daughter sporozoites (s).(E) Δpblap2 oocyst showing extensive degeneration and few nuclei (some of which are labelled n).(F) Degenerate Δpblap4 oocyst showing prominent melanization (m) of the extracellular oocyst wall (cw) which appears to spread into the mosquito basal lamina (bl).

Mentions: Light microscopy revealed the presence of two distinct populations of Δpblap1, Δpblap2, Δpblap4, and Δpblap6 oocysts: those that displayed a phenotype reminiscent of immature wt oocysts (i.e., non-sporulated), and those that appeared vacuolated/degenerate compared to wt (Figure 2). Transmission electron microscopy analysis of Δpblap1, Δpblap2, and Δpblap4 oocysts further confirmed these findings and revealed that oocysts of these parasites possessed an endoplasmic reticulum that was highly vacuolated compared to that of wt parasites (Figure 3). On day 13 p.i., the nuclear organization of Δpblap1, Δpblap2, and Δpblap4 oocysts appeared “immature” as indicated by the presence of few but large nuclei (Figure 3). By comparison, wt oocysts of the same age had formed sporozoites, each with their own (haploid) nucleus.


Female inheritance of malarial lap genes is essential for mosquito transmission.

Raine JD, Ecker A, Mendoza J, Tewari R, Stanway RR, Sinden RE - PLoS Pathog. (2007)

Transmission Electron Micrographs of wt and Δpblap OocystsAll images taken on day 13 p.i. unless otherwise indicated. Scale bar = 1 μm (A, B, F) or 5 μm (C–E). ep, midgut epithelium.(A) wt oocyst showing normal morphology of the endoplasmic reticulum (er).(B) Δpblap2 oocyst showing extensive expansion of the endoplasmic reticulum (er).(C) Δpblap1 oocyst (day 27 p.i.) showing extensive expansion of the endoplasmic reticulum (er) and some budding sporozoites (s).(D) wt oocyst showing normal morphology following cytokinesis to produce hundreds of daughter sporozoites (s).(E) Δpblap2 oocyst showing extensive degeneration and few nuclei (some of which are labelled n).(F) Degenerate Δpblap4 oocyst showing prominent melanization (m) of the extracellular oocyst wall (cw) which appears to spread into the mosquito basal lamina (bl).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1808070&req=5

ppat-0030030-g003: Transmission Electron Micrographs of wt and Δpblap OocystsAll images taken on day 13 p.i. unless otherwise indicated. Scale bar = 1 μm (A, B, F) or 5 μm (C–E). ep, midgut epithelium.(A) wt oocyst showing normal morphology of the endoplasmic reticulum (er).(B) Δpblap2 oocyst showing extensive expansion of the endoplasmic reticulum (er).(C) Δpblap1 oocyst (day 27 p.i.) showing extensive expansion of the endoplasmic reticulum (er) and some budding sporozoites (s).(D) wt oocyst showing normal morphology following cytokinesis to produce hundreds of daughter sporozoites (s).(E) Δpblap2 oocyst showing extensive degeneration and few nuclei (some of which are labelled n).(F) Degenerate Δpblap4 oocyst showing prominent melanization (m) of the extracellular oocyst wall (cw) which appears to spread into the mosquito basal lamina (bl).
Mentions: Light microscopy revealed the presence of two distinct populations of Δpblap1, Δpblap2, Δpblap4, and Δpblap6 oocysts: those that displayed a phenotype reminiscent of immature wt oocysts (i.e., non-sporulated), and those that appeared vacuolated/degenerate compared to wt (Figure 2). Transmission electron microscopy analysis of Δpblap1, Δpblap2, and Δpblap4 oocysts further confirmed these findings and revealed that oocysts of these parasites possessed an endoplasmic reticulum that was highly vacuolated compared to that of wt parasites (Figure 3). On day 13 p.i., the nuclear organization of Δpblap1, Δpblap2, and Δpblap4 oocysts appeared “immature” as indicated by the presence of few but large nuclei (Figure 3). By comparison, wt oocysts of the same age had formed sporozoites, each with their own (haploid) nucleus.

Bottom Line: Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Deltapblap sporozoites, but not if the individual Deltapblap parasite lines were crossed amongst each other.We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation.This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, London, United Kingdom.

ABSTRACT
Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite-mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Deltapblap sporozoites, but not if the individual Deltapblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Deltapbs47) and male-deficient (Deltapbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Deltapblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages.

Show MeSH
Related in: MedlinePlus