Parasite development in the mosquito.

<p>A) Exflagellation in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. The mean number of exflagellation centres was 7.0 for wild type and 6.8 for Pb<i>^pfpkg</i> parasites. Bar, mean ± SEM (Mann-Whitney U test: ns, not significant, p>0.05 compared to WT-GFP). B) Ookinete conversion in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type conversion was 63% and Pb<i>^pfpkg</i> conversion was 10%. Data shown as mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP). C) Gut oocyst numbers in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type infected guts contained 100 oocysts and Pb<i>^pfpkg</i> infected guts with less than 10 oocysts. Bar, mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP).</p

Generation of Pb<i>^pfpkg</i> parasites.

<p>A) Schematic diagram of the endogenous <i>pbpkg</i> locus, the targeting construct and the transgenic <i>pb^pfpkg</i> locus. Areas of 5′UTR and 3′UTR cloned into the targeting vector are indicated, S = spacer. Arrows 1–6 indicate binding sites for primers used in diagnostic PCR. Primers 1 and 2 were used to detect 5′ integration. Primers 3 and 4 were used to determine 3′ integration. Primers 5 and 6 bind specifically to the endogenous <i>pbpkg</i> and are used to confirm absence of the endogenous gene in the transgenic line. The area homologous to the probe used in Southern blotting and <i>Bcl</i>I restriction sites used for diagnostic digest are indicated. B) Diagnostic PCR used to determine integration of the targeting construct into the Pb<i>^pfpkg</i> transgenic line. C) Southern blot following <i>Bcl</i>I digest shows integration of the targeting construct as a specific 3.9 kb band and absence of the endogenous 5.1 kb band in the transgenic line (Pb<i>^pfpkg</i>) in comparison to wild type (WT-GFP). D) PFGE of wild type (WT-GFP) and mutant parasite (Pb<i>^pfpkg</i>) confirms integration into the correct chromosome. E) Western blot of asexual blood and ookinete stages confirm expression of PfPKG in the transgenic line (the transgenic PfPKG bands are 25.8% and 21.8% of the PbPKG band in the WT-GFP line in asexual blood and ookinete stages respectively).</p

Phenotypic analyses of ΔRep and ΔNΔRep mutants in the mosquito.

<p><b>A.</b> Oocyst numbers. On day 14 post-infection, midguts from 20–30 mosquitoes were scored for number of oocysts by phase or fluorescence microscopy. Shown is the mean ± SEM for each line. This analysis was performed 3 times with different batches of mosquitoes and a representative experiment is shown. <b>B.</b> Number of sporozoites per oocyst. On the indicated day post-infective blood meal, equal numbers of 10–20 mosquito midguts were collected and used either to count oocysts or were homogenized and sporozoites were counted. The number of sporozoites was then divided by the number of oocysts. Each point represents the mean ± SEM of 4 independent experiments. ΔNΔRep parasites did not produce sporozoites. <b>C.</b> Midgut sporozoite numbers. At each of the indicated days post-infective blood meal, midguts were dissected from 10–20 mosquitoes per parasite line, sporozoites were counted and the number of sporozoites per mosquito was calculated. Shown is the mean ± SEM of pooled data from 4 independent experiments. No sporozoites could be detected by light microscopy in the ΔNΔRep line. <b>D.</b> Hemolymph sporozoite numbers. On days 16 and 19 post-infective blood meal, hemolymph was collected from 15 mosquitoes and sporozoites were counted. Shown is the mean ± SEM of three independent experiments. No hemolymph sporozoites were observed in ΔRep infected mosquitoes. <b>E.</b> Salivary gland sporozoite numbers. On day 21 post-infective blood meal, salivary glands from 20 mosquitoes were dissected and sporozoites were counted. Shown is the mean ± SEM of 3 independent experiments. No salivary gland sporozoites were ever observed in ΔRep and ΔNΔRep infected mosquitoes. <b>F.</b> Representative differential interference contrast (DIC) microscopy images of oocysts from wild type, ΔRep and ΔNΔRep infected mosquitoes at the indicated days post infection. Bars represent 10 µm.</p

Pantagruel

Conjunt de particel·les dels següents instruments: 1r violí, 2n violí, viola, contrabaix, flauta, 1r clarinet, 2n clarinet, trompes, 1r cornetí, 2n cornetí, 1r trombó, 2n trombó, 3r trombó, fiscorn, caixa, bomboHi figura escrita a mà la data 1886Rigodo

Electron micrographs showing unusual aspect of inner membrane complex development of the ΔNΔRep mutant.

<p><b>A.</b> Low power of a mid-stage oocyst showing the retracted plasmalemma and areas of IMC invagination into the cytoplasmic mass (arrows). Bar is 1 µm. <b>B.</b> Detail of the surface of an early oocyst showing extensive growth of the IMC (arrows) but no evidence of budding. Bar is 100 nm. <b>C.</b> Detail of a more advanced stage in development showing areas of abnormal IMC/plasmalemma formation and invagination into the cytoplasmic mass of the sporoblasts (arrows). Bar is 100 nm. <b>D.</b> Cross section through two sporozoites showing loss of shape, adhesion, and folding of the plasmalemma of the sporozoites (arrows). R – rhoptry; Mt - microtubule. Bar is 100 nm. <b>E, F.</b> Enlargement of cross sections through ΔNΔRep (E) and WT (F) parasites, showing the relative distance between the plasmalemma of adjacent sporozoites. Note in the ΔNΔRep mutant the plasma membranes appeared tightly adhered (similar to that between the IMC membranes) (<b>E</b>) compared to the significantly wider space observed in the WT (<b>F</b>). I – IMC; Mt - sub-pellicular microtubules; P – plasmalemma. Bar is 100 nm.</p

Electron micrographs illustrating the process of cell death observed in oocysts of the ΔRep and ΔNΔRep mutants.

<p><b>A.</b> Low power of a ΔNΔRep oocyst with a degenerating, undifferentiated central cytoplasmic mass. Bar is 10 µm. <b>B.</b> Detail from the degeneration of a ΔNΔRep oocyst similar to that in <b>A</b> showing a dilated nuclear membrane containing a number of nuclei (N) exhibiting peripheral chromatin condensation. Bar is 100 nm. <b>C.</b> Low power of a ΔRep oocyst in which sporozoite formation had occurred but now exhibited features of cell degeneration. Bar is 10 µm. <b>D.</b> Detail of the nuclei observed in an intact ΔNΔRep oocyst showing the absence of any peripheral nuclear condensation. Bar is 100 nm. <b>E.</b> Longitudinal section through a sporozoite showing the nucleus with peripheral chromatin condensation and dilatation of the nuclear membranes. N – nucleus. Bar is 500 nm. <b>F.</b> Low power of a ΔRep oocyst in which there is a cross section of a central mass of degenerating sporozoites (S). Bar is 10 µm. <b>G.</b> Histogram comparing the relative number of immature mature and degenerate oocysts at two time points (12–14 days and 18–21 days) for WT, ΔRep and ΔNΔRep oocysts. (Based on EM examination of multiple midguts from multiple experiments – number of oocysts evaluated: 405 wild type; 236 ΔRep mutant; 165 ΔNΔRep mutant).</p

Generation of CSP repeatless mutants.

<p><b>A.</b> Schematic representation of CSP structure in wild type and mutant parasites ΔRep and ΔNΔRep. Region I is shown as hatched, repeat region as light grey and the TSR domain as dark grey. <b>B.</b> Western blot analysis of wild type (WT), WT-GFP and RCon as control parasites and the two repeat mutants: ΔRep and ΔNΔRep. Lysates from midgut sporozoites or infected midguts were probed using antisera specific for each of the three CSP domains: polyclonal antisera specific for the CSP NH₂-terminus, anti-repeat region (mAb 3D11) and polyclonal antisera specific for the CSP COOH-terminus. Molecular weight markers (kDa) shown on the left of each gel photograph.</p

Light microscopy analysis of cell death in ΔRep oocysts.

<p><b>A.</b> Photographs of representative mosquito midguts on days 14 and 21 post infection. Control and ΔRep parasites express GFP in most oocysts at day 14 post infection whereas by day 21 most of the ΔRep oocysts have lost the GFP fluorescence. Top panel shows a mosquito midgut and lower panel shows representative oocysts at higher magnification with degenerated features and absence of GFP fluorescence due to loss of plasmalemma integrity at day 21 post infection in ΔRep oocysts. <b>B.</b> Quantification of GFP positive and GFP negative oocysts at 14, 16, 18, and 21 days post infection for control and ΔRep oocysts.</p

Electron micrographs of sporogony in WT, ΔRep and ΔNΔRep mutants.

<p>A series of electron micrographs of oocysts illustrating the progressive stages in the sporogonic process undergone by WT, ΔRep, and ΔNΔRep oocysts in the mosquito midgut. The structure of the oocysts at the end of the growth phase was similar for WT (<b>A</b>) and both mutants. (<b>B, C</b>) The initiation of sporozoite formation with retraction of the plasmalemma was also similar (<b>D-F</b>). However, while sporozoite formation continued by a budding process in both WT (<b>G</b>) and the ΔRep mutant (<b>H</b>) there was no budding seen in the ΔNΔRep mutant (<b>I</b>). This budding process continued until the sporozoites were fully formed in the WT (<b>J</b>) and ΔRep (<b>K</b>). In contrast the mature oocyst of the ΔNΔRep mutant contained a tightly adhered mass of sporozoites (<b>L</b>). Bars represent 10 µm.</p

Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite

The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite