Recruitment of host organelles by <i>Toxoplasma</i> under adverse host conditions.

(A–B) Fluorescence microscopy of host ER with anti-calnexin antibody (green in [A]) and host mitochondria with mito-Tracker (red in B) or anti-Tom20 antibody (green in [B]). Nuclei are DAPI-stained. (A) Host ER association with the PV (identified by arrows on phase-contrast images or stained with anti-GRA7 antibody in red) in control mammalian cells, enucleated cells, or cells treated with aerolysin, which induces the vacuolization of host ER [41]. For this last condition, Vero cells were preincubated with 0.38-nM aerolysin for 2 h and infected for 10 min without toxin, then re-exposed to 0.38-nM aerolysin for 30 min. Despite extensive deformation of the host ER with the toxin, the parasite is able to attract this organelle, as exemplified by the intense zones of contact between the PV and host ER “bubbles,” clearly visible in the inset from another aerolysin-treated cell. (B) Host mitochondria association with the PV (identified by arrows on phase-contrast images) in control mammalian cells, enucleated cells, or cells coinfected with RFP-Toxoplasma (arrow) and Chlamydia psittaci (Cp) for 24 h. C. psittaci is notorious for attracting host mitochondria to its inclusion [42]. These bacteria multiply faster than Toxoplasma, occupying a large portion of the host cytoplasm. Despite these physical constraints, Toxoplasma manages to attract host mitochondria to the PV to a similar extent as Chlamydia. anti-Tom20, mitochondrial translocase of outer membrane; Cp, Chlamydia psittaci; GRA7, Toxoplasma parasitophorous vacuole membrane protein; ER, endoplasmic reticulum; PV, parasitophorous vacuole; RFP, red fluorescent protein.</p

Intraluminal structures in the <i>Toxoplasma</i> PV.

<p><b>(A-G)</b> Transmission EM of intracellular <i>Toxoplasma</i>. <b>(A)</b> Thin tubules formed by the parasite (P) 20 min post-invasion and packaged within a vesicle (panel a; arrowhead) before being discharged at the basal end of the parasite (panel b; arrows) and spread within the vacuole to form the IVN. <b>(B)</b> Host MT–based invaginations of the PV membrane (arrowheads). <b>(C)</b> Long microfilaments narrower than the IVN tubules (arrowheads). <b>(D)</b> IVN tubules and microtubular structures are coated by e-dense material. <b>(E)</b> Host endocytic organelles containing LDL-gold particles (arrowheads) surrounded by the PV membrane. <b>(F)</b> RB of the mother cells identified by discarded organelles such as the ER, either still attached to daughter cells or free in the PV lumen (arrowheads). <b>(G)</b> Unknown membrane-bound structures containing fibrillary material (arrowheads) accumulated between parasites (panel a), close to the PV membrane (panel b), or appending to the PV membrane (panel c). All scale bars, 500 nm. e-dense, electron-dense; EM, electron microscopy; ER, endoplasmic reticulum; hc, host cell; IVN, intravacuolar network; LDL, low-density lipoprotein; MT, microtubule; P, parasite; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; RB, residual body.</p

Comparison of WT and PbDmc1 KO oocysts in <i>An. stephensi</i> mosquitoes in four independent experiments (Experiments I–III evaluated one of the PbDmc 1 KO clones and Exp. IV represents data for another independent PbDmc 1 KO clone).

<p>Horizontal lines represent the median values. Shown below is N, the number of mosquitoes dissected, asexual parasitemia, gametocytemia, the rate of infection (percent of infected mosquitoes). The Fisher’s exact test was used to compare prevalence, i.e the rate of infection between WT and KO and indicated as significant (p<0.05) or not significant (ns). (*Median oocyst numbers of WT were significantly higher than those of PbDmc1 KO parasites, <i>p</i><0.001, Mann-Whitney test).</p

Morphology of WT (A,C,E) and Dmc1 KO (B, D, F) oocyts in the mosquito midgut on day 10 (A, B), 12 (C,D) and 14 (E,F) post blood feeding.

<p>Arrows indicate representative oocysts. Scale bar represents 10 µm.</p

In vitro ookinete counts/ml (mean ± SD) for WT and PbDmc1 KO parasites.

<p>Ookinetes cultures were setup from WT and PbDmc1 KO infected mice (n = 3) that had equivalent gametocytemia. Mean ookinete counts were compared using the <i>t</i>-test and they were not statistically significant (p>0.05) (A). Morphological comparison by light microscopy of Giemsa stained smears (Upper panel in B) and by TEM of cross-sections of ookinete nuclei showing differences in the chromatin structure (lower panel in B). Fifteen to 22 parasite nuclei were analyzed. n, nucleus. Bars are 100 nm.</p

Ultrastructural analysis of WT and Dmc1 KO oocysts 6 days post-infection. TEM of WT (A–C) and Dmc1 KO (D–I) parasites.

<p>WT parasites exhibit normal sporogony and contain several nuclei, homogenous in size. By contrast, PbDmc1 KO parasites were smaller in size and show fewer but larger nuclei with aberrant nuclear scission profiles (arrows; inset in H). Accumulation of unknown electron-dense structures as seen in panel E and abnormal capsule morphology in panel I were also characteristics of Dmc1 KO oocysts. c, capsule; ER, endoplasmic reticulum; m, mitochondrion; n, nucleus. Bars are 100 nm.</p

Kinetics of sporozoite development in midguts and salivary glands of mosquitoes.

<p>On each day indicated above, pools of 15–30 mosquitoes were dissected and sporozoite counts made on a hemocytometer. nd, not determined.</p

Schematic diagram (not drawn to scale) showing the PbDmc1 targeting disruption plasmid and the resulting locus following double homologous recombination (A).

<p>Restriction enzymes used in generating the targeting plasmids are Kpn I (K), Hind III (H), BamHI (B) and Not I (N). The 5′ targeting sequence comprised of exons 1 to 3 and at the end of the exon 3, a stop codon (TAA) was added. The 3′ targeting sequence comprised of exons 4 to 6. The bottom panel shows the resulting locus after recombination events (shown by the X ), where Dmc1 is disrupted with the insertion of the TgDHFR backbone. (B) Southern blot hybridization demonstrated integration at the appropriate locus. The Dmc1 probe hybridized to a 1.5 kb fragment in Dmc1 KO whereas in WT parasites it hybridized to a 1.7 kb fragment following FokI restriction digestion. The DHFR probe hybridized to DNA from Dmc1 KO parasites and not in WT parasites and the Rad51 probe bound to a 6 kb fragment after FokI digestion in both WT and Dmc1 KO parasites suggesting that the Rad51 locus was not affected by disruption of the Dmc1 locus. (C) PCR evidence suggests 5′ and 3′ integration in Dmc1 KO parasites and absence of full length Dmc1. Rad51(control) was present in both WT and Dmc1 KO parasites. L, 1 kb DNA ladder. (D) RT-PCR demonstrated that disruption of Dmc1 abolished expression of full length Dmc1 without any effect on Rad51 expression. Minus reverse transcriptase reactions were negative (data not shown).</p

Ultrastructural analysis of WT and PbDmc1 KO oocysts 13 days post-infection TEM of WT (A,B) and mutant (C–F) parasites.

<p>From WT oocysts, mature sporozoites were visible and ready to escape midgut epithelial cells after rupture of the capsule (arrow in A) before migration to the salivary glands. Few PbDmc1 KO were also able to form sporozoites (panel C), but a majority of the oocysts were still undergoing sporogony (panel D–F) and some oocysts looked highly vacuolated (panel F). Note the increasing number of electron-dense structures in panels D to F and the abnormal shape of the capsules for some oocysts (panel E) as observed 6 days post-infection.c, capsule; DG, dense granule; mi, microneme; n, nucleus. Bars are 2 µm (A,C), 1 µm in B and 250 nm in D–F.</p

Asexual growth kinetics and male to female gametocyte ratios.

<p>Asexual growth kinetics (A) Mice (n = 5) were inoculated with 10⁵ parasites <i>i.v</i> and parasitemia (percent infected erythrocytes) is expressed as Mean ± SD. Asexual parasitemias between WT and KO were compared by regression analysis and they were not statistically significant (p = 0.71). (B) Male to female gametocyte ratios of PbDmc1 KO parasites compared to WT parasites when mosquito transmission experiments were done. Gametocytes were counted per 1000 rbc and data shown is from two independent experiments, N is the total number of gametocytes analyzed. Male to female ratios between WT and KO were compared using the Fisher’s exact test and they were not statistically significant (p = 0.99) for both experiments.</p