Susceptibility of PbDmc1 KO parasites to the DNA alkylating agent bizelesin.

<p>Swiss Webster mice were infected with WT or PbDmc1 KO parasites and 12 hours prior to transmission, mice were treated with varying concentrations of bizelesin and starved mosquitoes fed on the mice. Shown are the median, range (min and max values), 25 and 75 percentiles and % inhibition which was calculated by dividing median number of oocysts at 0.1, 0.5, 1 and 10 µg/kg Bizelesin by the number of oocysts from mice that received vehicle only. N represents total number of mosquitoes analyzed. (* Differences in the oocyst numbers between WT and PbDmc1 KO parasites were statistically significantly, <i>p</i><0.05, Kruskal-Wallis test).</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

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

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

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

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

Infectivity of WT and PbDmc1 KO sporozoites in mice.

<p>Data for mouse mosquito infections is pooled for three independent experiments and data for mouse i.v inoculations is pooled for two independent experiments.</p>a<p>Mice were exposed to 100 infected mosquitoes for 45 minutes.</p>b<p>Mice were injected i.v (tail vein) with 4,000 salivary gland sporozoites.</p