A Cysteine Protease Inhibitor of <i>Plasmodium berghei</i> Is Essential for Exo-erythrocytic Development

<div><p><i>Plasmodium</i> parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the <i>Plasmodium berghei</i> ICP (PbICP). Excision of the <i>pbicb</i> gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a <i>pbicp-gfp</i> construct fully reversed these defects. Taken together, in <i>P. berghei</i> this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.</p></div

PbICP is not essential for parasite blood stage development.

<p>(A) Assessment of SSR at the <i>pbicp</i> locus in <i>pbicp</i>-transgenic parasites (PbICP_cond, PbICP_KO, and PbICP_comp) by PCR of genomic DNA using primers P1 and P3 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336.s001" target="_blank">Figure S1</a>). PbICP_KO erythrocytic stages were generated by subcloning of PbICP_cond parasites via single merosome injection into mice. PbICP_comp erythrocytic stages were generated by transfection of PbICP_KO parasites with the pL0017-<i>pbicp</i>-<i>gfp</i> plasmid <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336-Rennenberg1" target="_blank">[29]</a> as a complementation (add-back) for <i>pbicp</i>. The sizes of the DNA fragments amplified from <i>pbicp</i> excised (SSR+) or non-excised (SSR−) loci are shown. As a control, primers specific for <i>pbicp</i> were used (bottom panel). (B) Western blot analysis of extracts from blood stage parasites. <i>In vitro</i> cultured parasites were collected at the schizont stage. Analysis included the following strains: PbICP_control (UIS4/Flp(−)), PbICP_cond, PbICP_KO, and PbICP_comp parasites. PbICP-C was detected using a specific mouse anti-PbICP-C antiserum <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336-Rennenberg1" target="_blank">[29]</a>. Rat anti-MSP1 was used as a control. Molecular masses are indicated in kDa. The expected mass of full-length PbICP is 55 kDa in PbICP_control and 57 kDa in PbICP_cond parasites and 23 kDa after processing (PbICP-C). PbICP-GFP has an expected molecular mass of 81 kDa and 49 kDa after processing (PbICP-C-GFP). (*: non-specific protein bands, probably Ig subunits remaining in the blood culture detected by the secondary HRP-labeled anti-mouse antibody). (C) Blood stage development of PbICP_control (UIS4/Flp(−)), PbICP_KO and PbICP_comp parasites. Mice were infected by i.p. injection of 100 µl blood from infected mice, adjusted to a parasitemia of 5% with PBS. The onset and development of a blood stage infection was determined by observation of blood smears. The two graphs represent two separate sets of experiments. In the left graph, parasitemia of PbICP_control and PbICP_KO parasites were compared and, in the right graph, parasitemia of PbICP_control and PbICP_comp parasites were compared. For statistical evaluation of the difference in parasitemia at day 3 see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004336#ppat.1004336.s002" target="_blank">Figure S2D</a>.</p

PbICP is Important for EEF Maturation.

<p>(A) IFA of infected, detached cells 65 hpi (mature EEFs) of either PbICP_control (first panel), PbICP_cond (second panel), or PbICP_comp (third panel) parasites. Cells were stained with mouse anti-MSP1 or mouse anti-GFP (green), rat anti-PbICP-C (red), and chicken anti-ExpI (cyan). Secondary antibodies were: anti-mouse Alexa488, anti-rat Alexa594, or anti-chicken Cy5. DNA was stained with DAPI (blue), Scale bars: 10 µm. (B) Quantification of infected, detached cells 65 hpi (mature EEFs) of the different parasite strains in relation to infected cells 48 hpi. HepG2 cells were infected with either PbICP_control (control), PbICP_cond (cond), or PbICP_comp (comp) sporozoites and infected cells were quantified 48 hpi (values normalized to 100%). At 65 hpi, supernatant was collected and stained with Hoechst 33342. The number of infected, detached cells was quantified and the ratio between infected cells at 48 hpi and detached cells at 65 hpi was calculated. Results are the means ± S.D. from three independent measurements. Differences between PbICP_control and <i>pbicp</i>-transgenic parasites (PbICP_cond, PbICP_comp) were compared using Student's t test (**** = P<0.0001; ns, not significant). PbICP_KO parasites were excluded from this experiment as no invasion could be achieved. n.a.: not applicable.</p

PbICP is important for effective invasion of and development within hepatocytes.

<p>(A) Quantification of infected HepG2 cells incubated with either 1×10⁴ PbICP_control (UIS4/Flp(−)), PbICP_cond, PbICP_KO, or PbICP_comp parasites at 30 hpi is shown. Suitable amounts of PbICP_KO sporozoites, could be harvested from salivary glands at day 24–26 after blood feeding only. For consistency, also mosquitoes infected with the other parasite strains were also used at day 24–26 after blood feeding. Differentiation of PbICP-C-positive (striped bars) or PbICP-C-negative (black bars) EEFs was quantified by IFA. Results are the means ± standard deviation (S.D.) from three independent experiments. The relative infection rate (rel. inf. rate) of PbICP_control, PbICP_cond, PbICP_KO, and PbICP_comp parasites is shown below the histogram. Differences between PbICP_control and <i>pbicp</i>-transgenic parasites (PbICP_cond, PbICP_KO, and PbICP_comp) were compared using Student's t test (* = P<0.05, **** = P<0.0001). (B) Parasite size during exo-erythrocytic development (at 30 and 60 hpi) in PbICP_cond parasites compared with PbICP_control parasites, as determined using density slicing (OpenLab). Results are the means ± S.D. from three independent measurements. Differences between PbICP_control and PbICP_cond parasites were compared using Student's t test (* = P<0.05; **** = P<0.0001).</p

PbICP is essential for parasite development <i>in vivo</i>.

<p><i>In vivo</i> infectivity of <i>pbicp</i>-parasites was determined by parasite prepatency. Mice (5–10 per treatment group) were infected with 5×10³ PbICP_control, PbICP_cond, PbICP_KO, or PbICP_comp salivary gland sporozoites by intravenous (i.v.) inoculation. The onset of a blood stage infection (prepatency) was determined by observation of blood smears up to 20 days post-infection. Non-infected mice were not considered in the prepatency calculations.</p><p>PbICP is essential for parasite development <i>in vivo</i>.</p