SPCLIP1 and TEP1 interact after challenge with <i>E.</i><i>coli</i> bioparticles.

<p>IP beads containing SPCLIP1 antibody or control beads were used to capture proteins from hemolymph 15 min after injection with <i>E. coli</i> bioparticles (+) or PBS (−). The beads were separated and samples of the unbound and bound fractions were analyzed by western blot under reducing and non-reducing conditions for TEP1 and SPCLIP1, respectively. Images are representative of two independent biological replicates.</p

SPCLIP1 is required for triggering the melanization cascade.

<p>(<b>A</b>) Reducing western blot analysis of CLIPA8 in hemolymph collected from control <i>LacZ</i> dsRNA-injected (top) and <i>SPCLIP1</i> kd mosquitoes (bottom) after injection with <i>E. coli</i> bioparticles. Full-length and cleaved CLIPA8 are labeled CLIPA8-F and CLIPA8-C, respectively. Images are representative of two independent biological replicates. (<b>B</b>) PO activity measured in hemolymph samples collected from ds<i>LacZ</i>, ds<i>SPCLIP1</i> and ds<i>CLIPA8</i> treated mosquitoes 6 h after injection with bacteria. Data are representative of two independent biological replicates. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003623#ppat.1003623.s002" target="_blank">Figure S2</a>. (<b>C</b>) GFP-expressing <i>P. berghei</i> oocysts (green circles) and melanized ookinetes (gray squares) in ds<i>LacZ</i>, ds<i>SPCLIP1</i>, ds<i>CTL4</i> and ds<i>CTL4</i>/ds<i>SPCLIP1</i> injected mosquitoes 7 days post infection were counted. Lines indicate median infection intensity values. Data were combined from three independent biological replicates. For statistical analysis, ds<i>CTL4</i> and ds<i>SPCLIP1</i> injected mosquitoes were compared to ds<i>LacZ</i> while ds<i>CTL4</i>/ds<i>SPCLIP1</i> injected mosquitoes were compared to ds<i>CTL4</i>. Asterisks indicate Kruskal-Wallis P-values<0.01.</p

TEP1 and SPCLIP1 localization on dead parasites is mutually dependent.

<p>(<b>A</b>) TEP1 immunolocalization on the surface of GFP-expressing <i>P. berghei</i> parasites invading the mosquito midgut 26 h after infection. TEP1 positive parasites (arrows) do not express GFP and appear fragmented indicating that they are killed, while TEP1 negative parasites express GFP and are considered live. There is a dramatic reduction in TEP1 signal in mosquitoes treated with ds<i>SPCLIP1</i>. Lack of TEP1 signal in ds<i>TEP1</i> treated mosquitoes confirms the specificity of the antibody. A rare TEP1, GFP double positive parasite is visible in the upper left panel of the ds<i>LacZ</i> control. (<b>B</b>) SPCLIP1 immunolocalization on the surface of GFP-expressing <i>P. berghei</i> parasites invading the mosquito midgut epithelium 26 h after infection. SPCLIP1 positive parasites (arrows) are fragmented and lack GFP signal indicating they are dead. There is a dramatic reduction in SPCLIP1 signal in mosquitoes treated with ds<i>TEP1</i>. Lack of SPCLIP1 signal in the ds<i>SPCLIP1</i> treated mosquitoes confirms the specificity of the antibody. The background staining observed in all panels is non-specific antibody trapping by the trachea and muscle fibers present on the basolateral surface of the mosquito midgut. For both TEP1 and SPCLIP1 immunolocalization assays two independent biological replicates were performed with 5–10 midguts for each dsRNA. Panels are representative confocal projections of an approximately 20 µm thick section of the midgut basolateral surface. The scale bar is 10 µm.</p

SPCLIP1 is a component of the mosquito complement cascade.

<p>(<b>A</b>) Western analysis of mosquito hemolymph collected 4 days after injection with <i>LacZ</i> or <i>SPCLIP1</i> dsRNA. The blot was initially probed with a polyclonal antibody raised against recombinant SPCLIP1 (top panel) and re-probed with an APL1C antibody (bottom panel) to confirm equal loading. (<b>B</b>)(<b>C</b>) Mosquito hemolymph collected 4 days after injection of <i>LacZ</i> dsRNA or silencing <i>SPCLIP1</i>, <i>LRIM1</i> or <i>TEP1</i> (or combinations of those) was analyzed by western blot using SPCLIP1, APL1C and TEP1 antibodies. Blots were re-probed with SRPN3 and PPO6 antibodies to confirm equal loading. The labels on the right indicate protein or complex detected. Images are representative of three independent biological replicates.</p

SPCLIP1 is required for the utilization of TEP1-F.

<p>(<b>A</b>) Western blot analysis of hemolymph collected from mosquitoes after injection with PBS (left) or <i>E. coli</i> bioparticles (right) using a panel of different antibodies. Full-length and processed TEP1 are indicated as TEP1-F and TEP1_cut, respectively. Re-probing with SRPN3 was used to confirm equal loading. (<b>B</b>) Western blot analysis of hemolymph collected from control <i>LacZ</i> dsRNA-injected (left) and <i>SPCLIP1</i> kd (right) mosquitoes after injection with <i>E. coli</i> bioparticles. Re-probing with PPO6 was used to confirm equal loading. Images are representative of three independent biological replicates.</p

Model of TEP1 convertase formation.

<p>In steady state hemolymph a pool of TEP1-F is processed by an unknown protease to generate TEP1_cut, which interacts and circulates with the LRIM1/APL1C complex. Recognition of microbial surfaces leads to deposition of LRIM1/APL1C and TEP1_cut and subsequent recruitment of SPCLIP1. An unknown catalytically active protease is then recruited generating the mature TEP1 convertase, which processes TEP1-F causing it to rapidly interact with nearby surfaces. Steady state processing of TEP1-F and that performed by the TEP1 convertase are distinct, as only the latter requires SPCLIP1. Formation of the TEP1 convertase is required for phagocytosis, lysis, or CLIPA8 cleavage by an unknown protease and subsequent activation of the melanization cascade.</p

The association between the number of salivary gland sporozoites and the duration of the pre-patent period in humans (A,B,C) and mice (D,E,F).

<p>In panels A,B,D,E unvaccinated/untreated control infections are denoted by a solid line (and darker shade of colour) whilst humans given a pre-erythrocytic vaccine (PEV) candidate or mice given an anti-circumsporozoite protein antibody (PEV) are shown by a dashed line (lighter shade of colour). Panels (A) and (D) show the number of sporozoites in the salivary glands following blood-feeding used in the different experimental infections. Panels (B) and (E) show the survival curves predicted by the model for the time between sporozoite inoculation and patent parasitaemia. Grey, green, blue and red lines shows predictions for a vertebrate host with 5 bites from mosquitoes with 1–10, 11–100, 101–1000 and >1000 salivary gland sporozoites after blood-feeding, respectively (grey line is omitted from B as no human volunteers received bites solely from mosquitoes with 1–10 residual-sporozoites). Panels (C) and (F) show the ability of the best fit model to predict the time to patency for humans and mice. Each point gives the prediction for an individual volunteer according to the residual-sporozoite score of biting mosquitoes and whether they received a PEV (orange dots) or were untreated (blue dots).</p

The frequency and impact on the time to patency in humans of mosquito bites with ≤10 residual-sporozoites.

<p>Grey bars show how many volunteers received bites from mosquitoes with ≤10 salivary gland sporozoites. Black dots (and line) show model predictions for the mean time to patency for volunteers with different number of (≤10) bites. As the impact of the lightly infected bites will depend on the number of residual-sporozoites in the bites the volunteer received, in this example all volunteer receive 5 bites from mosquitoes with 101–1000 residual-sporozoites in addition to the extra mosquito bites with ≤10 residual-sporozoites.</p

The association between the number of salivary gland sporozoites and the probability of infection in humans (A,B,C) and mice (D,E,F).

<p>In panels A,B,D,E naïve infections are denoted by a darker shade of colour whilst hosts given an anti-circumsporozoite protein vaccine or antibody (Pre-Erythrocytic Vaccine -PEV) are a lighter shade. Unvaccinated control humans are excluded from the analysis in (A-C) as all received very high levels of parasite challenge and all developed infection (so there is no variability to test the association with residual-sporozoite load). Panels (A) and (D) show the number of sporozoites in the salivary glands following blood-feeding used in the different experimental infections (be it 0 (orange), 1–10 (purple), 11–100 (green), 101–1000 (blue) or >1000 (red). Panels (B) and (E) show model predictions of the probability that a single bite from a mosquito with a given number of salivary gland sporozoites following blood-feeding will induce patent infection (black lines show 95% confidence interval estimates on model predictions). Panels (C) and (F) show model predictions for overall probability of infection for each individual human and mouse and as estimated using the best fit model. Each point gives the prediction for an individual volunteer according to the number of bites they receive, the residual-sporozoite score of those individual bites (taken from panel (B) and (E) and whether they received a PEV (orange dots) or were untreated (blue dots).</p