Figure 1.
SPCLIP1 is a component of the mosquito complement cascade.
(A) Western analysis of mosquito hemolymph collected 4 days after injection with LacZ or SPCLIP1 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)(C) Mosquito hemolymph collected 4 days after injection of LacZ dsRNA or silencing SPCLIP1, LRIM1 or TEP1 (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.
Figure 2.
TEP1 and SPCLIP1 localization on dead parasites is mutually dependent.
(A) TEP1 immunolocalization on the surface of GFP-expressing P. berghei 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 dsSPCLIP1. Lack of TEP1 signal in dsTEP1 treated mosquitoes confirms the specificity of the antibody. A rare TEP1, GFP double positive parasite is visible in the upper left panel of the dsLacZ control. (B) SPCLIP1 immunolocalization on the surface of GFP-expressing P. berghei 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 dsTEP1. Lack of SPCLIP1 signal in the dsSPCLIP1 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.
Figure 3.
SPCLIP1 is required for the utilization of TEP1-F.
(A) Western blot analysis of hemolymph collected from mosquitoes after injection with PBS (left) or E. coli bioparticles (right) using a panel of different antibodies. Full-length and processed TEP1 are indicated as TEP1-F and TEP1cut, respectively. Re-probing with SRPN3 was used to confirm equal loading. (B) Western blot analysis of hemolymph collected from control LacZ dsRNA-injected (left) and SPCLIP1 kd (right) mosquitoes after injection with E. coli bioparticles. Re-probing with PPO6 was used to confirm equal loading. Images are representative of three independent biological replicates.
Figure 4.
SPCLIP1 and TEP1cut are localized on the surface of E. coli bioparticles.
(A) Schematic overview of sample preparation. Hemolymph containing E. coli bioparticles was recovered 15 min after injection into mosquitoes after gene silencing. The bacteria were separated by centrifugation and the soluble fraction was collected. The bacterial pellet was washed with buffer and extracted for analysis. (B) Western blot analysis of soluble and bioparticle bound fractions using antibodies against TEP1 and SPCLIP1. Images are representative of two independent biological replicates.
Figure 5.
SPCLIP1 and TEP1 interact after challenge with E. coli bioparticles.
IP beads containing SPCLIP1 antibody or control beads were used to capture proteins from hemolymph 15 min after injection with E. coli 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.
Figure 6.
SPCLIP1 is required for triggering the melanization cascade.
(A) Reducing western blot analysis of CLIPA8 in hemolymph collected from control LacZ dsRNA-injected (top) and SPCLIP1 kd mosquitoes (bottom) after injection with E. coli bioparticles. Full-length and cleaved CLIPA8 are labeled CLIPA8-F and CLIPA8-C, respectively. Images are representative of two independent biological replicates. (B) PO activity measured in hemolymph samples collected from dsLacZ, dsSPCLIP1 and dsCLIPA8 treated mosquitoes 6 h after injection with bacteria. Data are representative of two independent biological replicates. See also Figure S2. (C) GFP-expressing P. berghei oocysts (green circles) and melanized ookinetes (gray squares) in dsLacZ, dsSPCLIP1, dsCTL4 and dsCTL4/dsSPCLIP1 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, dsCTL4 and dsSPCLIP1 injected mosquitoes were compared to dsLacZ while dsCTL4/dsSPCLIP1 injected mosquitoes were compared to dsCTL4. Asterisks indicate Kruskal-Wallis P-values<0.01.
Figure 7.
Model of TEP1 convertase formation.
In steady state hemolymph a pool of TEP1-F is processed by an unknown protease to generate TEP1cut, which interacts and circulates with the LRIM1/APL1C complex. Recognition of microbial surfaces leads to deposition of LRIM1/APL1C and TEP1cut 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.