Stimulation of a protease targeting the LRIM1/APL1C complex reveals specificity in complement-like pathway activation in Anopheles gambiae

Abstract

The complement-like pathway of the African malaria mosquito Anopheles gambiae provides protection against infection by diverse pathogens. A functional requirement for a core set of proteins during infections by rodent and human malaria parasites, bacteria, and fungi suggests a similar mechanism operates against different pathogens. However, the extent to which the molecular mechanisms are conserved is unknown. In this study we probed the biochemical responses of complement-like pathway to challenge by the Gram-positive bacterium Staphyloccocus aureus . Western blot analysis of the hemolymph revealed that S . aureus challenge activates a TEP1 convertase-like activity and promotes the depletion of the protein SPCLIP1. S . aureus challenge did not lead to an apparent change in the abundance of the LRIM1/APL1C complex compared to challenge by the Gram-negative bacterium, Escherichia coli . Following up on this observation using a panel of LRIM1 and APL1C antibodies, we found that E . coli challenge, but not S . aureus , specifically activates a protease that cleaves the C-terminus of APL1C. Inhibitor studies in vivo and in vitro protease assays suggest that a serine protease is responsible for APL1C cleavage. This study reveals that despite different challenges converging on activation of a TEP1 convertase-like activity, the mosquito complement-like pathway also includes pathogen-specific reactions.

The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae

“…The p12 fragment was consistently apparent following extended blot exposures and the uncropped image is provided to the right of the compilation. CLIPC9 cleavage following E. coli challenge coincided with reduction/loss of the Anopheles Plasmodium -responsive leucine-rich repeat 1-C (APL1C) protein western signal, which occurs due to limited proteolysis, as previously reported [52]. Only full-length CLIPC9 is observed under non-reducing western analysis of these samples, confirming the disulfide linkage joining the p30 and p12 fragments (Fig S5).…”

“…SPCLIP1 (also called CLIPA30) localizes to microbial surfaces in a TEP1-dependent manner and positively regulates melanization by promoting TEP1 convertase activity [46, 51]. SPCLIP1 is also required for the cleavage-induced activation of CLIPA8 and CLIPA28, which collectively form the core of a sequentially activated CLIP-SPH cascade essential for melanization [46, 52, 53]. The cleavages of CLIPA8 and CLIPA28 are inhibited by SRPN2 and silencing this negative regulator results in the spontaneous melanization of self-tissue [40, 53, 54].…”

Leucine-Rich Immune Factor APL1 Is Associated With Specific Modulation of Enteric Microbiome Taxa in the Asian Malaria Mosquito Anopheles stephensi

“…Because the presence of the immune complex has only been detected in A. gambiae and A. coluzzii, it cannot be excluded that APL1 could function very differently in A. stephensi. Nevertheless, a recent report indicated that A. coluzzii APL1 was processed by a limited C-terminal cleavage specifically after mosquito injection with the Enterobacteriaceae, Escherichia coli, but not with the Gram positive Staphylococcus aureus (Reyes Ruiz et al, 2019).…”

The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae

“…SPCLIP1 (also called CLIPA30) localizes to microbial surfaces in a TEP1-dependent manner and positively regulates melanization by promoting TEP1 convertase activity [ 46 , 51 ]. SPCLIP1 is also required for the cleavage-induced activation of CLIPA8 and CLIPA28, which collectively form the core of a sequentially activated CLIP-SPH cascade essential for melanization [ 46 , 52 , 53 ]. The cleavages of CLIPA8 and CLIPA28 are inhibited by SRPN2 and silencing this negative regulator results in the spontaneous melanization of self-tissue [ 40 , 53 , 54 ].…”