Comprehensive proteolytic profiling ofAedes aegyptimosquito midgut extracts: Unraveling the blood meal protein digestion system
Authors: O’Donoghue Anthony J. , Lui Chenxi , Simington Carter J. , Montermoso Saira , Moreno-Galvez Elizabeth , Serafim Mateus Sá M , Burata Olive E. , Lucero Rachael M. , Nguyen James T. , Fong Daniel , Tran Khanh , Millan Neomi , Gallimore Jamie M. , Parungao Kamille , Fong Jonathan , Suzuki Brian M. , Jiang Zhenze , Isoe Jun , Rascón Alberto A. 
Published: September 2024

To sustain the gonotrophic cycle, theAedes aegyptimosquito must acquire a blood meal from a human or other vertebrate host. However, in the process of blood feeding, the mosquito may facilitate the transmission of several bloodborne viral pathogens (e.g., dengue, Zika, and chikungunya). The blood meal is essential as it contains proteins that are digested into polypeptides and amino acid nutrients that are eventually used for egg production. These proteins are digested by several midgut proteolytic enzymes. As such, the female mosquito’s reliance on blood may serve as a potential target for vector and viral transmission control. However, this strategy may prove to be challenging since midgut proteolytic activity is a complex process dependent on several exo- and endo-proteases. Therefore, to understand the complexity ofAe. aegyptiblood meal digestion, we used Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to generate global proteolytic profiles of sugar- and blood-fed midgut tissue extracts, along with substrate profiles of recombinantly expressed midgut proteases. Our results reveal a shift from high exoproteolytic activity in sugar-fed mosquitoes to an expressive increase in endoproteolytic activity in blood-fed mosquitoes. This approach allowed for the identification of 146 cleaved peptide bonds (by the combined 6 h and 24 h blood-fed samples) in the MSP-MS substrate library, and of these 146, 99 (68%) were cleaved by the five recombinant proteases evaluated. These reveal the individual contribution of each recombinant midgut protease to the overall blood meal digestion process of theAe. aegyptimosquito. Further, our molecular docking simulations support the substrate specificity of each recombinant protease. Therefore, the present study provides key information of midgut proteases and the blood meal digestion process in mosquitoes, which may be exploited for the development of potential inhibitor targets for vector and viral transmission control strategies.

Author Summary

TheAedes aegyptimosquito is a vector of viral pathogens that can be transmitted directly to humans. For instance, the transmission of dengue, Zika, or chikungunya viruses may happen during theAe. aegyptiacquisition of an infected blood meal. This blood meal is important for the anautogenous mosquito because without the digestion of blood proteins the mosquito will not obtain the necessary nutrients needed for egg production. After imbibing a blood meal, midgut digestive enzymes (proteases) are expressed and secreted into the lumen. To fully understand their roles in blood meal digestion, we used a special technique called Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS). This method allows us to generate global proteolytic activity profiles ofAe. aegyptimidgut tissue extracts that were fed with sugar or blood. In addition, we generated substrate cleavage profiles of recombinantly expressed midgut proteases allowing us to understand the enzyme preferences for blood proteins. Therefore, utilizing this approach, we found the contribution of each individual recombinant protease tested relative to the global activity profile of blood-fed midgut tissue extracts. This may be a starting point for the validation of midgut protease inhibition and the development of a new potential vector control strategy.