Molecular Characterization of Na+-dependent Cation-Chloride Coupled Cotransporters in the Yellow Fever Mosquito Aedes aegypti

Aedes aegypti transmits yellow fever and dengue, two of the most devastating mosquito-borne arboviral diseases. To overcome mosquito resistance to insecticides, new insecticides can potentially be produced to target important physiological processes such as osmoregulation. We have identified three cation chloride cotransporter (CCC) proteins important for osmoregulation in the yellow fever mosquito, namely aeCCC1, aeCCC2 and aeCCC3. In this study, we measured the effects of different biological and environmental factors on the gene expression levels of these proteins, using qPCR. AeCCC1 was expressed moderately through all life stages of the mosquito. In adult female mosquitoes, aeCCC1 was expressed highest in the head and MTs whereas it is expressed only highest in MTs in larvae. In adults, aeCCC2 showed 200-fold higher expression in the hindgut than in other regions whereas larvae expressed highest aeCCC2 levels in MTs. AeCCC3 is expressed most highly in the anal papillae of larvae (about 6,000 times higher than MTs). And, exposure to 30% seawater decreased this expression. In contrast, aeCCC3 expression increased in MTs due to seawater exposure. In an effort to begin functional characterization of the aeCCCs, we also utilized cation chloride chromatography to study the hemolymph ion composition of larvae raised in 30% seawater compared to freshwater. We found higher Na+ and NH4+ levels in seawater-exposed larvae compared to freshwater controls. The different aeCCC gene expression patterns suggest that aeCCC1 might be a secretory cotransporter, while aeCCC2 and aeCCC3 might be both a secretory and an absorptive cotransporter, depending on developmental stage and salinity

Catalytic activity and autoprocessing of murine caspase-11 mediate noncanonical inflammasome assembly in response to cytosolic LPS

Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammasomes. Inflammasomes recruit caspases to undergo proximity-induced autoprocessing into an enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high-molecular-weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Expression of fluorescently labeled Casp11 in macrophages revealed that cytosolic LPS induced Casp11 speck formation. Unexpectedly, catalytic activity and autoprocessing were required for Casp11 to form LPS-induced specks in macrophages. Furthermore, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and processing of Casp11 via ectopically expressed TEV protease was sufficient to induce Casp11 speck formation. These data reveal a previously undescribed role for Casp11 catalytic activity and autoprocessing in noncanonical inflammasome assembly, and shed new light on the molecular requirements for noncanonical inflammasome assembly in response to cytosolic LPS