Rab6 mediates lysosome biogenesis to regulate phagocytosis and bacterial clearance in the Chinese mitten crab...

Phagocytosis by circulating hemocytes is crucial for antibacterial defense in crustaceans, but how intracellular trafficking GTPases coordinate this process remains poorly understood. Here, we identified and characterized a Rab6 ortholog (EsRab6) from the Chinese mitten crab, Eriocheir sinensis, and defined its role in hemocyte phagocytosis and resistance to Vibrio parahaemolyticus. EsRab6 encodes a typical Rab6 GTPase with conserved nucleotide-binding and Rab family motifs and clusters within the invertebrate Rab6 clade. Transcripts were widely expressed, with higher levels in the hepatopancreas and eyestalk, yet EsRab6 was rapidly and transiently upregulated in hemocytes following V. parahaemolyticus challenge. RNA interference-mediated knockdown of EsRab6 resulted in a significant reduction in FITC-labeled bacterial uptake by hemocytes, as shown by microscopy and flow cytometry. Immunofluorescence and LysoTracker staining showed that EsRab6 relocates from a diffuse cytosolic pattern to punctate vesicles that colocalize with lysosomes during infection. Simultaneously, EsRab6 knockdown significantly decreased lysosome-associated signals, indicating a role for EsRab6 in lysosome formation or stability. In vivo, dsRNA-mediated silencing of EsRab6 results in higher hemolymph bacterial loads and significantly lower survival rates following V. parahaemolyticus infection. Overall, these findings identify EsRab6 as a conserved trafficking regulator that links phagosome-lysosome biogenesis to adequate bacterial clearance in crab hemocytes and suggest that Rab6-dependent pathways could be targeted to enhance disease resistance in crustacean aquaculture.