Rab5-mediated phagocytosis restricts Spiroplasma eriocheiris infection in crabs through a ubiquitination-dependent mechanism

Rab GTPases are widely expressed in eukaryotes and have been implicated in multiple intracellular transport pathways and pathogenic infection. Spiroplasma eriocheiris, an intracellular pathogen, can cause tremor disease in Eriocheir sinensis. However, the role of Rab GTPases in the transport mechanism during S. eriocheiris infection remains poorly understood. This study identified E. sinensis Rab5 (EsRab5), a member of the Rab GTPase family, as a key regulator of resistance to S. eriocheiris infection. Specifically, EsRab5 was upregulated during S. eriocheiris infection, while its deficiency significantly increased crab mortality and promoted the intracellular proliferation of S. eriocheiris. Additionally, EsRab5-depleted crabs exhibited impaired hemocyte phagocytic activity against S. eriocheiris. At the cellular level, overexpression of EsRab5 in Drosophila S2 cells potentiated the endocytosis of S. eriocheiris, leading to increased cell viability and decreased intracellular S. eriocheiris load. Ubiquitinated proteome analysis further revealed that the K134 site of EsRab5 was a target for polyubiquitination during S. eriocheiris infection. S. eriocheiris infection resulted in the degradation of ectopic expression of EsRab5 in Drosophila S2 cells via the ubiquitin-proteasome pathway, and this degradation was significantly inhibited by K134R mutation. Transfection of the EsRab5 K134R mutant significantly reduced the number of apoptotic cells, promoted host cell phagocytosis, and limited S. eriocheiris proliferation in S2 cells, which enhanced the immune function of EsRab5 upon S. eriocheiris infection. These findings support the critical role of Rab5 in the regulation of phagocytosis, with its function tightly regulated by ubiquitination, thereby expanding the understanding of innate immunity in crustaceans.