Abstract

The hemolysin co-regulator protein (Hcp) is a core virulence protein of the Type VI Secretion System (T6SS) in avian pathogenic Escherichia coli (APEC) and plays a critical role in host-pathogen interactions. While several bacterial effectors are known to subvert host immunity by modulating autophagy, whether and how the APEC Hcp2a protein influences autophagy in chicken macrophages (HD11) remains unclear. Here, we demonstrate that Hcp2a efficiently is internalized into HD11 cells and induces significant cytotoxicity. Notably, Hcp2a treatment elicited an autophagic response, as evidenced by elevated LC3-II levels and increased formation of autophagic vesicles. However, this was accompanied by p62/SQSTM1 aggregation and impaired autophagic flux, indicating a blockade in autophagosome-lysosome degradation. Quantitative proteomic analysis revealed significant down-regulation of the lysosomal pathway, particularly of key components involved in acidification and proteolysis. Functional assays confirmed that Hcp2a causes lysosomal dysfunction, characterized by the reduction in acidic lysosomal compartments and decreased levels of mature cathepsin D (CTSD). These defects ultimately impair the degradative capacity of lysosomes following autophagosome fusion, culminating in the block of autophagic flux. Our findings uncover a mechanism by which APEC Hcp2a interrupts host autophagy through lysosomal impairment, providing novel insights into APEC pathogenesis and bacterial immune evasion strategies.