Abstract

The secretion system of avian pathogenic Escherichia coli (APEC) plays a key role in bacterial colonization and invasion of the host. The core structural component of the type VI secretion system (T6SS), hemolysin-coregulatory protein (Hcp), functions both as a T6SS structural component and a secreted virulence effector. However, the pathogenic mechanisms by which Hcp2 affects host cell function remain poorly understood. In this study, we focused on the impact of Hcp2 on mitochondrial function in chicken tracheal mucosal epithelial (CTE) cells to reveal the mechanism of APEC-induced host cell damage. Hcp2 exposure led to significant mitochondrial dysfunction, as evidenced by elevated levels of reactive oxygen species, mitochondrial membrane potential depolarization, and intracellular calcium overload. These findings suggest that Hcp2 induces mitochondrial oxidative stress and disrupts cellular homeostasis. Notably, when mitochondrial function is impaired, cells initiate a selective autophagic mechanism, a process that may be closely related to the pathogenic mechanism of Hcp2 protein. Transmission electron microscopy (TEM) and immunofluorescence microscopy confirmed the formation of double-membraned autophagosomes. Western blot analysis further revealed increased conversion of LC3-I to LC3-II and a dynamic change in p62/SQSTM1 expression. Additionally, the degradation of mitochondrial proteins and the increased colocalization of mitochondria with autophagosomes and lysosomes confirmed the activation of mitophagy. Our study reveals that Hcp2 disrupts mitochondrial functional homeostasis and activates mitophagy in CTE cells.