Extracellular vesicles (EVs) carrying hemolysin HlyE variant (HlyE-V) from avian pathogenic Escherichia coli (APEC) induce mitochondria-dependent apoptosis in HD11 macrophages.

Abstract

Avian pathogenic Escherichia coli (APEC) is an important bacterial pathogen that causes systemic disease in poultry. Extracellular vesicles (EVs) released by APEC are involved in host-pathogen interactions, but the virulence factors they carry and their effects on avian immune cells are not fully understood. In this study, we examined whether the hemolysin variant HlyE (HlyE-V) is secreted via APEC EVs and evaluated its effects in chicken macrophage HD11 cells. EVs were isolated from the wild-type strain CBE59, the hlyE-V deletion mutant CBE59ΔhlyE-V, and the complemented strain CBE59CΔhlyE-V. EVs from all strains exhibited comparable particle sizes and protein concentrations. HlyE-V was detected in EVs from the wild-type and complemented strains but was absent from EVs of the mutant strain. EVs from all strains were internalized by HD11 cells, while only EVs carrying HlyE-V induced pronounced cytotoxicity and apoptosis. Treatment with APEC EVs increased the transcription levels of Bax, Caspase-3, and Caspase-9 and reduced Bcl-2 levels, indicating the activation of intrinsic, mitochondria-dependent apoptosis. Although EVs also upregulated the transcription levels of TLR4 and MyD88, the hlyE-V deletion did not affected this pathway. Together, these findings demonstrate that APEC EVs carry HlyE-V, thereby enhancing EV-mediated cytotoxicity and intrinsic apoptosis in HD11 cells. This study identifies HlyE-V as an EV-associated virulence factor that contributes to the interaction between APEC and HD11 chicken macrophages.