Three Hcp homologs with divergent extended loop regions exhibit different functions in avian pathogenic <i>Escherichia coli</i>

Type VI secretion systems (T6SSs) contribute to the pathogenicity of avian pathogenic <i>Escherichia coli</i> (APEC), one of the leading causative agents of sepsis and meningitis in poultry. The Hcp protein is a core component of the T6SS tail tube and acts as an exported receptor and a chaperone of effectors. In this study, four distinct Hcp types (Ia, Ib, IIa, and IIb) were designated in Gram-negative bacteria, three of which were widely distributed in APEC. We detected divergence in transcription levels among three <i>hcp</i> clusters in 50% duck serum and demonstrated that <i>hcp1</i> was upregulated by relieving Fur repression. Further analyses revealed that the host serum could activate the <i>hcp2B</i> operon by H-NS derepression to transcribe the downstream <i>xmtU/xmtV</i> pair for inter-bacterial antagonism. Notably, in a structural analysis based on the genetic classification, Hcp proteins exhibited significant differences in the extended loop regions, suggesting that these regions were related to their functional properties. Indeed, the variant region Vs2 (Loop L2, 3) in Hcp1 and Hcp2B was essential for the delivery of antibacterial effectors and the inhibition of macrophage phagocytosis. Further analyses using a duck model indicated that these Hcps play different roles in the pathogenic processes of APEC and immunoprotection. These results indicated that the functional differentiation of Hcp homologs was driven by differences in transcriptional regulation, extended loop regions, and effector delivery.