Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus Branchiostoma belcheri: Implications for a dual role of O-antigens in the resistance to bactericidal activity

The functional properties of complement in invertebrate deuterostomes are rather ill-defined. Here we showed that the humoral fluids from amphioxus Branchiostoma belcheri were capable of causing lysis of some Vibrio species including Vibrio alginolyticus HW284, Vibrio parahaemolyticus HW458 and Vibrio harvey SF-1, the first such data in the invertebrate deuterostomes. The fluid bacteriolytic activity was abolished by pre-incubation with heat-inactivated rabbit anti-human C3 serum, heating at 45 °C for 30 min, and repeated thawing and freezing. Additionally, the bacteriolytic activity was Mg2+-dependent and Ca2+-independent, and selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity. This strongly suggests that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity. It was also shown that some Vibrio species like Vibrio cincinnatiensis HW287 appeared resistant to the complement-mediated lysis. The LPS profiling revealed that the fluid-resistant V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands, whereas the fluid-sensitive V. alginolyticus HW284 had few HMW O-antigen bands, suggesting a positive correlation between O-antigen size and humoral fluid resistance. Moreover, complement consumption assays demonstrated that both V. alginolyticus HW284 and V. cincinnatiensis HW287 consumed complement, with the former consuming significantly higher complement than the latter. Overall, it is suggested that HMW O-antigens may protect the fluid-resistant Vibrio species by a dual act of avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.