Complement-mediated Killing Of Vibrio Species By The Humoral Fluids Of Amphioxus Branchiostoma Belcheri Tsingtauense And Vibrio Species Escape The Mechanisms Of Complement's Attacktion

The Phylogeny of the complement system during evolution has become one of the key problems in immunology. Comparative studies have suggested common ancestries of complement system in both vertebrates and invertebrates. In the past decade, the structurally and functionally similar complement components including C3 and factor B, have been identified in the invertebrate deuterostomes and even in the invertebrate protostome. However, the functions of complement in these animals are rather ill-defined. Amphioxus, a cephalochordate, is the closest living relative to vertebrate, and has been widely known as the most important animal to analyze the origin and evolution of vertebrates. Here, Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus is studied that attribute to better understand the phylogeny of the complement system during evolve of chordate. In addition, this study explores Vibrio species escape the mechanisms of complement's attacktion.In this study, the standard method of measuring bacteriolytic activity of serum of vertebrate is used in the study of humoral fluids bacteriolytic activity from amphioxus, the first such report in the invertebrate deuterostomes. Here, when humoral fluids from amphioxus Branchiostoma belcheri was interactive with Vibrio species, it was showed that the humoral fluids in vitro were capable of inhibitting growth of some Vibrio species including V. alginolyticus HW284, V. parahaemolyticus HW458 and V. harvey SF-1, only 42%, 28% and 59% of V. alginolyticus HW284, V. parahaemolyticus HW458 and V. harvey SF-1 survived incubation in amphioxus humoral fluids, respectively, the first such data in the invertebrate deuterostomes. Scanning electron microscopy examination definitely revealed the presence of the bacteriolytic activity of amphioxus humoral fluids. V. alginolyticus HW284 incubated in the humoral fluids was lysed, and the cell surface was apparently dissolved, whereas that incubated in the heated humoral fluids and sterilized seawater remained intact, and the cell surface was smooth. Pre-incubation of the heat-inactivated rabbit anti-human C3 serum with amphioxus humoral fluids abrogated the bacteriolytic activity of V. alginolyticus HW284, while pre-incubation with the heat-inactivated non-immune rabbit serum or with PBS did not impair the bacteriolytic activity. This strongly suggested a role of complement in the bacteriolytic activity of amphioxus humoral fluids. This was further supported by the experiments that heat treatment (45℃, 30 min) and repeated thaw and freezing of the humoral fluids markedly reduced the bacteriolytic activity of V. alginolyticus HW284, with 89% and 81% survival observed, respectively, contrasting to 48% survival in the control. 40 mM EGTA plus 4 mM MgCl2 had little effect on the bacteriolytic activity, and survival of V. alginolyticus HW284 (42%) remained comparable to that of the control. In agreement, addition of 10 mM CaCl2 did not enhance the bacteriolytic activity of the EGTA-treated humoral fluids. On the contrary, addition of 40 mM EDTA to the humoral fluids resulted in a significant loss of the bacteriolytic activity, with 86% survival observed, contrasting to 44% survival in the control. On the other hand, the bacteriolytic activity of the EDTA-treated humoral fluids was restored by adding 4 mM MgCl2,This shows strongly the bacteriolytic activity was Mg2+-dependant and Ca2+-independent. In addition, selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity, and resulted in an increased V. alginolyticus HW284 survival. On average, 82% V. alginolyticus HW284 cells survived exposure to a 1: 2 dilution of the humoral fluids previously incubated with zymosan A. In contrast, just 43% of the cells survived in the untreated humoral fluids. Therefore, these results strongly suggest that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity.It is of note that the four Vibrio species, V. furnissi HW293, V. cincinnatiensis HW287, V. anguillarum W-1, and V. harveyi Z3G2 out of the seven species tested appear resistant to the complement-mediated lysis. Here, the method of consumption of complement monitored in serum of vertebrate is used in the functional study of humoral fluids from amphioxus, the first such data in the invertebrate deuterostomes. LPS was extracted from V. alginolyticus HW284 and V. cincinnatiensis HW287 by a proteinase K method,then subjected to electrophoresis on 12% SDS-PAGE and silver stained. The LPS profiling revealed that V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands. In contrast, V. alginolyticus HW284 had few HMW O-antigen bands. Thus the lack of HMW O-antigen bands of V. alginolyticus HW284 coincided with sensitivity to the humoral fluids, whereas the humoral fluid-resistant V. cincinnatiensis HW287 had many HMW O-antigen bands, indicating the presence of a positive correlation between O-antigen size and humoral fluids resistance. Moreover, consumption of complement was monitored by measuring the hemolysis of RaRBC by amphioxus humoral fluids after the fluids had been pre-incubated with V. alginolyticus HW284 or V. cincinnatiensis HW287. It is showed that both Vibrio species tested consumed complement, but V. alginolyticus HW284 consumed significantly higher complement than V. cincinnatiensis HW287, agreeing well with the observation that the humoral fluids were much more bacteriolytic to V. alginolyticus HW284 than to V. cincinnatiensis HW287. All these results suggest that HMW O-antigens may protect the fluid-resistant Vibrio species including V. cincinnatiensis HW287 by a dual act of both avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.