Shicui Zhang

Vitellogenin Functions as a Multivalent Pattern Recognition Receptor with an Opsonic Activity

Background Vitellogenin (Vg), a major reproductive protein, has been associated with infection-resistant response in fish. However, the underlying mechanisms by which Vg is involved in anti-infectious response are not understood. Methodology/Results By both protein-microbe interaction analysis and enzyme-linked immunosorbent assay as well as phagocytosis test, we demonstrate for the first time that fish Vg acts as a pattern recognition molecule with multiple specificities that can recognize bacteria as well as fungus rather than self components from fish, and functions as an opsonin that can enhance macrophage phagocytosis. Conclusions This study shows that fish Vg plays an integrative function in regulating immunity via its pleiotropic effects on both recognizing pathogen-associated molecular patterns and promoting macrophage phagocytosis. It also supports the notion that factors normally involved in control of female reproduction are associated with immunity in organisms that rely on Vg for oocyte development.

Complement activity in the egg cytosol of zebrafish Danio rerio: evidence for the defense role of maternal complement components.

Most fish embryos that develop externally are exposed to an environment full of microbes. How they survive microbial attacks are not understood to date. Here we demonstrated that the egg cytosol prepared from the newly fertilized eggs of zebrafish Danio rerio is capable of killing the Gram-negative bacterium Escherichia coli, via in vitro assay system of the complement activity established. All findings indicate that it is the complement system operating via the alternative pathway that is attributable to the bacteriolytic activity. This is the first report providing the evidence for the functional role of the maternal complement components in fish eggs, paving the way for study of maternal immunity in other organisms whose eggs are fertilized in vitro.

Response of complement expression to challenge with lipopolysaccharide in embryos/larvae of zebrafish Danio rerio: acquisition of immunocompetent complement.

The development of complement in fish is poorly understood, and little information is available about the acquisition of full complement capacities. In this study we demonstrated the ontogenic and differential expression of complement key component genes (C3, C1r/s, C4, C6, Bf, MBL and MASP) of the classical pathway (CP), alternative pathway (AP), lectin pathway (LP) and lytic pathway, and their responses to challenge with LPS during development. All components, except C6, showed a similar trend of increasing expression after hatching. The contents of Bf and C3 transcripts were both markedly increased by challenge with LPS soon after hatching, while the expression levels of C1r/s and MASP transcripts were not elevated. The expression of C4 mRNA was first down-regulated by LPS challenge and then fluctuated. The expression of MBL mRNA was up-regulated by LPS challenge after 18 days post fertilization. In response to LPS challenge, C6 was peculiarly expressed: it was significantly decreased before hatching, followed by a significant up-regulation post hatching. Taken together, these suggest that the complement operating via the AP is already competent by responding to challenge with LPS in the hatched larvae of Danio rerio.

Characterization of a pattern recognition molecule vitellogenin from carp (Cyprinus carpio).

Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. To explore the role of vitellogenin (Vg) in fish innate immunity, we purified Vg from Carp by gel filtration combined with diethylaminoethyl (DEAE) chromatography. The purified Vg was confirmed by MALDI-TOF mass spectrometry. Antibacterial activity analysis showed that Vg inhibited bacterial activity to Escherichia coli and Staphylococcus aureus in a dose-dependent manner. Vg bound to the surface of Gram-negative and Gram-positive bacteria. It also agglutinated E. coli and S. aureus and weakly to Saccharomyces cerevisiae. Vg showed a strong binding activity to lipopolysaccharides from Gram-negative bacteria. Vg-treated macrophage enhanced phagocytosis to E. coli and S. aureus. Vg also bind with macrophage function as opsonins to promote phagocytosis. The results suggest that Vg serves as a pattern recognition molecule and opsonins in antibacterial defense and as an effector in fish innate immunity.

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 degrees C for 30 min, and repeated thawing and freezing. Additionally, the bacteriolytic activity was Mg(2+)-dependent and Ca(2+)-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.

Alternative complement activity in the egg cytosol of amphioxus Branchiostoma belcheri: evidence for the defense role of maternal complement components.

Background The eggs in most invertebrates are fertilized externally, and therefore their resulting embryos are exposed to an environment full of microbes, many of which are pathogens capable of killing other organisms. How the developing embryos of invertebrates defend themselves against pathogenic attacks is an intriguing question to biologists, and remains largely unknown. Methodology/Principal Findings Here we clearly demonstrated that the egg cytosol prepared from the newly fertilized eggs of amphioxus Branchiostoma belcheri, an invertebrate chordate, was able to inhibit the growth of both the Gram-negative bacterium Vibrio anguillarum and the Gram-positive bacterium Staphylococcus aureus. All findings point to that it is the complement system operating via the alternative pathway that is attributable to the bacteriostatic activity. Conclusions/Significance This appears to be the first report providing the evidence for the functional role of the maternal complement components in the eggs of invertebrate species, paving the way for the study of maternal immunity in other invertebrate organisms whose eggs are fertilized in vitro. It also supports the notion that the early developing embryos share some defense mechanisms common with the adult species.