Study Of Bacterial Clearance And Immunological Priming In Amphioxus Branchiostoma Belcheri

Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. Previously, amphioxus is widely used as the model organism for studying evolution and development. Recently, it has been used to explore the origin and evolution of immune system. This paper, deals with the bacterial clearance and immunological priming in amphioxus Branchiostoma belcheri.First, amphioxus was challenged with E.coli with transgenic green fluorescent protein (GFP) gene, and the numbers of E. coli in the humoral fluids were counted under a fluorescence microscope. Results indicated that amphioxus B. belcheri was able to efficiently clear the invading bacteria like E. coli from its humoral fluids. Phagocytosis by blood cells is regarded as an important way to remove bacteria in invertebrates. To find out the major elements responsible for the clearance of infected invaders, tissue sections were tested to search for the cells which may engulf E. coli in amphioxus. Not a single haemocyte-like cell has ever been seen among at least 2 000 samples examined, suggesting that free circulating macrophages not the main elements for the elimination of invading bacterium E. coli in amphioxus. Then the humoral fluids were tested for screening the factors attributable to the removal of invaders in amphioxus. The findings suggest that the humoral fluids can readily lyse the bacterium E. coli in vitro. The complement and lysozyme are both involved in the elimination in vivo of the invading bacterium in amphioxus, but the complement appears playing a stronger role than the lysozyme. Pre-incubation of amphioxus humoral fluids with the specific antibodies and inhibitor, results showed that the complement operating via the alternative pathway. Western blot showed that both C3αand lysozyme levels increase with the challenge with E. coli. This was further showed the complement and lysozyme were both involved in the elimination in vivo of the invading bacterium in amphioxus. Quantitative real-time PCR was employed to quantify the complement key gene expression in the gut and gut-free body of B. belcheri challenge with E.coli and V.anguillarum. Results showed that the patterns of the gene expression were similar after challenge with the both bacteria. Compared with the controls, Bf, C3 and C6 genes showed a similar up-regulation profile after infection. Their expression in the gut peaked at 3 h, while the expression in the gut-free body reached the highest levels at 6 h after E.coli challenge. Similarly, the expression of the genes in the gut maximized at 6 h, while their expression in the gut-free body reached maximum at 12 h after V.anguillarum challenge. The results indicated that the complement components play an important role in the acute phase of infection. In contrast, MASP expression decreased markedly after infection. This may be due to the bacterial counter-measure to avoid the activation of the complment. All these indicate that amphioxus digestive system rich in mucosa may be the major immune tissue responding to bacterial challenge and play an important role in the immune response.Finally, amphioxus was re-infected with E.coli and V.anguillarum in different ways, and quantitative real-time PCR was employed to study if there is immunological memory or priming in amphioxus Branchiostoma belcheri. Results indicated that the expression of Bf, C3 and C6 genes were significantly up-regulated and peaked earlier after secondary infection with the same bacteria. What's more, the gene expression kept at high levels for a longer time compared with the primary infection. In contrast, the expression levels of the genes in the animals infected with the different bacterium were lower than those in the animals infected with the same bacterium. These properties appear bearing resemblance to the immunological priming in vertebrate, suggesting the presence of some immunological priming in amphioxus.In summary, this study demonstrates that the circulating haemocyte-free amphioxus is able to eliminate invading bacteria like E. coli efficiently, both the complement and lysozyme are the main factors acting in concert to eliminate the invaders in vivo. Also, amphioxus appears to have immunological primed ability, the first such data in the protochordates.