The function of Eriocheir sinensis transferrin and iron in Spiroplasma eriocheiris infection.

Transferrin, a member of the iron binding superfamily protein, plays an extremely important role in the transport of iron in the biological process of cells. The result of preliminary proteomic study on E. sinensis hemocytes infected Spiroplasma eriocheiris showed the expression of transferrin (EsTF) and ferrin (EsFe) significantly changed. In addition, other reports have confirmed that transferrin, ferritin and iron are involved in the immune response of hosts. In order to validate the immune function of EsTF, the whole length of EsTF was successfully amplified by the gene cloning and RACE technique. The results showed that the full-length cDNA of the EsTF gene was 2748 bp, including a 2193 bp open reading frame which encodes 730 amino acids. The result of bioinformatics analysis showed EsTF contains two highly conserved TR_FER domains. Evolutionary analysis showed that EsTF has a close genetic relationship with other TFs of invertebrates. In addition, EsTF mRNA was highly transcripted in nerve and intestine tissues, followed by hemocytes. The expression of EsTF, EsFe1 and EsFe2 increased after exogenous supplemental of iron under the concentration of 100 nmol/L in water. After exogenous supplement of iron and injection with S. eriocheiris, these three gene transcription of mRNA levels were higher than that of PBS group, while lower than the S. eriocheiris group and the iron group. Besides, the copy number of S. eriocheiris in the experimental group was significantly reduced, and the death rate decreased. As can be seen, iron made transferrin and ferritin return to normal levels during the infection of S. eriocheiris and help the host maintain normal immunity levels to resist S. eriocheiris. These results further demonstrated that EsTF, EsFe1, EsFe2 and iron play a role in the immune defense mechanism of the crabs to resist S. eriocheiris infection.

Spiroplasma eriocheiris Adhesin-Like Protein (ALP) Interacts with Epidermal Growth Factor (EGF) Domain Proteins to Facilitate Infection.

Spiroplasma eriocheiris is a novel pathogen found in recent years, causing the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis. Like Spiroplasma mirum, S. eriocheiris infects the newborn mouse (adult mice are not infected) and can cause cataract. Adhesion-related protein is an important protein involved in the interaction between pathogen and host. In this study, the Adhesin-like Protein (ALP) of S. eriocheiris was detected on its outer membrane by using immune electron microscopy, and was found to be involved in the bacterium's infection of mouse embryo fibroblasts (3T6-Swiss albino). Yeast two-hybrid analysis demonstrated that ALP interacts with a diverse group of mouse proteins. The interactions between recombinant partial fibulin7 (FBLN7; including two epidermal growth factor [EGF] domains) and ALP were confirmed by Far-western blotting and colocalization. We synthetized the domains of FBLN7 [EGF domain: amino acids 136-172 and complement control protein (CCP) domain: 81-134 amino acids], and demonstrated that only EGF domain of FBLN7 can interact with ALP. Because the EGF domain has high degree of similarity to EGF, it can activate the downstream EGFR signaling pathway, in key site amino acids. The EGFR pathway in 3T6 cells was restrained after rALP stimulation resulting from competitive binding of ALP to EGF. The unborn mouse, newborn mouse, and the adult mouse with cataract have a small amount of expressed FBLN7; however, none was detected in the brain and very little expression was seen in the eye of normal adult mice. In short, ALP as a S. eriocheiris surface protein, is critical for infection and further supports the role of ALP in S. eriocheiris infection by competitive effection of the EGF/EGFR axis of the target cells.