Abstract
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.
Highlights
Spiroplasma eriocheiris is a causative agent of the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis (Wang et al, 2003, 2011), and a novel pathogen of aquatic crustaceans includingProcambarus clarki, Penaeus vannamei, Macrobrachium rosenbergii, and Macrobrachium nipponense (Bi et al, 2008; Liang et al, 2011; Xiu et al, 2015)
In agreement with TEM (Figure 1C), the results showed that the ALP signal was detected in the lanes of total protein from S. eriocheiris (T)
The results showed that the quantities of S. eriocheiris infection within the
Summary
We demonstrated that S. eriocheiris ALP is critical for the bacterial infection of host cells, and may be important in finding ways to prevent host cell infection. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), (Wisent, Canada) complete medium supplemented with 10% fetal bovine serum (FBS), 0.15% NaHCO3 , 0.45% glucose, 4 mM L-Glutamine (Wisent, Canada), and antibiotics S. eriocheiris (S. eriocheiris groups) for 36 h; (2) 3T6 cells were infected with S. eriocheiris, which was treated with anti-ALP (1:2000) for 1 h at 30◦ C (S. eriocheiris+Anti-ALP groups), and incubated for 36 h; (3) 3T6 cells were infected with S. eriocheiris, which was treated with pre-immume serum for 1 h at 30◦ C (S. eriocheiris+serum groups), and incubated for 36 h; (4) 3T6 cells were treated with recombinant ALP (5 μg/ml) for 0, 15, 30, 45, and 60 min (using 0 min as control); (5) 3T6 cells were treated with EGF (20 ng/ml) and using different concentrations of recombinant ALP (0, 0.5, 1, 5, and 10 μg/ml) for 15 min, cells without any treatment served as a control; (6) HeLa cells were co-transfected with plasmids encoding EGFP-ALP and DsRedFiblin, using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the supplier’s instructions. The all host cells were culture with same conditions 37◦ C and 5% CO2
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