Abstract
Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and β-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.
Highlights
Host-pathogen contact is a prerequisite for bacterial invasion and colonization [1, 2]
Our previous research [11] has shown that in M. rosenbergii hemocytes infected with S. eriocheiris, 69 differentially expressed proteins including LGBP were identified compared with control group injected R2 medium [8% sucrose, 2.5% heart infusion broth (HIB), 15% fetal bovine serum (FBS), 100 U ml−1 penicillin, and pH 7.20–7.40]
Proteins that mediate the interaction of spiroplasma with host cells likely play an important role in pathogenesis
Summary
Host-pathogen contact is a prerequisite for bacterial invasion and colonization [1, 2]. It is likely that proteins mediate the interaction between S. eriocheiris and hemocytes, permitting entry of spiroplasma into hemocytes. Our previous research [11] has shown that in M. rosenbergii hemocytes infected with S. eriocheiris, 69 differentially expressed proteins including LGBP were identified compared with control group injected R2 medium [8% sucrose, 2.5% heart infusion broth (HIB), 15% fetal bovine serum (FBS), 100 U ml−1 penicillin, and pH 7.20–7.40]. These results suggested that LGBP play an important role in pathogen invasion into host. The specific mechanism by which LGBP facilitates S. eriocheiris entry into M. rosenbergii has not been elucidated
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