A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of Vibrio alginolyticus in the Crab Scylla paramamosain.

Fangyi Chen,Lixia Dong,Jun Bo,Xiaowan Ma,Zhongguo Shan,Kejian Wang,Huiyun Chen,Chao Qian

doi:10.1371/journal.pone.0133443

Abstract

Pathogens can enter their host cells by way of endocytosis in which the membrane lipid raft gene flotillins are probably involved in the invasion process and this is an important way to cause infection. In this study, a new gene SpFLT-1 was identified in Scylla paramamosain, which shared high identity with the flotillin-1 of other species. The SpFLT-1 gene was widely distributed in tissues and showed the highest level of mRNA transcripts in the hemocytes. This gene might be a maternal gene based on the evident results that it was highly expressed in maternal ovaries and in the early developmental stages of the zygote and early embryo stage whereas it gradually decreased in zoea 1. SpFLT-1 positively responded to the challenge of Vibrio alginolyticus with a significantly increased level of mRNA expression in the hemocytes and gills at 3 hours post infection (hpi). The SpFLT-1 protein was detected densely in the same fraction layer where the Vibrio protein was most present in the hemocytes and gills at 3 hpi. Furthermore, it was found that the expression of SpFLT-1 decreased to the base level following disappearance of the Vibrio protein at 6 hpi in the gills. Silencing SpFLT-1 inhibited the endocytosis rate of V. alginolyticus but overexpression of the gene could facilitate bacterial entry into the epithelioma papulosum cyprinid cells. Our study indicated that SpFLT-1 may act as a key protein involved in the process of bacterial infection and this sheds light on clarifying the pathogenesis of pathogens infecting S. paramamosain.

Highlights

The mud crab, Scylla paramamosain, is one of the most important marine breeding crabs in China, with vital nutritional and economic value
A subtractive suppression hybridization (SSH) cDNA library was constructed and many interesting genes or proteins potentially involved in the innate immune defense of the crab were screened from the hemocytes of the crab S. paramamosain challenged with lipopolysaccharide (LPS) [7]
In the currently known flotillin-1, SpFLT-1 has the highest identity to yellow fever mosquito (A. aegypti), fruit fly (D. melanogaster), and honey bee (Apis mellifera) with identity to 74 or 75%

Summary

Introduction

The mud crab, Scylla paramamosain, is one of the most important marine breeding crabs in China, with vital nutritional and economic value. Invertebrate animals lack an adaptive immune system and they have to rely on their innate immune system in which immune-related components may play a key role [1]. A number of innate immune-related genes have been identified so far from S. paramamosain, including antimicrobial peptides [2,3,4] and pathogen recognition protein [5, 6], which are found to have potentially critical roles in defense against pathogens. A subtractive suppression hybridization (SSH) cDNA library was constructed and many interesting genes or proteins potentially involved in the innate immune defense of the crab were screened from the hemocytes of the crab S. paramamosain challenged with lipopolysaccharide (LPS) [7]. A new membrane lipid raft gene flotillin-1 was identified which was possibly involved in the pathogenesis of invading pathogens, and worth further study

Methods

Results

Conclusion