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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2014 Vol.15 No.9 P.776-787
A homolog of glyceraldehyde-3-phosphate dehydrogenase from Riemerella anatipestifer is an extracellular protein and exhibits biological activity
Abstract: Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer (RaGAPDH) has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genomic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligand-binding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coli (EHEC), belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestifer displayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism’s virulence.
Key words: Riemerella anatipestifer, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Extracellular protein
创新要点：首次证实鸭疫里默氏杆菌具有GAPDH的同源体酶（RaGAPDH）是一种无信号肽和跨膜区的胞外蛋白酶，具有将3-磷酸甘油醛转化为1,3-二磷酸甘油酸的活性，可与纤维蛋白溶酶原及纤维蛋白原发生结合，推测该酶可能是鸭疫里默氏杆菌的一个新发现的毒力因子。
研究方法：1．对分离自重庆、四川地区的鸭疫里默氏杆菌（表1）菌体细胞表面蛋白（图1a）和CZ2、SC12、YC1三株菌胞外蛋白（图1b）的GAPDH活性进行检测，对其编码基因进行PCR鉴定（图2）和克隆测序分析
（图3）；2．采用染色体步移技术获得CZ2的GAPDH编码基因进行原核表达（图4a和4c）；3．以获得的具有活性的重组GAPDH为抗原，制备鼠原多克隆抗体并采用Western-blot方法对鸭疫里默氏杆菌的胞外分泌蛋白进行检测分析（图4b）；4．采用固相配体结合试验检测RaGAPDH与纤维蛋白溶酶原、血纤维蛋白原、肌动蛋白和纤连蛋白的结合作用（图5）。
重要结论：鸭疫里默氏杆菌具有三磷酸甘油醛脱氢酶同源体，具有GAPDH活性，能与纤维蛋白溶酶原和血纤维蛋白原结合，可能是其重要的毒力因子。
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DOI:
10.1631/jzus.B1400023
CLC number:
Q936; S85
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On-line Access:
2014-06-03
Received:
2014-01-24
Revision Accepted:
2014-05-29
Crosschecked:
2014-08-21