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首页> 美国卫生研究院文献>Journal of Bacteriology >Mycoplasma hyopneumoniae p65 Surface Lipoprotein Is a Lipolytic Enzyme with a Preference for Shorter-Chain Fatty Acids
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Mycoplasma hyopneumoniae p65 Surface Lipoprotein Is a Lipolytic Enzyme with a Preference for Shorter-Chain Fatty Acids

机译：猪肺炎支原体p65表面脂蛋白是一种脂解酶首选短链脂肪酸

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Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39°C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.
机译：猪肺炎支原体是猪呼吸道最重要的细菌病原体。 p65是猪肺炎支原体的一种免疫优势表面脂蛋白，在疾病期间被特异性识别。分析编码p65的基因的翻译的氨基酸序列，发现其与脂解酶的GDSL家族相似。为了检验p65的脂解活性，在原核脂蛋白信号序列被截短并支原体TGA色氨酸密码子诱变后，将该基因克隆并在大肠杆菌中表达。用凝血酶处理后，重组谷胱甘肽S-转移酶（GST）-p65蛋白产生了一个66-kDa融合蛋白裂解产物，大小相当于成熟的p65蛋白。重组GST-p65的酯酶活性通过在三丁酸甘油酯琼脂板上形成一个透明区域以及己酸对硝基苯酯（pNPC）和棕榈酸酯对硝基苯酯（pNPP）的水解来表明。脂肪酶活性通过人工甘油三酸酯1,2-O-二月桂酰基-rac-甘油-3-戊二酸间苯二酚酯的水解来指示。使用pNPC和pNPP作为底物，重组GST-p65在pH 9.2和10.2之间以及高于39°C的温度下具有最佳活性。钙离子并未增加重组GST-p65的活性。兔抗p65抗体抑制了重组GST-p65的活性，并且还抑制了猪肺炎支原体的体外生长。检查重组GST-p65水解pNPC和pNPP的动力学参数表明，偏爱较短的pNPC脂肪酸链。支原体脂解酶的生理和/或致病作用尚未确定，但它们可能在支原体对长链脂肪酸的营养需求中起重要作用，并可能降低肺表面活性剂在支原体诱发的呼吸系统疾病中的功能。这是关于支原体脂质修饰的表面免疫原的脂解活性的首次报道。

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