肺炎克雷伯菌HdeA蛋白的抗酸理化性质鉴定

Characterization of the Klebsiella pneumoniae Hde A Protein under Acidic Condition

肺炎克雷伯菌是一种常见的院内条件致病菌,通常定植在人体的呼吸道和胃肠道。但肺炎克雷伯菌是怎样在胃液的强酸条件下存活并进入肠道尚不十分清楚。本研究发现,酸性条件能够诱导肺炎克雷伯菌Kp Hde A基因表达上调。克隆Kp Hde A基因并将其转化大肠杆菌,获得可溶性的Kp Hde A蛋白并利用镍离子亲和层析纯化得到目的蛋白。光散射分析证明,酸性胁迫下,Kp Hde A蛋白能够减少酸诱导的醇脱氢酶聚集。荧光实验证明,酸胁迫可以诱导Kp Hde A蛋白的疏水基团暴露。圆二色谱实验证明,酸性条件能够诱导Kp Hde A蛋白形成无序结构。此外Kp Hde A蛋白的表达能提高大肠杆菌的耐酸能力。故推测,Kp Hde A蛋白具有分子伴侣活性,并在肺炎克雷伯菌抵抗酸胁迫中起重要作用。

Klebsiella pneumoniae is an important conditional pathogen in hospital infection, which frequently colonizes the respiratory and intestinal tracts of human. How K. pneumoniae escapes the acid stress of gastric acid and then enters the intestinal remains unclear. The expression of Kp Hde A was upregulated in K. pneumoniae under acid stress. The coding sequence of Kp Hde A was cloned from K.pneumoniae and overexpressed in Escherichia coli. Soluble Kp Hde A was purified by Ni2 ＋-affinity chromatography with apparent homogeneity. The light scattering assay showed that the acid-induced alcohol dehydrogenase（ ADH） aggregation was attenuated by Kp Hde A. The fluorescence emission spectra indicated that the hydrophobic surfaces of Kp Hde A protein were exposed under acidic condition. CD spectra also indicated that Kp Hde A protein could form a disordered conformation when treated with acid.Additionally,over-expression of Kp Hde A could confer anti-acid capability on E. coli. It was concluded that Kp Hde A was a molecular chaperone and was crucial for acid resistance of K. pneumoniae.