St Geme J W, Grass S
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA.
Mol Microbiol. 1998 Feb;27(3):617-30. doi: 10.1046/j.1365-2958.1998.00711.x.
Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typeable H. influenzae HMW1 and HMW2 non-pilus adhesins mediate attachment to human epithelial cells, an essential step during colonization. In order to facilitate interaction with host cells, HMW1 and HMW2 are localized on the surface of the organism in a process that involves cleavage of a 441-amino-acid N-terminal fragment. In the present study, we investigated the pathway for the secretion of HMW1 and HMW2. Cell fractionation experiments and cryoimmunoelectron microscopy demonstrated that a periplasmic intermediate occurs, suggesting involvement of the Sec machinery. Additional analysis revealed that, ultimately, the proteins are partially released from the surface of the organism. Studies with Escherichia coli harbouring plasmid subclones extended earlier findings and suggested that the secretion of HMW1 requires accessory proteins designated HMW1B and HMW1C, while the secretion of HMW2 requires proteins called HMW2B and HMW2C. Further analysis established that HMW1B/HMW1C and HMW2B/HMW2C are interchangeable, an observation consistent with the high degree of homology between HMW1B and HMW2B and between HMW1C and HMW2C. Additional studies of the hmw1 locus indicated that HMW1B is located in the outer membrane and serves to translocate HMW1 across the outer membrane. In the absence of HMW1B, HMW1 remains unprocessed and is degraded in the periplasmic space, at least in part by the DegP protease. Mutagenesis of an HMW1 N-terminal motif shared with other secreted proteins resulted in diminished processing and extracellular release, suggesting interaction of this motif with the HMW1B protein. Continued investigation of the HMW1 and HMW2 adhesins may provide general insights into protein secretion and bacterial pathogenesis.
不可分型流感嗜血杆菌是人类疾病的常见病因，通过在上呼吸道定植引发感染。不可分型流感嗜血杆菌的高分子量1（HMW1）和高分子量2（HMW2）非菌毛黏附素介导与人类上皮细胞的黏附，这是定植过程中的关键步骤。为便于与宿主细胞相互作用，HMW1和HMW2通过一个涉及441个氨基酸的N端片段切割的过程定位于菌体表面。在本研究中，我们调查了HMW1和HMW2的分泌途径。细胞分级分离实验和冷冻免疫电子显微镜显示存在周质中间体，提示Sec机制参与其中。进一步分析表明，最终这些蛋白质会从菌体表面部分释放。对携带质粒亚克隆的大肠杆菌的研究扩展了早期发现，表明HMW1的分泌需要名为HMW1B和HMW1C的辅助蛋白，而HMW2的分泌需要名为HMW2B和HMW2C的蛋白。进一步分析确定HMW1B/HMW1C和HMW2B/HMW2C是可互换的，这一观察结果与HMW1B和HMW2B之间以及HMW1C和HMW2C之间的高度同源性一致。对hmw1基因座的进一步研究表明，HMW1B位于外膜，负责将HMW1转运穿过外膜。在没有HMW1B的情况下，HMW1保持未加工状态，并在周质空间中降解，至少部分是由DegP蛋白酶降解的。与其他分泌蛋白共有的HMW1 N端基序的诱变导致加工和细胞外释放减少，提示该基序与HMW1B蛋白相互作用。对HMW1和HMW2黏附素的持续研究可能会为蛋白质分泌和细菌发病机制提供一般性见解。
