Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites
Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites
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DOI：
10.1038/s41598-020-68054-7
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发表时间：
2020-07-09
影响因子：
4.6
通讯作者：
Imperi, Francesco
中科院分区：
文献类型：
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作者：
Scala, Romina;Di Matteo, Adele;Imperi, Francesco
脂多糖（LPS）是许多革兰氏阴性细菌外膜（OM）的关键成分。 LPS 通过 LPS 传输 (Lpt) 系统转移到 OM。在人类病原体铜绿假单胞菌中，周质 Lpt 成分 LptH 对于 LPS 运输、浮游和生物膜生长、OM 稳定性和感染性至关重要。 LptH 已被提议寡聚并形成蛋白质桥，在运输过程中容纳 LPS。基于已知的 LptH 晶体结构，我们通过计算机模拟预测了可能参与 LptH 寡聚化的五个不同位点。这些位点与 LptH 活性的相关性通过铜绿假单胞菌 lptH 条件突变体中位点特异性突变蛋白的质粒介导表达进行了验证。互补和蛋白质表达分析提供的证据表明所有突变位点对于体内 LptH 活性都很重要。据观察，lptH条件突变体通过基因组中的野生型lptH基因与质粒中的突变拷贝之间的RecA介导的同源重组克服了非功能性lptH变体的致死性。最后，对纯化的重组蛋白进行的生化测定表明，一些 LptH 变体确实在寡聚化过程中受到特定损害，而其他变体似乎在蛋白质折叠和/或稳定性方面存在缺陷。
Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gram-negative bacteria. LPS is translocated to the OM by the LPS transport (Lpt) system. In the human pathogen Pseudomonas aeruginosa, the periplasmic Lpt component, LptH, is essential for LPS transport, planktonic and biofilm growth, OM stability and infectivity. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization. The relevance of these sites for LptH activity was verified through plasmid-mediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. Complementation and protein expression analyses provided evidence that all mutated sites are important for LptH activity in vivo. It was observed that the lptH conditional mutant overcomes the lethality of nonfunctional lptH variants through RecA-mediated homologous recombination between the wild-type lptH gene in the genome and mutated copies in the plasmid. Finally, biochemical assays on purified recombinant proteins showed that some LptH variants are indeed specifically impaired in oligomerization, while others appear to have defects in protein folding and/or stability.