Expression and porin activity of p28 and OMP-1F during intracellular Ehrlichia chaffeensis development
Expression and porin activity of p28 and OMP-1F during intracellular Ehrlichia chaffeensis development
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DOI：
10.1128/jb.02017-07
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发表时间：
2008-05-01
影响因子：
3.2
通讯作者：
Rikihisa, Yasuko
中科院分区：
文献类型：
--
作者：
Kumagai, Yumi;Huang, Haibin;Rikihisa, Yasuko
查菲埃里希体是一种专性胞内革兰氏阴性细菌，由于缺乏许多参与代谢的基因，必须摄取各种营养物质和代谢化合物。革兰氏阴性菌主要通过细菌外膜中的孔或通道摄取营养物质。在这里，我们证明了孤立的E。Chaffeensis外膜具有孔蛋白活性，如通过蛋白脂质体溶胀测定所测定的。该活性被识别两种最丰富的外膜蛋白P28/OMP-19和OMP-1F/OMP-18的抗体部分阻断。预测这两种蛋白质具有孔蛋白的结构特征，包括由两亲性和反平行P-链组成的12个跨膜区段。两种蛋白质的十二烷基硫酸钠稳定性与P-桶结构一致。分离的天然P28和OMP-1F表现出孔蛋白活性，其孔径分别类似于和大于OprF的孔径，OprF是迄今为止已知的具有最大孔径的孔蛋白。E. chaffeensis在通过蜱传播期间经历温度变化。在E.在chaffeensis中，P28和OMP-1F都主要在37 ℃的指数生长中期和28 ℃的指数生长晚期表达。在28 ℃和37 ℃的中期和晚期指数生长期，用来自细菌的外膜组分的蛋白质重构的脂蛋白体的孔蛋白活性与P28和OMP-1F的表达水平相关。这些结果表明，P28和OMP-1F作为具有大孔径的孔蛋白发挥功能，这两种蛋白的差异表达可能调节细胞内E. chaffeensis发育在这两个温度。
Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gram-negative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and anti-parallel P-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a P-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37 degrees C and the late-exponential growth phase at 28 degrees C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid- and late-exponential growth phases at 28 degrees C and 37 degrees C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.