Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.
Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.
复制标题
睡眠嗜组织菌 IbpA DR2/Fic 对天然宿主肺泡上皮屏障的毒力和免疫保护作用。
DOI：
10.1128/iai.01277-09
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发表时间：
2010
影响因子：
3.1
通讯作者：
Corbeil,LynetteB
中科院分区：
文献类型：
--
作者：
Zekarias,Bereket;Mattoo,Seema;Worby,Carolyn;Lehmann,Jason;Rosenbusch,RicardoF;Corbeil,LynetteB
新认识的Fic家族毒力蛋白可能在许多细菌病原体中是重要的。为了研究嗜组织菌睡眠免疫球蛋白结合蛋白A（HibpA）直接重复序列2 Fic结构域（DR 2/Fic）对宿主靶细胞的细胞毒性，探讨其与免疫保护和发病机制的关系。产IbpA活毒H.睡眠菌株2336、该菌株的无细胞培养上清液（CCS）或重组DR 2/Fic（rDR 2/Fic）引起牛肺泡2型（BAT 2）上皮细胞的显著收缩和变圆。IbpA缺乏。睡眠菌株129 Pt和Fic基序His 298 Ala突变体rDR 2/Fic蛋白没有细胞毒性。通过在[α-32 P]ATP存在下将BAT 2细胞裂解物与菌株2336 CCS或rDR 2/Fic孵育，导致Rho GTP酶腺苷酸化和细胞骨架破坏，证明了DR 2/Fic细胞毒性的细胞机制。由于IbpA不被III型或IV型分泌系统分泌，我们确定DR 2/Fic是否进入宿主细胞质以接近其Rho GT3靶标。虽然。Somni不能侵入BAT 2细胞，DR 2/Fic被H处理的细胞内化。somni、CCS或rDR 2/Fic蛋白，如通过共聚焦免疫显微镜所示。Transwell细菌迁移试验表明，大量菌株2336细菌在缩回的BAT 2细胞之间迁移，但IbpA缺陷型菌株129 Pt不穿过单层，除非用菌株2336 CCS或rDR 2/Fic蛋白预处理单层。抗rDR 2/Fic抗体或被动保护性恢复期血清可阻断IbpA介导的细胞毒作用，抑制H.这证实了DR 2/Fic在发病机制中的作用，并与先前动物研究中的体内保护结果相对应。
Newly recognized Fic family virulence proteins may be important in many bacterial pathogens. To relate cellular mechanisms to pathogenesis and immune protection, we studied the cytotoxicity of theHistophilus somniimmunoglobulin-binding protein A (IbpA) direct repeat 2 Fic domain (DR2/Fic) for natural host target cells. Live virulent IbpA-producingH. somnistrain 2336, a cell-free culture supernatant (CCS) of this strain, or recombinant DR2/Fic (rDR2/Fic) caused dramatic retraction and rounding of bovine alveolar type 2 (BAT2) epithelial cells. IbpA-deficientH. somnistrain 129Pt and a Fic motif His298Ala mutant rDR2/Fic protein were not cytotoxic. The cellular mechanism of DR2/Fic cytotoxicity was demonstrated by incubation of BAT2 cell lysates with strain 2336 CCS or rDR2/Fic in the presence of [α-32P]ATP, which resulted in adenylylation of Rho GTPases and cytoskeletal disruption. Since IbpA is not secreted by type III or type IV secretion systems, we determined whether DR2/Fic entered the host cytoplasm to access its Rho GTPase targets. AlthoughH. somnidid not invade BAT2 cells, DR2/Fic was internalized by cells treated withH. somni, CCS, or the rDR2/Fic protein, as shown by confocal immunomicroscopy. Transwell bacterial migration assays showed that large numbers of strain 2336 bacteria migrated between retracted BAT2 cells, but IbpA-deficient strain 129Pt did not cross a monolayer unless the monolayer was pretreated with strain 2336 CCS or rDR2/Fic protein. Antibody to rDR2/Fic or passively protective convalescent-phase serum blocked IbpA-mediated cytotoxicity and inhibitedH. somnitransmigration across BAT2 monolayers, confirming the role of DR2/Fic in pathogenesis and corresponding to the results forin vivoprotection in previous animal studies.