The Bacterial Alarmone (p) ppGpp Activates the Type III Secretion System in Erwinia amylovora
The Bacterial Alarmone (p) ppGpp Activates the Type III Secretion System in Erwinia amylovora
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DOI：
10.1128/jb.02551-14
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发表时间：
2015-04-01
影响因子：
3.2
通讯作者：
Zhao, Youfu
中科院分区：
文献类型：
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作者：
Ancona, Veronica;Lee, Jae Hoon;Zhao, Youfu
过敏反应和致病性（hrp）III型分泌系统（T3SS）是梨火疫病菌的关键致病因子。先前的研究表明，梨火蚁中的 T3SS 受到 sigma 因子级联的转录调节。在本研究中，使用 Red 重组酶克隆产生的 ppGpp 突变体研究了细菌报警素 ppGpp 在激活 T3SS 和梨火疫病菌毒力中的作用。 ppGpp 缺陷突变体 (ppGpp(0)) 以及梨火疫病菌 dksA 突变体的毒力完全受损，并且细菌生长显着减少，表明 ppGpp 是梨火疫病菌完全毒力所必需的。 T3SS 基因的表达在 ppGpp(0) 和 dksA 突变体中大大下调。 Western blotting显示，ppGpp(0)和dksA突变体中HrpA蛋白的积累分别约为野生型菌株的10%和4%。此外，较高水平的 ppGpp 导致梨火蚁细胞尺寸减小。此外，丝氨酸异羟肟酸和α-甲基葡萄糖苷分别诱导氨基酸和碳饥饿，在hrp诱导基本培养基中激活hrpA和hrpL启动子活性。这些结果表明，ppGpp 和 DksA 在梨火疫病菌毒力中发挥核心作用，并表明梨火疫病菌利用 ppGpp 作为内部信使来感知环境/营养刺激，从而调节 T3SS 和毒力。 重要性 III 型分泌系统 (T3SS) 是革兰氏阴性菌的关键致病因子。充分阐明T3SS如何激活对于全面了解T3SS的功能、细菌发病机制和应激条件下的生存至关重要。在这项研究中，我们提出了第一个证据，证明细菌报警素 ppGpp 介导的严格反应通过 sigma 因子级联激活 T3SS，表明 ppGpp 作为内部信使来感知环境/营养刺激，从而调节 T3SS 和植物病原细菌的毒力。此外，spoT 无效突变体的恢复显示出非常独特的表型，表明含有单个 ppGpp 水解酶结构域的小蛋白是有功能的。
The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp(0)) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp(0) and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp(0) and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and alpha-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence.IMPORTANCEThe type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.