Su Marcia Shu-Wei, Dickins Benjamin, Kiang Fang Yie, Tsai Wei-Jiun, Chen Yueh-Lin, Chen Jenn-Wei, Wang Shuying, Tsai Pei-Jane, Wu Jiunn-Jong
Department of Medical Laboratory Science and Biotechnology, College of Medical and Health Sciences, Asia University, Taichung, Taiwan.
Department of Biotechnology and Laboratory Science in Medicine, College of Biomedical Science and Engineering, National Yang Ming Chiao Tung University, Taipei, Taiwan.
Helicobacter. 2025 Mar-Apr;30(2):e70019. doi: 10.1111/hel.70019.
Helicobacter pylori colonizes the human stomach as a dominant member of the gastric microbiota and constitutively expresses flagellar motility for survival. Carbon storage regulator A (CsrA) is a posttranscriptional global regulator and a critical determinant of H. pylori's motility and pathogenicity. The regulation of H. pylori CsrA is still uncertain although in other species CsrA is reported to be antagonized by small RNAs and proteins. In this study, we attempted to unveil how CsrA is regulated and hypothesized that H. pylori CsrA activity is antagonized by a flagellar assembly factor, FliW2, via protein allosteric obstruction.
Multiple sequence comparisons indicated that, along its length and in contrast to fliW1, the fliW2 of H. pylori J99 is conserved. We then generated an isogenic ΔfliW2 strain whose function was characterized using phenotypic and biochemical approaches. We also applied a machine learning approach (AlphaFold2) to predict FliW2-CsrA binding domains and investigated the FliW2-CsrA interaction using pull-down assays and in vivo bacterial two-hybrid systems.
We observed the reduced expression of major flagellin FlaA and impaired flagellar filaments that attenuated the motility of the ΔfliW2 strain. Furthermore, a direct interaction between FliW2 and CsrA was demonstrated, and a novel region of the C-terminal extension of CsrA was suggested to be crucial for CsrA interacting with FliW2. Based on our AlphaFold2 prediction, this C-terminal region of FliW2-CsrA interaction does not overlap with CsrA's N-terminal RNA binding domain, implying that FliW2 allosterically antagonizes CsrA activity and restricts CsrA's binding to flaA mRNAs.
Our data points to novel regulatory roles that the H. pylori flagellar assembly factor FliW2 has in obstructing CsrA activity, and thus FliW2 may indirectly antagonize CsrA's regulation of flaA mRNA processing and translation. Our findings reveal a new regulatory mechanism of flagellar motility in H. pylori.
幽门螺杆菌作为胃微生物群的主要成员定殖于人类胃部，并持续表达鞭毛运动以生存。碳储存调节因子A（CsrA）是一种转录后全局调节因子，也是幽门螺杆菌运动性和致病性的关键决定因素。尽管在其他物种中据报道CsrA会被小RNA和蛋白质拮抗，但幽门螺杆菌中CsrA的调节机制仍不确定。在本研究中，我们试图揭示CsrA是如何被调节的，并假设幽门螺杆菌的CsrA活性会被一种鞭毛组装因子FliW2通过蛋白质变构阻碍作用所拮抗。
多序列比对表明，与fliW1不同，幽门螺杆菌J99的fliW2在其全长范围内是保守的。然后我们构建了一个同基因的ΔfliW2菌株，并使用表型和生化方法对其功能进行了表征。我们还应用机器学习方法（AlphaFold2）预测FliW2-CsrA结合结构域，并使用下拉试验和体内细菌双杂交系统研究FliW2-CsrA相互作用。
我们观察到主要鞭毛蛋白FlaA的表达降低，鞭毛丝受损，这减弱了ΔfliW2菌株的运动性。此外，证实了FliW2与CsrA之间存在直接相互作用，并且CsrA C末端延伸的一个新区域被认为对CsrA与FliW2相互作用至关重要。基于我们的AlphaFold2预测，FliW2-CsrA相互作用的这个C末端区域与CsrA的N末端RNA结合结构域不重叠，这意味着FliW2通过变构拮抗CsrA活性并限制CsrA与flaA mRNA的结合。
我们的数据表明幽门螺杆菌鞭毛组装因子FliW2在阻碍CsrA活性方面具有新的调节作用，因此FliW2可能间接拮抗CsrA对flaA mRNA加工和翻译的调节。我们的发现揭示了幽门螺杆菌鞭毛运动的一种新调节机制。
