Mazzantini Diletta, Fonnesu Rossella, Celandroni Francesco, Calvigioni Marco, Vecchione Alessandra, Mrusek Devid, Bange Gert, Ghelardi Emilia
Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy.
Center for Synthetic Microbiology (SYNMIKRO) and Department of Chemistry, Philipps University, Marburg, Germany.
Front Microbiol. 2020 May 6;11:879. doi: 10.3389/fmicb.2020.00879. eCollection 2020.
The multidomain (B-NG) protein FlhF, a flagellar biogenesis regulator in several bacteria, is the third paralog of the signal recognition particle (SRP)-GTPases Ffh and FtsY, which are known to drive protein-delivery to the plasma membrane. Previously, we showed that FlhF is required for pathogenicity in an insect model of infection, being essential for physiological peritrichous flagellation, for motility, and for the secretion of virulence proteins. Among these proteins, we found that the L component of hemolysin BL, one of the most powerful toxins produces, was drastically reduced by the FlhF depletion. Herein, we demonstrate that FlhF forms GTP-dependent homodimers since the replacement of residues critical for their GTP-dependent homodimerization alters this ability. The protein directly or indirectly controls flagellation by affecting flagellin-gene transcription and its overproduction leads to a hyperflagellated phenotype. On the other hand, FlhF does not affect the expression of the L-encoding gene (), but physically binds L when in its homodimeric form, recruiting the protein to the plasma membrane for secretion. We additionally show that FlhF overproduction increases L secretion and that the FlhF/L interaction requires the NG domain of FlhF. Our findings demonstrate the peculiar behavior of FlhF, which is required for the correct flagellar pattern and acts as SRP-GTPase in the secretion of a bacterial toxin subunit.
多结构域（B-NG）蛋白FlhF是几种细菌中鞭毛生物合成的调节因子，是信号识别颗粒（SRP）-GTP酶Ffh和FtsY的第三个旁系同源物，已知这两种酶可驱动蛋白质向质膜的转运。此前，我们发现FlhF在昆虫感染模型的致病性中是必需的，对生理性周生鞭毛形成、运动性以及毒力蛋白的分泌至关重要。在这些蛋白中，我们发现溶血素BL（其产生的最强大毒素之一）的L组分在FlhF缺失时大幅减少。在此，我们证明FlhF形成GTP依赖性同源二聚体，因为替换对其GTP依赖性同源二聚化至关重要的残基会改变这种能力。该蛋白通过影响鞭毛蛋白基因转录直接或间接控制鞭毛形成，其过量产生会导致多鞭毛表型。另一方面，FlhF不影响L编码基因（）的表达，但在其同源二聚体形式时会与L物理结合，将该蛋白募集到质膜进行分泌。我们还表明FlhF过量产生会增加L的分泌，并且FlhF/L相互作用需要FlhF的NG结构域。我们的研究结果证明了FlhF的特殊行为，它是正确鞭毛模式所必需的，并且在细菌毒素亚基的分泌中充当SRP-GTP酶。
