Duong-Nu Tra-My, Jeong Kwangjoon, Hong Seol Hee, Nguyen Hong-Vu, Ngo Van-Hoan, Min Jung-Joon, Lee Shee Eun, Rhee Joon Haeng
Clinical Vaccine R&D Center, Chonnam National University, Gwangju, Republic of Korea Department of Molecular Medicine, Graduate School, Chonnam National University, Gwangju, Republic of Korea.
Clinical Vaccine R&D Center, Chonnam National University, Gwangju, Republic of Korea Department of Microbiology, Chonnam National University Medical School, Gwangju, Republic of Korea.
Infect Immun. 2015 Nov 2;84(1):254-65. doi: 10.1128/IAI.00821-15. Print 2016 Jan.
TonB systems actively transport iron-bound substrates across the outer membranes of Gram-negative bacteria. Vibrio vulnificus CMCP6, which causes fatal septicemia and necrotizing wound infections, possesses three active TonB systems. It is not known why V. vulnificus CMCP6 has maintained three TonB systems throughout its evolution. The TonB1 and TonB2 systems are relatively well characterized, while the pathophysiological function of the TonB3 system is still elusive. A reverse transcription-PCR (RT-PCR) study showed that the tonB1 and tonB2 genes are preferentially induced in vivo, whereas tonB3 is persistently transcribed, albeit at low expression levels, under both in vitro and in vivo conditions. The goal of the present study was to elucidate the raison d'être of these three TonB systems. In contrast to previous studies, we constructed in-frame single-, double-, and triple-deletion mutants of the entire structural genes in TonB loci, and the changes in various virulence-related phenotypes were evaluated. Surprisingly, only the tonB123 mutant exhibited a significant delay in killing eukaryotic cells, which was complemented in trans with any TonB operon. Very interestingly, we discovered that flagellum biogenesis was defective in the tonB123 mutant. The loss of flagellation contributed to severe defects in motility and adhesion of the mutant. Because of the difficulty of making contact with host cells, the mutant manifested defective RtxA1 toxin production, which resulted in impaired invasiveness, delayed cytotoxicity, and decreased lethality for mice. Taken together, these results indicate that a series of virulence defects in all three TonB systems of V. vulnificus CMCP6 coordinately complement each other for iron assimilation and full virulence expression by ensuring flagellar biogenesis.
托蛋白（TonB）系统可将铁结合底物主动转运穿过革兰氏阴性菌的外膜。创伤弧菌CMCP6可引发致命的败血症和坏死性伤口感染，它拥有三个活性托蛋白系统。目前尚不清楚为何创伤弧菌CMCP6在整个进化过程中保留了三个托蛋白系统。托蛋白1（TonB1）系统和托蛋白2（TonB2）系统的特征相对较为明确，而托蛋白3（TonB3）系统的病理生理功能仍不清楚。一项逆转录聚合酶链反应（RT-PCR）研究表明，tonB1和tonB2基因在体内优先被诱导表达，而tonB3在体外和体内条件下均持续转录，尽管表达水平较低。本研究的目的是阐明这三个托蛋白系统存在的原因。与之前的研究不同，我们构建了托蛋白基因座中整个结构基因的框内单缺失、双缺失和三缺失突变体，并评估了各种毒力相关表型的变化。令人惊讶的是，只有tonB123突变体在杀死真核细胞方面表现出显著延迟，用任何托蛋白操纵子进行反式互补均可恢复。非常有趣的是，我们发现tonB123突变体的鞭毛生物合成存在缺陷。鞭毛缺失导致突变体的运动性和黏附性严重受损。由于难以与宿主细胞接触，该突变体表现出RtxA1毒素产生缺陷，从而导致侵袭性受损、细胞毒性延迟以及对小鼠的致死率降低。综上所述，这些结果表明，创伤弧菌CMCP6的所有三个托蛋白系统中的一系列毒力缺陷通过确保鞭毛生物合成，在铁同化和完全毒力表达方面相互协调互补。
