Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.
Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.
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DOI：
10.1128/mbio.00360-18
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发表时间：
2018-04-24
期刊：
影响因子：
6.4
通讯作者：
Ton-That H
中科院分区：
文献类型：
--
作者：
Wu C;Al Mamun AAM;Luong TT;Hu B;Gu J;Lee JH;D'Amore M;Das A;Ton-That H
具核梭杆菌是人类口腔生物膜的重要成员。它也与早产和结直肠癌有关。为了促进梭菌毒力的基础研究，我们在这里描述了一个通用的转座子诱变程序和生物膜形成缺陷的突变体的试点筛选。在分离的10个独立的生物膜缺陷突变体中，受影响的基因包括大肠杆菌细胞分裂蛋白FtsX和EnvC的同源物、电子传递蛋白RnfA和四种功能未知的蛋白质。接下来，一种简单的新基因缺失方法证明，ftsX或envC的非极性、框内缺失产生了由于细胞分裂缺陷而高度丝状的活细菌。透射电子显微镜和冷冻电子显微镜显示，ΔftsX和ΔenvC突变体细胞仍然连接有明显的收缩，扫描电子显微镜（EM）发现细胞表面光滑，没有野生型细胞中存在的微折叠。FtsX和EnvC蛋白相互作用以及一组共同的相互作用伴侣，许多功能未知。最后，当细胞分裂被MinC过量生产阻断时，生物膜的发育会改变;然而，与ΔftsX和ΔenvC突变体的表型不同，形成了弱粘附的生物膜，并保持了野生型粗糙的细胞表面。因此，FtsX和EnvC可能在梭杆菌细胞生物学中发挥新的功能。这是第一次报告的一个公正的方法来揭示梭菌生物膜发展的遗传决定因素。它指出了胞质分裂、细胞表面动力学和生物膜形成之间的有趣联系，其分子基础仍有待阐明。对具核梭菌的毒力机制和相关因子知之甚少，主要是由于缺乏方便的遗传工具。我们采用了两种有效的遗传策略来鉴定具核梭菌生物膜缺陷型突变体，揭示了七种生物膜相关因子中的FtsX和EnvC。电子显微镜检查证实了ΔftsX和ΔenvC突变体的细胞分裂缺陷，并伴有光滑的细胞表面，这与野生型细菌的微折叠、凹凸不平的外观不同。蛋白质组学研究表明，FtsX和EnvC相互作用，以及一组共同的和独特的相互作用的蛋白质，许多未知的功能。重要的是，通过MinC过量生产阻断细胞分裂导致形成弱粘附的生物膜，而不改变野生型细胞表面。因此，这项工作将细胞分裂和表面动力学与生物膜的发育联系起来，并为这种临床重要病原体的基本细胞过程的未来遗传和生化研究奠定了基础。
Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated. Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen.