Abstract：
The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp), and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB\'s role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro. Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1, ΔtonB2, ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain, which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1, ΔtonB2, ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo. The genetic organization of tonB1, tonB2, tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space-in contrast to an oxidative environment in aerobes-it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.
摘要：
机会主义者,厌氧病原体和人类大肠的共生，脆弱拟杆菌菌株638R，包含六种预测的TonB蛋白，称为TonB1-6，四个Exbb直系同源物，ExbB1-4和五个ExbDs直系同源物，ExbD1-5.内膜TonB/ExbB/ExbD复合物从质子动力（Δp）中获取能量，TonBC末端结构域与外膜TonB依赖性转运蛋白（TBDTs）相互作用并将能量转导至外膜。然而,在肠道定植和肠外感染期间，TonB在激活近一百个TBDTs以获取脆弱芽孢杆菌营养中的作用尚未确定。在这项研究中,我们表明，当血红素时，ΔtonB3突变体的生长被取消，维生素B12，Fe（III）-铁，淀粉，粘蛋白-聚糖，或N-连接的聚糖用作体外生长的底物。ΔtonB3突变体与tonB3基因的遗传互补恢复了这些底物上的生长。ΔtonB1，ΔtonB2，ΔtonB4，ΔtonB5和ΔtonB6单突变体未显示生长缺陷。这表明没有对TonB3的缺乏进行功能补偿，并且表明仅TonB3，驱动参与必需营养素运输的TBDT。与亲本菌株相比，ΔtonB3突变体在小鼠肠道定殖模型中具有严重的生长缺陷。这种肠道生长缺陷在ΔtonB3ΔtonB6双突变菌株中得到增强，与亲本菌株相比，它完全失去了在小鼠肠道定殖的能力。ΔtonB1，ΔtonB2，ΔtonB4和ΔtonB5突变体对肠定植没有显着影响。此外，在腹腔感染的大鼠模型中，ΔtonB3突变株的存活被完全根除。一起来看,这些发现表明，TonB3对于体内存活至关重要。tonB1，tonB2，tonB4，tonB5和tonB6基因直向同源物的遗传组织表明它们可能与周质和非受体外膜蛋白相互作用，但这种生理相关性尚未确定。由于厌氧发酵代谢产生的Δp比有氧呼吸低，并且与需氧菌中的氧化环境相反，脆弱芽孢杆菌在其周质空间中的氧化还原状态降低，因此尚待确定的是，由脆弱芽孢杆菌编码的TonB/ExbB/ExbD直系同源物具有对PMF（相对于需氧菌）的敏感性增加，以允许在厌氧条件下收集能量。
