Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA.
mBio. 2013 Aug 6;4(4):e00360-13. doi: 10.1128/mBio.00360-13.
Acinetobacter baumannii is a Gram-negative, opportunistic pathogen. Recently, multiple A. baumannii genomes have been sequenced; these data have led to the identification of many genes predicted to encode proteins required for the biogenesis of type IV pili (TFP). However, there is no experimental evidence demonstrating that A. baumannii strains actually produce functional TFP. Here, we demonstrated that A. baumannii strain M2 is naturally transformable and capable of twitching motility, two classical TFP-associated phenotypes. Strains were constructed with mutations in pilA, pilD, and pilT, genes whose products have been well characterized in other systems. These mutants were no longer naturally transformable and did not exhibit twitching motility. These TFP-associated phenotypes were restored when these mutations were complemented. More PilA was detected on the surface of the pilT mutant than the parental strain, and TFP were visualized on the pilT mutant by transmission electron microscopy. Thus, A. baumannii produces functional TFP and utilizes TFP for both natural transformation and twitching motility. Several investigators have hypothesized that TFP might be responsible, in part, for the flagellum-independent surface-associated motility exhibited by many A. baumannii clinical isolates. We demonstrated that surface-associated motility was not dependent on the products of the pilA, pilD, and pilT genes and, by correlation, TFP. The identification of functional TFP in A. baumannii lays the foundation for future work determining the role of TFP in models of virulence that partially recapitulate human disease.
Several investigators have documented the presence of genes predicted to encode proteins required for the biogenesis of TFP in many A. baumannii genomes. Furthermore, some have speculated that TFP may play a role in the unique surface-associated motility phenotype exhibited by many A. baumannii clinical isolates, yet there has been no experimental evidence to prove this. Unfortunately, progress in understanding the biology and virulence of A. baumannii has been slowed by the difficulty of constructing and complementing mutations in this species. Strain M2, a recently characterized clinical isolate, is amenable to genetic manipulation. We have established a reproducible system for the generation of marked and/or unmarked mutations using a modified recombineering strategy as well as a genetic complementation system utilizing a modified mini-Tn7 element in strain M2. Using this strategy, we demonstrated that strain M2 produces TFP and that TFP are not required for surface-associated motility exhibited by strain M2.
鲍曼不动杆菌是一种革兰氏阴性、机会性病原体。最近，已经对多个鲍曼不动杆菌基因组进行了测序；这些数据导致了许多基因的鉴定，这些基因预测编码了 IV 型菌毛（TFP）生物发生所需的蛋白质。然而，目前还没有实验证据证明鲍曼不动杆菌菌株实际上产生了功能性的 TFP。在这里，我们证明了鲍曼不动杆菌 M2 株是自然可转化的，并且能够进行菌毛滑动运动，这是两种经典的 TFP 相关表型。构建了 pilA、pilD 和 pilT 基因的突变菌株，这些基因的产物在其他系统中已经得到了很好的描述。这些突变体不再自然可转化，也没有表现出菌毛滑动运动。当这些突变得到互补时，这些 TFP 相关表型得到了恢复。在 pilT 突变体上检测到的 PilA 比亲本菌株更多，并且通过透射电子显微镜在 pilT 突变体上观察到 TFP。因此，鲍曼不动杆菌产生功能性的 TFP，并利用 TFP 进行自然转化和菌毛滑动运动。一些研究人员假设，TFP 可能部分负责许多鲍曼不动杆菌临床分离株表现出的鞭毛独立表面相关运动。我们证明了表面相关运动不依赖于 pilA、pilD 和 pilT 基因的产物，并且通过相关性，不依赖于 TFP。在鲍曼不动杆菌中鉴定出功能性的 TFP 为未来确定 TFP 在部分模拟人类疾病的毒力模型中的作用奠定了基础。
一些研究人员已经在许多鲍曼不动杆菌基因组中记录了预测编码 TFP 生物发生所需蛋白质的基因的存在。此外，一些人推测 TFP 可能在许多鲍曼不动杆菌临床分离株表现出的独特表面相关运动表型中发挥作用，但目前还没有实验证据证明这一点。不幸的是，由于难以在该物种中构建和互补突变，鲍曼不动杆菌的生物学和毒力的理解进展已经放缓。最近被描述的临床分离株 M2 株易于遗传操作。我们已经建立了一种使用改良重组策略和 M2 株中改良的 mini-Tn7 元件的遗传互补系统来生成标记和/或非标记突变的可重复系统。使用这种策略，我们证明了 M2 株产生 TFP，并且 TFP 不是 M2 株表现出的表面相关运动所必需的。
