Department of Biology, Indiana University, Bloomington, Indiana, USA.
Biology Department and Graduate Center, City University of New York, Brooklyn, New York, USA.
Appl Environ Microbiol. 2021 Jun 25;87(14):e0047821. doi: 10.1128/AEM.00478-21.
Bacteria utilize dynamic appendages, called type IV pili (T4P), to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can occur with the aid of an ATPase motor or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remain poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. Extracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions; however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.
细菌利用称为 IV 型菌毛（T4P）的动态附属物与环境相互作用，并介导多种功能。所有 T4P 中，菌毛的延伸由一个延伸 ATP 酶马达介导，通常称为 PilB。然而，菌毛的回缩可以在 ATP 酶马达的帮助下发生，也可以在没有回缩马达的情况下发生。虽然已经投入大量精力研究依赖于马达的回缩，但马达独立回缩的机制和调节仍未得到很好的描述。我们之前已经证明，霍乱弧菌感受态 T4P 在缺乏专用回缩 ATP 酶 PilT 和 PilU 的情况下会发生马达独立回缩。在这里，我们利用这个模型系统来描述影响马达独立回缩的因素。我们发现，刚延伸的菌毛经常会发生马达独立回缩，但如果这些菌毛不能立即回缩，它们就会在细胞表面静态延伸。重要的是，我们表明，通过 PilT 的紧密调控异位表达，这些静态菌毛仍然可以通过依赖于马达的回缩，这表明这些 T4P 没有断裂，只是不能进行马达独立回缩。通过对菌毛进行额外的遗传和生物物理特性分析，我们认为菌毛丝在动态延伸和回缩过程中会发生构象变化。我们提出，只有某些构象，如刚延伸的菌毛所采用的构象，才能进行马达独立回缩。总之，这些数据突出了调节 T4P 动态活性的多种机制，并为马达独立回缩通过自发解聚发生的长期假设提供了额外的支持。 细胞外菌毛纤维对许多致病性细菌的毒力和持久性至关重要。大多数菌毛的一个关键功能是能够从细胞表面动态延伸和回缩。抑制这种动态菌毛活性是一种有吸引力的治疗干预方法；然而，目前对这一过程的详细机制知之甚少。在这里，我们使用霍乱弧菌的感受态菌毛来研究在没有专用回缩马达的情况下菌毛是如何回缩的。我们的结果揭示了一种新的菌毛回缩调节机制，这是菌毛丝的固有特性。因此，了解菌毛在不同条件下采用的构象变化可能对开发旨在靶向这些结构动态活性的新型疗法至关重要。
