Regulation of Spore Germination in Clostridioides difficile

萌发是许多芽胞形成细菌引发疾病的关键。医院内病原菌的孢子 艰难梭状芽孢杆菌感觉胆汁酸(萌发物)与氨基酸结合时可诱导萌发 和/或Ca~(2+)(共发芽)。艰难梭菌感知这些小分子的机制与 以前研究过的孢子形成者，因为它缺乏所有其他基因编码的跨膜萌发受体 孢子形成者。艰难梭菌使用的是梭状芽胞杆菌丝氨酸蛋白酶家族的两种可溶性蛋白：CSPC和CSPC CSPA，以感知萌发和共生的信号。虽然大多数CSP是活性蛋白酶，但艰难梭菌CSPC 和CSPA是假蛋白水解酶，因为它们的催化三联体发生了突变。尽管它们缺乏催化活性， 在萌发过程中，CSPC和CSPA假蛋白水解酶控制一种相关的蛋白水解酶CSPB的活性。而当 CSPC和CSPA分别被鉴定为可能的胆汁酸萌发受体和共萌发受体。 通过基因筛查，我们在过去的资助期意外发现，CSPC不仅能感觉到胆汁 酸性发芽也是共发芽的信号。这些发现提出了一个问题，即CSPC和CSPA如何 整合来自不同小分子的信号，特别是因为我们缺乏生化证据 这些蛋白质中的任何一个都能与这些信号分子结合。 我们最近通过证明CSPC和CSPA直接 互动。我们还通过溶解CSPA的晶体确定了CSPA与CSPC相比形成了同源二聚体 结构。有趣的是，当我们在未发表的CSPA结构上模拟CSPC时，CSPC残基控制 艰难梭菌孢子对萌发菌和/或共萌发菌的敏感性聚集到预测的CSPC：CSPA 异二聚化界面。这些数据让我们假设萌发和共萌发的信号会改变 CSPC：CSPA异二聚体和CSPA均二聚体之间的平衡。因为CSPC也需要CSPA 为了稳定地结合到孢子中，我们提出了一种伙伴互换机制来调节艰难梭菌的孢子。 萌发。根据这一模型，CSPC：CSPA异二聚化允许CSPC稳定地并入 休眠的孢子。不活跃的异源二聚体在萌发和共萌发加成时被破坏，这触发了 可能通过促进CSPA同源二聚而萌发。我们将通过评估函数来测试此模型 CSPC的意义：CSPA异二聚化和CSPA同源二聚化(目标1)和发芽剂的影响 以及这些蛋白质上的共同萌发物：蛋白质相互作用(目标2)。我们还将确定CSPA的角色 非结构原结构域在调节CSPA同源二聚体和CSPC：CSPA异源二聚体之间平衡中的作用 在有萌芽和共萌发信号的情况下(目标3)。总的来说，这些分析将提供原子- 深入了解艰难梭菌孢子如何转导萌发和共萌发信号以诱导萌发。 它们还将揭示假酶的新机制，这种假酶普遍存在并在 生命的这三个领域都控制着同源酶对小分子的反应活性。

Germination is essential for many spore-forming bacteria to initiate disease. Spores of the nosocomial pathogen Clostridioides difficile induce germination when they sense bile acid (germinants) combined with amino acids and/or Ca2+ (co-germinants). C. difficile senses these small molecules using a mechanism that is distinct from previously studied spore-formers because it lacks the transmembrane germinant receptors encoded by all other spore-formers. Instead, C. difficile uses two soluble proteins of the clostridial serine protease family, CspC and CspA, to sense germinant and co-germinant signals. While most Csps are active proteases, C. difficile CspC and CspA are pseudoproteases due to mutations in their catalytic triads. Despite their lack of catalytic activity, the CspC and CspA pseudoproteases control the activity of a related protease, CspB, during germination. While CspC and CspA were identified as the likely bile acid germinant and co-germinant receptors, respectively, through genetic screens, we unexpectedly discovered in the past funding period that CspC not only senses bile acid germinant but also co-germinant signals. These findings raise the question as to how CspC and CspA integrate signals from such different small molecules, especially since we lack biochemical evidence that either of these proteins binds any of these signaling molecules. We recently gained biochemical insight into this question by demonstrating that CspC and CspA directly interact. We also determined that CspA forms a homodimer, in contrast with CspC, by solving CspA’s crystal structure. Intriguingly, when we model CspC onto our unpublished structure of CspA, CspC residues that control the sensitivity of C. difficile spores to germinants and/or co-germinants cluster to the predicted CspC:CspA heterodimerization interface. These data lead us to hypothesize that germinant and co-germinant signals alter the equilibrium between CspC:CspA heterodimers and CspA homodimers. Since CspA is also needed for CspC to be stably incorporated into spores, we propose that a partner-swap mechanism regulates C. difficile spore germination. According to this model, CspC:CspA heterodimerization allows CspC to be stably incorporated into dormant spores. The inactive heterodimer is disrupted upon germinant and co-germinant addition, which triggers germination potentially by promoting CspA homodimerization. We will test this model by evaluating the functional significance of CspC:CspA heterodimerization and CspA homodimerization (Aim 1) and the impact of germinants and co-germinants on these protein:protein interactions (Aim 2). We will also determine the role of CspA’s unstructured prodomain in regulating the equilibrium between CspA homodimers and CspC:CspA heterodimers in the presence of germinant and co-germinant signals (Aim 3). Collectively, these analyses will provide atomic- level insight into how C. difficile spores transduce germinant and co-germinant signals to induce germination. They will also reveal novel mechanisms for how pseudoenzymes, which are ubiquitous and conserved across all three domains of life, control the activity of cognate enzymes in response to small molecules.