Regulation of Spore Germination in Clostridioides difficile

萌发是许多孢子形成细菌引发疾病所必需的。医院内病原体孢子 当艰难梭菌感受到胆汁酸（发芽剂）与氨基酸结合时， 和/或Ca 2+（共萌发剂）。C. difficile使用一种不同于 以前研究的孢子形成，因为它缺乏跨膜萌发受体编码的所有其他 孢子形成者相反，C.艰难梭菌使用梭菌丝氨酸蛋白酶家族的两种可溶性蛋白质，CspC和 CspA，用于感测萌发和共萌发信号。虽然大多数Csps是活性蛋白酶，但C.艰难梭菌 和CspA是假蛋白酶，这是由于它们的催化三联体中的突变。尽管它们缺乏催化活性， CspC和CspA假蛋白酶在萌发过程中控制相关蛋白酶CspB的活性。而 CspC和CspA分别被鉴定为可能的胆汁酸萌发和共萌发受体， 通过基因筛选，我们在过去的资助期间意外地发现，CspC不仅能感知胆汁， 酸性萌发剂，但也是共萌发剂信号。这些发现提出了CspC和CspA如何 整合来自这些不同小分子的信号，特别是因为我们缺乏生物化学证据， 这些蛋白质结合任何这些信号分子。 我们最近通过证明CspC和CspA直接作用于细胞， 互动.我们还确定了CspA形成同源二聚体，与CspC相反，通过解CspA的晶体， 结构有趣的是，当我们将CspC模型化到我们未发表的CspA结构上时， C.艰难梭菌孢子到萌发体和/或共萌发体簇集到预测的CspC：CspA 异二聚化界面这些数据使我们假设，萌发和共萌发信号改变 CspC：CspA异二聚体和CspA同二聚体之间的平衡。由于CspC也需要CspA， 为了稳定地整合到孢子中，我们提出了一种伴侣交换机制调节C。艰难梭菌抱子 萌发根据该模型，CspC：CspA异源二聚化允许CspC稳定地掺入到细胞中。 休眠孢子无活性的异二聚体在萌发剂和共萌发剂添加后被破坏，这引发了 通过促进CspA同源二聚化潜在地萌发。我们将通过评估函数来测试这个模型。 CspC的意义：CspA异源二聚化和CspA同源二聚化（目的1）以及萌发剂的影响 以及这些蛋白质：蛋白质相互作用的共同萌发物（Aim 2）。我们还将确定CspA的作用 非结构化前结构域在调节CspA同源二聚体和CspC：CspA异源二聚体之间的平衡中的作用 在存在萌发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C.艰难梭菌孢子发出萌发和共萌发信号以诱导萌发。 他们还将揭示新的机制，如何假酶，这是普遍存在的和保守的跨 生命的所有三个领域都控制着响应小分子的同源酶的活性。

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.