Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, GA, USA. Electronic address: https://twitter.com/mdicandia.
Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, GA, USA.
J Mol Biol. 2022 Jul 15;434(13):167641. doi: 10.1016/j.jmb.2022.167641. Epub 2022 May 18.
Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To survive in the environment and spread to new hosts, C. difficile must form metabolically dormant spores. The formation of spores requires activation of the transcription factor Spo0A, which is the master regulator of sporulation in all endospore-forming bacteria. Though the sporulation initiation pathway has been delineated in the Bacilli, including the model spore-former Bacillus subtilis, the direct regulators of Spo0A in C. difficile remain undefined. C. difficile Spo0A shares highly conserved protein interaction regions with the B. subtilis sporulation proteins Spo0F and Spo0A, although many of the interacting factors present in B. subtilis are not encoded in C. difficile. To determine if comparable Spo0A residues are important for C. difficile sporulation initiation, site-directed mutagenesis was performed at conserved receiver domain residues and the effects on sporulation were examined. Mutation of residues important for homodimerization and interaction with positive and negative regulators of B. subtilis Spo0A and Spo0F impacted C. difficile Spo0A function. The data also demonstrated that mutation of many additional conserved residues altered C. difficile Spo0A activity, even when the corresponding Bacillus interacting proteins are not apparent in the C. difficile genome. Finally, the conserved aspartate residue at position 56 of C. difficile Spo0A was determined to be the phosphorylation site that is necessary for Spo0A activation. The finding that Spo0A interacting motifs maintain functionality suggests that C. difficile Spo0A interacts with yet unidentified proteins that regulate its activity and control spore formation.
艰难梭菌是一种厌氧的革兰氏阳性病原体，它是艰难梭菌感染（CDI）的元凶。为了在环境中生存并传播到新宿主，艰难梭菌必须形成代谢休眠的孢子。孢子的形成需要激活转录因子 Spo0A，它是所有产芽孢细菌中芽孢形成的主要调节剂。虽然芽孢形成起始途径在芽孢杆菌中已经被描绘出来，包括模式芽孢形成菌枯草芽孢杆菌，但艰难梭菌中 Spo0A 的直接调控因子仍未定义。艰难梭菌 Spo0A 与枯草芽孢杆菌的 Spo0F 和 Spo0A 等芽孢形成蛋白共享高度保守的蛋白相互作用区域，尽管枯草芽孢杆菌中存在许多相互作用因子在艰难梭菌中并未编码。为了确定与枯草芽孢杆菌 Spo0A 相比，艰难梭菌 Spo0A 中类似的残基是否对其芽孢形成起始很重要，我们对保守的受体结构域残基进行了定点突变，并研究了其对芽孢形成的影响。突变对于同源二聚体形成以及与枯草芽孢杆菌 Spo0A 和 Spo0F 的正、负调节剂相互作用很重要的残基会影响艰难梭菌 Spo0A 的功能。数据还表明，即使在艰难梭菌基因组中没有明显的相应芽孢杆菌相互作用蛋白，突变许多其他保守残基也会改变艰难梭菌 Spo0A 的活性。最后，确定了艰难梭菌 Spo0A 位置 56 的保守天冬氨酸残基是磷酸化位点，该位点对于 Spo0A 的激活是必需的。发现 Spo0A 相互作用基序保持功能，这表明艰难梭菌 Spo0A 与尚未鉴定的调节其活性和控制孢子形成的蛋白相互作用。
