Mebus Viktor H, De Supradipta, Busch Larissa M, Gesell Salazar Manuela, Schlüter Rabea, Völker Uwe, Hammerschmidt Sven, Frees Dorte
Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, Capital Region of Denmark, Denmark.
Research Unit of Molecular Microbiology, University of Southern Denmark, Odense, Region Syddanmark, Denmark.
Microbiol Spectr. 2025 Jul;13(7):e0080425. doi: 10.1128/spectrum.00804-25. Epub 2025 May 23.
In all living cells, molecular chaperones and ATP-dependent proteases are essential for protein homeostasis. The ClpXP protease is a chaperone-protease complex conserved between bacteria and mitochondria. Proteolytic activity resides in the caseinolytic protease (ClpP) subunits that associate to form a tetradecameric serine protease that degrades substrates recognized and unfolded by the ClpX ATPase. In the important human pathogen (), ClpX but not ClpP was proposed to be essential for viability. We here follow up on this finding by characterizing phenotypes associated with depriving D39V of ClpX unfoldase or ClpXP activity. Virulence was examined in the infection model and was found to be severely attenuated for both mutants, suggesting that ClpXP is essential for virulence. Inactivation of ClpXP also resulted in elevated extracellular levels of LytA and early onset of LytA-dependent autolysis, aberrant localization of teichoic acids, and diminished cell size, while depletion of ClpX resulted in defective division septa, heterogeneous cell sizes, elongated cell chains, and cell lysis. Accordingly, proteomic analysis revealed that ClpXP directly or indirectly controls proteins involved in teichoic acid synthesis and cell division and elongation. In summary, we show that ClpX alone and in complex with ClpP control critical processes such as virulence and cell division.IMPORTANCEThe human nose is colonized by the opportunistic pathogenic bacterium , a leading cause of community-acquired pneumonia and a common cause of meningitis and sepsis. In , the highly conserved ClpP protease is essential for virulence, and we here show that ClpP controls virulence through associating with the ClpX unfoldase. The essentiality of ClpX has hampered its characterization. By constructing mutants expressing a variant of ClpX that cannot interact with ClpP, we further show that the ClpXP protease controls vital processes such as cell size and autolytic activity. Depletion of ClpX resulted in even more severe phenotypes, suggesting that ClpX unfoldase activity independently of ClpP plays a more fundamental role in coordinating cell division and cell elongation. In summary, the presented work adds to our understanding of how a highly conserved chaperone-protease complex contributes to the growth and pathogenicity of a prominent bacterial pathogen.
在所有活细胞中，分子伴侣和ATP依赖性蛋白酶对于蛋白质稳态至关重要。ClpXP蛋白酶是一种在细菌和线粒体之间保守的伴侣蛋白酶复合物。蛋白水解活性存在于酪蛋白水解蛋白酶（ClpP）亚基中，这些亚基结合形成十四聚体丝氨酸蛋白酶，可降解由ClpX ATP酶识别并展开的底物。在重要的人类病原体肺炎链球菌中，有人提出ClpX而非ClpP对其生存能力至关重要。我们在此通过表征与剥夺肺炎链球菌D39V菌株的ClpX解折叠酶或ClpXP活性相关的表型来跟进这一发现。在肺炎链球菌感染模型中检测了毒力，发现两种突变体的毒力均严重减弱，这表明ClpXP对毒力至关重要。ClpXP的失活还导致细胞外LytA水平升高以及LytA依赖性自溶提前发生、磷壁酸定位异常和细胞大小减小，而ClpX的缺失则导致分裂隔膜缺陷、细胞大小不均一、细胞链伸长和细胞裂解。因此，蛋白质组学分析表明，ClpXP直接或间接控制参与磷壁酸合成以及细胞分裂和伸长的蛋白质。总之，我们表明单独的ClpX以及与ClpP形成的复合物控制着诸如毒力和细胞分裂等关键过程。重要性人类鼻腔被机会致病菌肺炎链球菌定殖，它是社区获得性肺炎的主要病因以及脑膜炎和败血症的常见病因。在肺炎链球菌中，高度保守的ClpP蛋白酶对毒力至关重要，我们在此表明ClpP通过与ClpX解折叠酶结合来控制毒力。ClpX的必要性阻碍了对其特性的表征。通过构建表达不能与ClpP相互作用的ClpX变体的肺炎链球菌突变体，我们进一步表明ClpXP蛋白酶控制着诸如细胞大小和自溶活性等重要过程。ClpX的缺失导致了更严重的表型，这表明独立于ClpP的ClpX解折叠酶活性在协调细胞分裂和细胞伸长方面发挥着更基本的作用。总之，所呈现的工作增进了我们对一种高度保守的伴侣蛋白酶复合物如何促进一种重要细菌病原体的生长和致病性的理解。
