Zmuda Martin, Sedlackova Eliska, Pravdova Barbora, Cizkova Monika, Dalecka Marketa, Cerny Ondrej, Allsop Tania Romero, Grousl Tomas, Malcova Ivana, Kamanova Jana
Laboratory of Infection Biology, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic.
Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic.
mBio. 2024 Dec 11;15(12):e0192524. doi: 10.1128/mbio.01925-24. Epub 2024 Nov 21.
is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens and exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.
是人类百日咳的病原体，这种疾病最近有所回升。相比之下，感染各种哺乳动物物种的呼吸道，引起从无症状慢性携带到急性疾病的一系列症状。两种病原体都利用III型分泌系统（T3SS）将效应蛋白BteA递送到宿主细胞中。一旦注入，BteA通过一种尚未完全理解的机制引发一系列导致不依赖半胱天冬酶1的坏死的事件。我们证明，BteA诱导的细胞死亡的特征是细胞内质网和线粒体的碎片化、坏死性气球样突起的形成以及质膜通透性增加。重要的是，针对19050个基因的全基因组CRISPR-Cas9筛选未能鉴定出BteA细胞毒性所需的任何宿主因子，这表明BteA的细胞毒性不需要单个非必需宿主因子。我们进一步揭示，BteA触发钙离子的快速和持续内流，这与细胞器碎片化和质膜通透性增加有关。胞质钙水平的持续升高导致线粒体钙超载、线粒体肿胀、嵴溶解和线粒体膜电位丧失。用2-APB抑制钙通道可延迟钙内流和BteA诱导的细胞死亡。我们的研究结果表明，BteA利用宿主的基本过程和/或冗余途径来破坏钙稳态和线粒体功能，最终导致宿主细胞死亡。重要性呼吸道病原体和对多种哺乳动物细胞表现出细胞毒性，这取决于III型分泌效应物BteA。此外，的毒力增加与T3SS和BteA的表达增强有关。然而，BteA细胞毒性的分子机制尚不清楚。在这项研究中，我们进行了CRISPR-Cas9筛选，揭示BteA诱导的细胞死亡取决于宿主的基本或冗余过程。此外，我们证明BteA破坏钙稳态，导致线粒体功能障碍和细胞死亡。这些发现有助于填补我们对BteA靶向的信号级联理解上的空白。
