Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid and IRYCIS, Madrid, Spain.
Laboratory of Molecular Microbiology and Structural Biochemistry, Centre National de la Recherche Scientifique UMR5086, Université de Lyon, Lyon, France.
PLoS Pathog. 2020 Apr 16;16(4):e1007979. doi: 10.1371/journal.ppat.1007979. eCollection 2020 Apr.
Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.
布鲁氏菌属是与动物和人类健康有关的兼性胞内革兰氏阴性细菌。它们能够在细胞内建立小生境并颠覆宿主细胞途径以利于自己，这取决于通过 IV 型分泌系统将细菌效应蛋白递送到细胞内。布鲁氏菌 Toll/白细胞介素-1 受体（TIR）结构域蛋白 BtpA（也称为 TcpB）和 BtpB 就是这样的效应物之一。尽管它们在一级序列上存在差异，但它们干扰 Toll 样受体（TLR）信号传导以抑制先天免疫反应。然而，所涉及的分子机制仍不清楚。为了深入了解 BtpA 和 BtpB 的功能，我们在出芽酵母酿酒酵母中表达了它们作为真核细胞模型。我们发现这两种效应物都具有细胞毒性，并且它们各自的 TIR 结构域对于酵母生长抑制是必需且充分的。生长抑制伴随着肌动蛋白解聚、内吞作用阻断和细胞内激酶活性的普遍下降，表明能量代谢出现故障。事实上，在表达 BtpA 和 BtpB TIR 结构域的酵母细胞中，ATP 和 NAD+ 的水平较低，这与最近描述的一些 TIR 结构域作为 NAD+ 水解酶的酶活性一致。在人上皮细胞中，BtpA 和 BtpB 的表达均降低了细胞内总 NAD 水平。在感染细胞中，BtpA 和 BtpB 均有助于降低总 NAD，表明它们的 NAD+ 水解酶功能在感染期间在细胞内是活跃的。总的来说，结合酵母模型以及哺乳动物细胞和感染研究，我们的结果表明，BtpA 和 BtpB 通过 NAD+ 水解来调节宿主细胞的能量代谢，为这些含有 TIR 结构域的效应物在布鲁氏菌病发病机制中的作用赋予了新的角色。
