The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism

Author summary Brucella is a genus of zoonotic bacteria that cause severe disease in a variety of mammals, ranging from farm animals (as bovines, swine and ovine) to marine mammals. Transmission to humans, often by ingestion of non-treated dairy products, leads to serious systemic infection. Brucella abortus invades host cells and replicates intracellularly. Such behavior relies on the injection of bacterial proteins into the host cytoplasm via specialized secretion systems. Our work focuses on the study of two of these factors, BtpA and BtpB, previously described to contain Toll/Interleukin-1 Receptor (TIR)-domains that modulate innate immunity. We use here two biological models: the yeast Saccharomyces cerevisiae and human cell lines. We found that the TIR domains of both Brucella proteins were necessary and sufficient to collapse energy metabolism in yeast by depleting ATP and NAD(+). This result was translatable to higher cells and consistent with the recently described NADase activity of some TIR domains both in mammalian and bacterial proteins. Importantly, we demonstrate that Brucella down-regulates total NAD levels in host cells by using both BtpA and BtpB effectors. Our results show that NAD(+) is targeted by Brucella during infection, which may constitute a novel mechanism for its pathogenicity. 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.