The TIR-domain containing effectors BtpA and BtpB fromBrucella abortusblock energy metabolism

Abstract

27Brucella species are facultative intracellular Gram-negative bacteria relevant to animal 28 and human health. Their ability to establish an intracellular niche and subvert host cell 29 pathways to their advantage depends on the delivery of bacterial effector proteins 30 through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-31 containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. 32Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) 33 signaling to inhibit the innate immune responses. However, the molecular mechanisms 34 implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we 35 expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell 36 model. We found that both effectors were cytotoxic and that their respective TIR 37 domains were necessary and sufficient for yeast growth inhibition. Growth arrest was 38 concomitant with actin depolymerization, endocytic block and a general decrease in 39 kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of 40 ATP and NAD + were low in yeast cells expressing BtpA and BtpB TIR domains, 41 consistent with the recently described enzymatic activity of some TIR domains as 42 NAD + hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced 43 intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to 44 reduction of total NAD, indicating that their NAD + hydrolase functions are active 45 intracellularly during infection. Overall, combining the yeast model together with 46 mammalian cells and infection studies our results show that BtpA and BtpB modulate 47 AUTHOR SUMMARY 51Brucella is a genus of zoonotic bacteria that cause severe disease in a variety of 52 mammals, ranging from farm animals (as bovines, swine and ovine) to marine 53 mammals. Transmission to humans, often by ingestion of non-treated dairy products, 54 leads to serious systemic infection. Brucella abortus invades host cells and replicates 55intracellularly. Such behavior relies on the injection of bacterial proteins into the host 56 cytoplasm via specialized secretion systems. Our work focuses on the study of two of 57 these factors, BtpA and BtpB, previously described to contain Toll/Interleukin-1 58Receptor (TIR)-domains that modulate innate immunity. We use here two biological 59 models: the yeast Saccharomyces cerevisiae and human cell lines. We found that the 60 TIR domains of both Brucella proteins were necessary and sufficient to collapse energy 61 metabolism in yeast by depleting ATP and NAD + . This result was translatable to higher 62 cells and consistent with the recently described NADase activity of some TIR domains 63 both in mammalian and bacterial proteins. Importantly, we demonstrate that Brucella 64 down-regulates total NAD levels in host cells by using both BtpA and BtpB effectors. 65Our results show that NAD + is targeted by Brucella during infection, which may 66 cons...

Enzymatic Functions for Toll/Interleukin-1 Receptor Domain Proteins in the Plant Immune System

“…Numerous bacterial and archaeal species encode TIR-domain containing proteins, primarily of unknown function (Spear et al, 2009;Doron et al, 2018;Essuman et al, 2018). However, some prokaryotic TIRs are reported to function in anti-phage immunity, while other TIRs may act as virulence factors which manipulate host responses ( Figure 4B) (Alaidarous et al, 2014;Doron et al, 2018;Coronas-Serna et al, 2019). TIRdomains encoded by Brucella and Paracoccus are reported to mimic animal TIR-adaptors and disrupt TLR immune signaling, potentially via physical interactions with animal TIR domains (Chan et al, 2009;Alaidarous et al, 2014;Snyder et al, 2014).…”