Abstract
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.
Highlights
Several bacterial pathogens can circumvent host innate immune responses during infection, often by injecting effector proteins into host cells that target components of innate immune pathways
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 found that the TIR domains of both Brucella proteins were necessary and sufficient to collapse energy metabolism in yeast by depleting ATP and NAD+
Summary
Several bacterial pathogens can circumvent host innate immune responses during infection, often by injecting effector proteins into host cells that target components of innate immune pathways. In many cases, these effectors contain eukaryotic-like domains capable of modulating receptor proximal events. These effectors contain eukaryotic-like domains capable of modulating receptor proximal events This is the case of Toll/interleukin 1 receptor (TIR) domains present on the cytosolic faces of all Toll-like receptors (TLRs) and corresponding adaptor proteins, enabling the formation of a scaffold for the assembly of intricate protein signaling complexes [1]. The observation that expression of TirS from Staphylococcus aureus, present in a multi-drug resistant (MDR) island of numerous clinical isolates, is induced by specific antibiotic treatment [7] raises the possibility that these bacterial proteins may be tightly regulated, enhancing virulence, persistence or dissemination in particular clinical contexts such as exposure to selective pressure
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