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Abstract
Mycobacterium bovis (M. bovis) is highly adapted to macrophages and has developed multiple mechanisms to resist intracellular assaults. However, the host cells in turn deploy a multipronged defense mechanism to control bacterial infection. Endoplasmic reticulum (ER) stress-mediated apoptosis is one such primary defense mechanism. However, the role of interferon regulatory factor 3 (IRF3) between ER stress and apoptosis during M. bovis infection is unknown. Here, we demonstrate that M. bovis effectively induced apoptosis in murine macrophages. Caspase-12, caspase-9, and caspase-3 were activated over a 48 h infection period. The splicing of XBP-1 mRNA and the level of phosphorylation of eIF2α, indicators of ER stress, significantly increased at early time points after M. bovis infection. The expansion of the ER compartment, a morphological hallmark of ER stress, was observed at 6 h. Pre-treatment of Raw 264.7 cells with 4-PBA (an ER stress-inhibitor) reduced the activation of the ER stress indicators, caspase activation and its downstream poly (ADP-ribose) polymerase (PARP) cleavage, phosphorylation of TBK1 and IRF3 and cytoplasmic co-localization of STING and TBK1. M. bovis infection led to the interaction of activated IRF3 and cytoplasmic Bax leading to mitochondrial damage. Role of IRF3 in apoptosis was further confirmed by blocking this molecule with BX-795 that showed significant reduction expression of caspase-8 and caspase-3. Intracellular survival of M. bovis increased in response to 4-PBA and BX-795. These findings indicate that STING-TBK1-IRF3 pathway mediates a crosstalk between ER stress and apoptosis during M. bovis infection, which can effectively control intracellular bacteria.
Highlights
It is more than a hundred years since Mycobacterium tuberculosis (Mtb) was first isolated by Robert Koch (Cui et al, 2016)
Our studies show that phosphorylation of interferon regulatory factor 3 (IRF3) ensued M. bovis infection at 24 h pi (Figures 4A–D)
Raw 264.7 cells were treated with 5 mM 4-phenyl butyric acid (4-PBA) before M. bovis infection to verify that phosphorylation of IRF3 was mediated by Endoplasmic reticulum (ER) stress
Summary
It is more than a hundred years since Mycobacterium tuberculosis (Mtb) was first isolated by Robert Koch (Cui et al, 2016). Mycobacterium bovis (M. bovis), a species belonging to Mycobacterium tuberculosis Complex, is the main cause of TB in cattle, deer and other mammals and it can infect people by drinking or eating contaminated, unpasteurized dairy products or close contact with infected animals (Arap et al, 2004; Waters and Palmer, 2015). Macrophages are the major cell type infected by Mtb and M. bovis. Host innate immune mechanisms have co-evolved to better counter mycobacterial infections (Aldwell et al, 1996). Host macrophages induce an apoptotic signal to control bacterial infection (Rodrigues et al, 2013; Srinivasan et al, 2014). Understanding the mechanism of apoptosis induced by M. bovis infection will lead to better delineate host immune responses that could be exploited to control infection
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