Protein Kinase R Restricts the Intracellular Survival of Mycobacterium tuberculosis by Promoting Selective Autophagy

Abstract

Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). The identification of macrophage signaling proteins exploited by Mtb during infection will enable the development of alternative host-directed therapies (HDT) for TB. HDT strategies will boost host immunity to restrict the intracellular replication of Mtb and therefore hold promise to overcome antimicrobial resistance, a growing crisis in TB therapy. Protein Kinase R (PKR) is a key host sensor that functions in the cellular antiviral response. However, its role in defense against intracellular bacterial pathogens is not clearly defined. Herein, we demonstrate that expression and activation of PKR is upregulated in macrophages infected with Mtb. Immunological profiling of human THP-1 macrophages that overexpress PKR (THP-PKR) showed increased production of IP-10 and reduced production of IL-6, two cytokines that are reported to activate and inhibit IFNγ-dependent autophagy, respectively. Indeed, sustained expression and activation of PKR reduced the intracellular survival of Mtb, an effect that could be enhanced by IFNγ treatment. We further demonstrate that the enhanced anti-mycobacterial activity of THP-PKR macrophages is mediated by a mechanism dependent on selective autophagy, as indicated by increased levels of LC3B-II that colocalize with intracellular Mtb. Consistent with this mechanism, inhibition of autophagolysosome maturation with bafilomycin A1 abrogated the ability of THP-PKR macrophages to limit replication of Mtb, whereas pharmacological activation of autophagy enhanced the anti-mycobacterial effect of PKR overexpression. As such, PKR represents a novel and attractive host target for development of HDT for TB, and our data suggest value in the design of more specific and potent activators of PKR.

Protein Kinase R in Bacterial Infections: Friend or Foe?

“…It is worth noting that the overall effect of PKR during M. tuberculosis infection may be context dependent. Although our findings and the results from Ranjbar et al indicate that PKR limits M. tuberculosis survival in vitro (97,98), a recent report using sst1-susceptible mice suggests that PKR contributes to macrophage necrosis in TB granulomas (119). sst1-susceptible mice develop necrotic inflammatory lung lesions similar to human TB granulomas, whereas their congenic B6 counterparts do not (120).…”

“…Salmonella Typhimurium can infect both humans and animals, and infection is commonly acquired by consuming contaminated food products. S. Typhimurium infection has been observed to increase mRNA levels of EIF2AK2 in MEFs ( 108 ), and we and others have reported that S. Typhimurium increases total and phosphorylated PKR protein levels in macrophages ( 87 , 98 ). These findings suggest that PKR plays a role in the antibacterial response to Salmonella .…”

“…Most studies thus far have focused on the role of PKR in cell death during bacterial infection. Although it is important to investigate the function of PKR during cell death, we contend that this should not be the sole focus, since PKR has recently been shown to induce selective autophagy during bacterial and parasitic infection (77,98). As such, it will be important for future studies to investigate the function of PKR in selective autophagy of intracellular bacteria such as L. monocytogenes and S. enterica, since autophagy is reported to play a role in antibacterial defense against these pathogens (196)(197)(198).…”

“…However, MAPK p38 and JNK have been shown to be important for induction of autophagy during M. tuberculosis infection, and IL-6 inhibits MAPK phosphorylation to block autophagy in M. tuberculosis-infected macrophages (118). Importantly, PKR activates p38 and JNK (25)(26)(27), and macrophages overexpressing PKR had reduced production of IL-6 (98). As such, it is possible that autophagy induction by PKR is dependent on a mechanism involving MAPK, whether by a direct effect of PKR on MAPK activation or an indirect effect from decreased IL-6 production.…”

“…Expression of a non-phosphorylatable mutant of EIF2α in MEFs resulted in increased bacterial invasion, suggesting an important role for EIF2α kinases in the antibacterial response to Listeria ( 103 ). Indeed, our group observed that L. monocytogenes infection triggers increased levels of total and phosphorylated PKR protein in the human macrophage cell-line THP-1 ( 98 ). Since L. monocytogenes is established to invade the cytosol and initiate a type I IFN response, and type I IFN signaling induces PKR transcription, Valderrama and colleagues examined the effect of L. monocytogenes infection on EIF2AK2 (PKR) mRNA levels ( 89 ).…”