Mycobacterium tuberculosis RipA Dampens TLR4-Mediated Host Protective Response Using a Multi-Pronged Approach Involving Autophagy, Apoptosis, Metabolic Repurposing, and Immune Modulation

Abstract

Reductive evolution has endowed Mycobacterium tuberculosis (M. tb) with moonlighting in protein functions. We demonstrate that RipA (Rv1477), a peptidoglycan hydrolase, activates the NFκB signaling pathway and elicits the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12, through the activation of an innate immune-receptor, toll-like receptor (TLR)4. RipA also induces an enhanced expression of macrophage activation markers MHC-II, CD80, and CD86, suggestive of M1 polarization. RipA harbors LC3 (Microtubule-associated protein 1A/1B-light chain 3) motifs known to be involved in autophagy regulation and indeed alters the levels of autophagy markers LC3BII and P62/SQSTM1 (Sequestosome-1), along with an increase in the ratio of P62/Beclin1, a hallmark of autophagy inhibition. The use of pharmacological agents, rapamycin and bafilomycin A1, reveals that RipA activates PI3K-AKT-mTORC1 signaling cascade that ultimately culminates in the inhibition of autophagy initiating kinase ULK1 (Unc-51 like autophagy activating kinase). This inhibition of autophagy translates into efficient intracellular survival, within macrophages, of recombinant Mycobacterium smegmatis expressing M. tb RipA. RipA, which also localizes into mitochondria, inhibits the production of oxidative phosphorylation enzymes to promote a Warburg-like phenotype in macrophages that favors bacterial replication. Furthermore, RipA also inhibited caspase-dependent programed cell death in macrophages, thus hindering an efficient innate antibacterial response. Collectively, our results highlight the role of an endopeptidase to create a permissive replication niche in host cells by inducing the repression of autophagy and apoptosis, along with metabolic reprogramming, and pointing to the role of RipA in disease pathogenesis.

Mycobacterium tuberculosis Protein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival

“…The enhanced intracellular survival (Eis) protein of M. tb suppresses innate immunity by inhibiting autophagy and producing proinflammatory cytokines TNF-α and IL-6 and caspase-independent cell death through a redox-dependent mechanism. RipA, a secretory endopeptidase, dampens both autophagy and apoptosis in macrophage cells for intracellular survival and virulence ( 21 , 22 ). M. tb is known to inhibit autophagy by suppressing autophagosomes’ fusion to lysosomes.…”

Autophagy and Mitochondrial Homeostasis During Infection: A Double-Edged Sword

“…MAPK signaling affects the maturation step of autophagy via mTOR signaling (Zhou et al, 2018). TLRs can affect Akt phosphorylation which regulates the formation of autophagosome via PI3K and mTOR signaling (Shariq et al, 2021). These studies suggest that TLR signaling is an important bridge between autophagy and mitochondrial function during infection (Figure 2).…”

Mechanisms of TLR4-Mediated Autophagy and Nitroxidative Stress

“…Generally, nitroxidative stress affects the phosphorylation of AMPK and PI3K signaling, which then influence autophagy (Hinchy et al, 2018;Mistry et al, 2019). Mechanistically, bacteria activate mTORC1 via the upstream TLR4-PI3K-Akt signaling cascade, which inhibits autophagy by suppressing autophagy initiating kinase ULK1 (Shariq et al, 2021). Down regulating TLR4 and MyD88 promoted autophagy by suppressing the phosphorylation of MAPK, mTOR and p65.…”