The coordinated outcome of STIM1-Orai1 and superoxide signalling is crucial for headkidney macrophage apoptosis and clearance of Mycobacterium fortuitum

Abstract

The mechanisms underlying M. fortuitum-induced pathogenesis remains elusive. Using headkidney macrophages (HKM) from Clarias gariepinus, we report that TLR-2-mediated internalization of M. fortuitum is imperative to the induction of pathogenic effects. Inhibiting TLR-2 signalling alleviated HKM apoptosis, thereby favouring bacterial survival. Additionally, TLR-2-mediated cytosolic calcium (Ca²⁺)_c elevation was instrumental for eliciting ER-stress in infected HKM. ER-stress triggered the activation of membrane-proximal calcium entry channels comprising stromal interaction molecule 1 (STIM1) and calcium-release activated calcium channel 1 (Orai1). RNAi studies suggested STIM1-Orai1 signalling initiate calpain-mediated cleavage of nitric oxide synthase interacting protein, prompting the release of pro-apoptotic nitric oxide. Inhibiting STIM1-Orai1 signalling attenuated superoxide production (O₂^•-) and vice versa. We conclude, TLR-2-induced ER-stress triggers STIM1/Orai1 expression and that the reciprocal association between STIM1-Orai1 signalling and oxidative stress is critical for sustaining (Ca²⁺)_c level, thereby prolonging ER-stress and maintenance of pro-oxidant rich environment to induce HKM apoptosis and bacterial clearance.

Keywords: Calpain; ER-Stress; NOSIP; SOCE; Superoxide; TLR-2.