Abstract
The implications of TLR-2 mediated alterations in cytosolic-Ca2+((Ca2+)c) levels in M. smegmatis infections is not well known. Using headkidney macrophages (HKM) from Clarias gariepinus, we observed TLR-2 signalling is required in the phagocytosis of M. smegmatis. M. smegmatis induced caspase-dependent HKM apoptosis in MOI, time and growth-phase dependent manner. RNAi and inhibitor studies demonstrated critical role of TLR-2 in eliciting (Ca2+)c-surge and c-Src-PI3K-PLC axis playing an intermediary role in the process. The (Ca2+)c-surge triggered downstream ER-stress and superoxide (O2−) generation. The cross-talk between ER-stress and O2− amplified TNF-α production, which led to HKM apoptosis and bacterial clearance. Release of nitric oxide (NO) was also observed and silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase activity. Pre-treatment with diphenyleneidonium chloride inhibited NO production implicating O2−–NO axis imperative in M. smegmatis-induced HKM apoptosis. NO positively impacted CHOP expression and TNF-α production in infected HKM. We conclude that, TLR-2 induced (Ca2+)c-surge and ensuing cross-talk between ER-stress and O2− potentiates HKM pathology by amplifying pro-inflammatory TNF-α production. Moreover, the pro-oxidant environment triggers NO release which prolonged ER-stress and TNF-α production, culminating in HKM apoptosis and bacterial clearance. Together, our study suggests HKM an alternate model to study macrophage-mycobacteria interactions.
Highlights
M. smegmatis though considered saprophytic, incidences of animal infections in natural and laboratory conditions has been reported[5,6], a suitable model is warranted to study the bacterium
We studied the effect of M. smegmatis infection on headkidney macrophages (HKM)
We observed HKM cytotoxicity was significantly attenuated on treatment with dead bacteria obtained from different treatment regimens (Fig. 1b) clearly suggesting that metabolically active M. smegmatis are proficient in inducing cytotoxicity
Summary
M. smegmatis though considered saprophytic, incidences of animal infections in natural and laboratory conditions has been reported[5,6], a suitable model is warranted to study the bacterium. Earlier studies suggested TLR-2 signalling induces apoptosis in mycobacteria-infected macrophages[13]. ER-stress and ROS production has been observed in mycobacterial pathogenesis, the cross-talk between the two molecular events has not been reported. The apical role of TLR-2 in TNF-α production has been observed in M. tuberculosis infections[23], it is yet to be reported against M. smegmatis. The fast growth rate, less stringent bio-safety requirements coupled with close homology with many of the M. tuberculosis virulence genes suggests M. smegmatis to be an appropriate model to study mechanisms underlying mycobacterial pathogenesis[27] and in identifying novel intervention strategies[28]. We developed an infection model for M. smegmatis using HKM from Clarias gariepinus This fish is available round the year, can adapt to laboratory conditions and have identifiable immune organs. Our earlier studies have established that C. gariepinius-HKM are inherently phagocytic and serve as an excellent model for studying the pathogenesis of M. fortuitum and A. hydrophila[15,32]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call