Involvement of Sphingosine Kinase (SK1) during autophagosome formation plays a critical role in controlling the growth of <i>Mycobacterium tuberculosis</i> within Human Mesenchymal Stem Cells

Abstract Mycobacterium tuberculosis (Mtb) evades host defense mechanisms by infecting and persisting in Mesenchymal Stem Cells (MSCs). Thus, MSCs contain M. tuberculosis DNA and harbor viable bacilli, and our recent study showed that autophagy plays a critical role in restricting the growth of Mtb in human MSCs. Activation of human MSCs using rapamycin enhanced the bactericidal function of MSCs. Here, we examined the autophagy pathway associated with the control of Mtb growth within MSCs. When compared to human macrophages, MSCs had increased expression of Sphingosine Kinase 1 (SK1) during Mtb infection, which was also found to be associated with overall increase in autophagic flux and LC3B positive puncta. SK1 also more often co-localized with gfpMtb containing autophagosomes within MSCs when compared to human macrophages. Percentage of SK1 and LC3B double positive M. tuberculosis containing autophagosomes were also significantly higher in MSCs compared to macrophages suggesting that Mtb induces SK1 mediated autophagy in MSCs during infection. To validate the role of SK1 mediated autophagy in inhibition of Mtb growth within MSCs, we knocked down SK1 using siRNA and scrambled control and examined the survival of M. tuberculosis. SK1-siRNA treated MSCs showed an enhanced survival of Mtb, and replication compared to control siRNA treated cells over 21 days of incubation. These results together indicate that SK1 mediates autophagy, autophagosome formation and autophagy dependent inhibition of Mtb growth within MSCs.