Endonuclease G takes part in AIF-mediated caspase-independent apoptosis in Mycobacterium bovis-infected bovine macrophages

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host-pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10: 1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.