Modulation of host protein phosphorylation upon mycobacterial infection

Tuberculosis, caused by Mycobacterium tuberculosis, remains the number one killer in the world today due to a single bacterial infection. M. tuberculosis primarily infects and resides within the human macrophage where it resists the host killing mechanisms. Based on the hypothesis that mycobacteria actively interfere with host signaling pathways to promote their own survival, we have employed a novel technique in proteomics to screen for simultaneous changes in host protein phosphorylation upon infection. The human monocytic cell line, THP-1, was differentiated with PMA and used as a macrophage model for infection with live or heat-killed M. bovis BCG or treated with the cell wall glycolipid lipoarabinomannan (LAM), known as a potential mycobacterial virulence factor. An infection or treatment time of 24 hours was chosen to study late signaling events associated with the progression of the pathogen within the host cell, as opposed to early events, shared by most microbial and inert particles during early stages of their uptake. Cell lysates were analyzed by employing an array of 31 phospho-specific antibodies covering kinases and other signaling elements from the major eukaryotic signaling networks known to date. Based upon two separate screens, we have identified changes in host signaling pathways that have not previously been described in mycobacterial infection. Six host proteins involved in regulation of apoptotic pathways, cytoskeletal arrangement, calcium signaling and macrophage activation have been identified. One of the major findings of the screens was an increased phosphorylation of the cytoskeletal protein a-adducin upon mycobacterial infection. a-Adducin binds to actin and spectrin and plays an important role in actin filament rearrangement in eukaryotic cells. Using classical Western blot techniques, we demonstrated that a-adducin undergoes increased phosphorylation in cells infected with live bacteria compared to those infected with heat-killed bacteria. Furthermore, adducin phosphorylation increases as function of time to reach a maximum at 24 hours. Interestingly, the surface glycolipid LAM was shown to trigger a dose-dependent increase in a-adducin phosphorylation. Moreover, immunostaining experiments and fluorescence microscopy in combination with Western blots have shown that adducin translocates from the cell membrane to the cytosol upon infection. We have also shown that live mycobacteria, in contrast to dead mycobacteria, prevent the assembly of actin around phagosomes during infection. These findings show an interesting and potentially important relationship between mycobacterial infection and a-adducin activity within the host cell. Given the importance of adducin in regulating actin rearrangements, mycobacterial-induced changes in a-adducin phosphorylation may be involved in the observed inhibition in phagosomal actin assembly around phagosomes containing live bacteria. Thus, ot-adducin phosphorylation may play a role in the mechanisms allowing mycobacteria to survive within the host cell.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat