Programmed cell death in Legionella infection

Legionella ssp. are the causative agents of severe inflammatory pneumonia, known as Legionnaires’ Disease, which is fatal in up to 30 % of cases. Legionella replicate within alveolar macrophages by hijacking host cell pathways to establish a unique vacuolar niche. This includes the regulation of programmed host cell death factors, as Legionella must first prevent, and then induce, host cell death to promote bacterial growth and egress, respectively. However, the molecular mechanisms involved in toggling “off” and “on” host cell death signalling pathways remain undetermined. The major focus of the work described in this thesis was the delineation of the role that programmed host cell death pathways play in Legionella infection. To do this, a novel live-cell imaging technique was employed to visualise the intracellular life-cycle of Legionella and to monitor macrophage health in real-time. Using this method, I was able to confirm that wild-type Legionella induce a rapid form of cell death, termed pyroptosis, which is dependent on bacterial flagellin and the host protease, caspase-1. While flagellin/caspase-1-mediated pyroptosis prevents bacterial replication, I have identified that aflagellated Legionella also induce caspase-11-dependent pyroptosis. In contrast to caspase-1, caspase-11-mediated pyroptosis is induced in the late stages of infection, concomitant with Legionella egress, and does not interfere with intracellular bacterial replication. Legionella are also thought to induce other forms of host cell death, however, genetic ablation of mitochondrial apoptosis (BAX and BAK deletion), caspase-independent necroptotic cell death (RIPK3 and MLKL deletion), or BNIP3 and BCL-RAMBO, the putative targets of the effector protein SidF, did not affect Legionella replication or the killing of host macrophages. Legionella must prevent the activation of host cell death signalling to allow for efficient replication. While down-regulation of flagellin enables intracellular growth in the presence of caspase-1, little is known about how Legionella might evade apoptotic cell death. Using live-cell imaging, I have now shown that Legionella-infected macrophages depend critically upon the anti-apoptotic activity of host cell BCL-XL, but not other BCL-2 family members, for viability. In the absence of BCL-XL, Legionella-infected cells underwent apoptosis, which abolished bacterial replication and dissemination. Legionella infection could be fully restored by inhibiting mitochondrial apoptosis, either via BAX/BAK deletion or caspase inhibition. A single dose of BCL-XL-targeted BH3-mimetic therapy significantly reduced Legionella burden in the lungs of mice and prevented lethal bacterial infection. Mechanistically, I identified that Legionella infection inhibits host protein synthesis, which sensitises macrophages to BCL-XL loss or inhibition, via depletion of another anti-apoptotic BCL-2 family protein, MCL-1. Together, these results demonstrate that Legionella- infected macrophages are specifically and acutely sensitive to apoptotic cell death following the loss, or inhibition, of BCL-XL. Thus, the re-purposing of existing drugs, such as chemotherapeutic BH3-mimetics, to target host, rather than bacterial, pathways represents a novel and promising strategy for the treatment of intracellular pathogens that show increased, and often rapidly acquired, antibiotic resistance