Legionella pneumophila Strain 130b Evades Macrophage Cell Death Independent of the Effector SidF in the Absence of Flagellin.

Mary Speir,Qingqing Han,Azadeh Seidi,Seth L Masters,Thomas Naderer,Richard A Flavell,Stéphane Hunot,James E Vince,Gerald W Dorn,Adam Vogrin,Gilu Abraham

doi:10.3389/fcimb.2017.00035

Abstract

The human pathogen Legionella pneumophila must evade host cell death signaling to enable replication in lung macrophages and to cause disease. After bacterial growth, however, L. pneumophila is thought to induce apoptosis during egress from macrophages. The bacterial effector protein, SidF, has been shown to control host cell survival and death by inhibiting pro-apoptotic BNIP3 and BCL-RAMBO signaling. Using live-cell imaging to follow the L. pneumophila-macrophage interaction, we now demonstrate that L. pneumophila evades host cell apoptosis independent of SidF. In the absence of SidF, L. pneumophila was able to replicate, cause loss of mitochondria membrane potential, kill macrophages, and establish infections in lungs of mice. Consistent with this, deletion of BNIP3 and BCL-RAMBO did not affect intracellular L. pneumophila replication, macrophage death rates, and in vivo bacterial virulence. Abrogating mitochondrial cell death by genetic deletion of the effectors of intrinsic apoptosis, BAX, and BAK, or the regulator of mitochondrial permeability transition pore formation, cyclophilin-D, did not affect bacterial growth or the initial killing of macrophages. Loss of BAX and BAK only marginally limited the ability of L. pneumophila to efficiently kill all macrophages over extended periods. L. pneumophila induced killing of macrophages was delayed in the absence of capsase-11 mediated pyroptosis. Together, our data demonstrate that L. pneumophila evades host cell death responses independently of SidF during replication and can induce pyroptosis to kill macrophages in a timely manner.

Highlights

Legionella pneumophila is the aetiological agent of Legionnaires’ Disease, a potentially life-threatening form of pneumonia in the elderly and immuno-compromised individuals (Cunha et al, 2016)
To examine L. pneumophila infection of bone marrow-derived macrophages (BMDMs) in real-time and to monitor their viability, infected cells were stained with the cell-permeable fluorescent dye, tetramethylrhodamine methyl ester (TMRM), which is sequestered by active mitochondria, depending on the inner membrane potential
The BMDMs infected with WT L. pneumophila died more rapidly than those infected with the flagellin-deficient strain, flaA, consistent with a flagellin/caspase-1-mediated pyroptotic cell death

Summary

Introduction

Legionella pneumophila is the aetiological agent of Legionnaires’ Disease, a potentially life-threatening form of pneumonia in the elderly and immuno-compromised individuals (Cunha et al, 2016). In the case of intrinsic apoptosis, the pro- and anti-apoptotic members of the BCL-2 protein family control the activity of the sentinel cell death regulators, BAX, and BAK (Czabotar et al, 2014). Cellular stresses, including bacterial infections, can promote activation of the pro-apoptotic BH3only proteins that either directly, or indirectly, induce BAX/BAKmediated apoptosis (Chipuk et al, 2010). This effectively controls intracellular pathogens by compromising their replicative niche and triggering bacterial clearance in a cell autonomous manner (Chow et al, 2016)

Methods

Results

Conclusion