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pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes
K E Beauregard1, K D Lee, R J Collier
1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.
Insights
Acidic conditions enable the pore-forming toxin listeriolysin O (LLO) to perforate phagocytic vacuoles, facilitating Listeria monocytogenes escape. This pH-dependent vacuolar damage is crucial for bacterial virulence.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Listeria monocytogenes is an intracellular pathogen that escapes host cells.
- Listeriolysin O (LLO) is a key virulence factor enabling bacterial escape from phagocytic vacuoles.
- Understanding LLO's mechanism is vital for developing anti-listerial strategies.
Purpose of the Study:
- To investigate the pH-dependence of vacuolar perforation by LLO.
- To elucidate the role of acidic vacuolar pH in LLO-mediated bacterial escape.
Main Methods:
- Utilized mouse bone marrow-derived macrophages.
- Employed the pH-sensitive fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor vacuolar pH and integrity.
- Compared wild-type L. monocytogenes with LLO-deficient mutants.
Main Results:
- Vacuolar perforation by LLO was observed at acidic pH (4.9-6.7).
- Perforation correlated with sudden pH increases and HPTS release into the cytosol.
- Treatment with lysosomotropic agents (ammonium chloride, bafilomycin A1) reduced or prevented perforation.
- LLO-deficient mutants did not induce vacuolar changes.
Conclusions:
- Acidic pH is a critical facilitator of LLO activity and vacuolar perforation in vivo.
- LLO-mediated vacuolar escape is dependent on the acidic environment within phagocytic vacuoles.