Oral Presentation The 3rd Prato Conference on Pore Forming Proteins 2015

The staphylococcal Panton and Valentine Leukocidin and γ-haemolysin HlgC/HlgB share C5aR as a receptor, but operate diverse intracellular activities in human polymorphonuclear neutrophils (#23)

Gaëlle Zimmermann-Meisse 1 , Mira Y. Tawk 1 , Jean-Louis Bossu 2 , Cristina Potrich 3 , Tristan Bourcier 1 , Mauro Dalla Serra 3 , Bernard Poulain 2 , Gilles Prevost 1 , Emmanuel Jover 1
  1. Institut de Bactériologie, Unité : EA-7290 Virulence bactérienne précoce, Strasbourg, France
  2. INCI – UPR-CNRS 3212, Université de Strasbourg, Strasbourg, France
  3. Institute of Biophysics & Bruno Kessler Foundation, National Research Council of Italy, Trento, Italy

The pathogenicity of S. aureus is defined by a number of virulence factors, among which bicomponent leukotoxins convey an important contribution. A better understanding of leukotoxin cellular activity would help to understand health threats and to improve care of nosocomial and community infections associated to S. aureus. The C5a receptor of the complement act as host target for LukS-PV and HlgC. The receptor mediates both leukotoxin binding and cytotoxicity (1,2) on human immune cells such as polymorphonuclear neutrophils (hPMNs) or macrophages.

Microfluorimetry revealed diverse intracellular calcium variations induced through C5aR activation by PVL and HlgC/HlgB, compared with the natural C5a peptide activation. Determination by flow cytometry of leukotoxins binding parameters revealed a good correlation with the toxin concentrations releasing intracellular calcium. However, lacks of correlation appear with experimental conditions needed to determine plasma membrane permeabilization (EtBr). Immunocytochemistry and confocal microscopy techniques were engaged to evaluate the cellular localization of leukotoxins in parallel with their cellular effect.

Physiological calcium concentration is needed for the internalization of the two leukotoxin components (S- and F-subunits) and to trigger [Ca2+]i mobilization. HlgC/HlgB releases Ca2+ from acidic intracellular stores, while PVL only operates on neutral reticular calcium stores. The addressing specificity to a particular compartment is given by the F-subunit identity. Electrophysiological recordings on living cells demonstrated that PVL does not alter the membrane resistance of C5aR-expressing cells. However, HlgC/HlgB operates both intracellularly and on the plasma membrane.

In conclusion, low concentrations of PVL and HlgC/HlgB specifically alter distinct intracellular compartments while preserving the cellular integrity. This sublytical cellular effect may have a clinical significance when considering S. aureus pathogenicity.

1. Spaan, A.N., et al.  (2013) Cell Host Microbe 13, 584-594  2. Spaan, A.N., et al. (2014) Nat Commun 5