Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages

L Senerovic,C Goosmann,S P Tsunoda,V Brinkmann,F Meissner,M Kolbe,A Zychlinsky

doi:10.1038/cddis.2012.124

Abstract

The Gram-negative bacterium Shigella flexneri invades the colonic epithelium and causes bacillary dysentery. S. flexneri requires the virulence factor invasion plasmid antigen B (IpaB) to invade host cells, escape from the phagosome and induce macrophage cell death. The mechanism by which IpaB functions remains unclear. Here, we show that purified IpaB spontaneously oligomerizes and inserts into the plasma membrane of target cells forming cation selective ion channels. After internalization, IpaB channels permit potassium influx within endolysosomal compartments inducing vacuolar destabilization. Endolysosomal leakage is followed by an ICE protease-activating factor-dependent activation of Caspase-1 in macrophages and cell death. Our results provide a mechanism for how the effector protein IpaB with its ion channel activity causes phagosomal destabilization and induces macrophage death. These data may explain how S. flexneri uses secreted IpaB to escape phagosome and kill the host cells during infection and, may be extended to homologs from other medically important enteropathogenic bacteria.

Highlights

Invasion plasmid antigen B (IpaB) is a 62 kDa protein and one of the first effectors secreted by S. flexneri
It is stored in the bacterial cytoplasm bound to its chaperone invasion plasmid gene C (IpgC), which is released before IpaB secretion.[6,7]
To understand the role of secreted IpaB, we studied the function of the purified protein

Summary

Introduction

Invasion plasmid antigen B (IpaB) is a 62 kDa protein and one of the first effectors secreted by S. flexneri It is stored in the bacterial cytoplasm bound to its chaperone invasion plasmid gene C (IpgC), which is released before IpaB secretion.[6,7] IpaB is essential for S. flexneri virulence as bacteria lacking ipaB fail to invade epithelial cells and to activate Caspase-1 in macrophages.[8,9] The current model of. S. flexneri infection postulates that secreted IpaB, together with IpaC, forms a translocon that transports bacterial effectors across the host cell membrane.[10] It was assumed that S. flexneri requires IpaB to lyse the phagosomal membrane based on the results showing that IpaB alone can destabilize model membranes,[11] and that strains where ipaB was deleted do not have contact hemolytic activity.[10,12] The previous data suggest a dual function for IpaB, one in concert with IpaC facilitating the transport of other Shigella virulence factors in host cells and the second inducing pyroptosis in infected cells without binding to IpaC. Indirect evidence suggests that bacterial ipaB expression is required for the activation of the IPAF inflammasome,[9,21] the underlying mechanism of S. flexneriinduced cytotoxicity remains unclear

Methods

Results

Conclusion