Shigella flexneri, the causative agent of bacillar dystentery, invades the colonic mucosa where it elicits an intense inflammatory reaction responsible for destruction of the epithelium. During cell invasion, contact with host cells activates the type-III secretion of the Shigella IpaB and IpaC proteins. IpaB and IpaC are inserted into host cell plasma membranes and trigger initial signals that result in actin polymerization, while allowing cytosolic access of other bacterial effectors that further reorganize the cytoskeleton. After internalization, Shigella moves intracellularly and forms protrusions that infect neighbouring cells, promoting bacterial dissemination across the epithelium. Here, we show that during cell invasion, Shigella induces transient peaks in intracellular calcium concentration that are dependent on a functional type-III secretory apparatus. In addition, Shigella invasion induces the opening of Connexin 26 (Cx26) hemichannels in an actin- and phospholipase-C-dependent manner, allowing release of ATP into the medium. The released ATP, in turn, increases bacterial invasion and spreading, as well as calcium signalling induced by Shigella. These results provide evidence that pathogen-induced opening of connexin channels promotes signalling events that favour bacterial invasion and dissemination.
SummaryEscape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for highcontent and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.
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