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The role of Shigella T3S-dependent modulation of extracellular ATP release and calcium signalling in neutrophil migration

Final Report Summary - ATP (The role of Shigella T3S-dependent modulation of extracellular ATP release and calcium signalling in neutrophil migration)

Infection with the enteric pathogen Shigella is marked by inflammatory tissue destruction (Phalipon and Sansonetti, 2007). After ingestion Shigella invades the intestinal mucosa by means of injection of type III secretion (T3S) effectors (Phalipon and Sansonetti, 2007), which also triggers opening of connexin hemichannels and ensuing release of ATP (Tran Van Nhieu et al., 2003).

Regulation of hemichannel activity appears complex and remains elusive (Evans et al., 2006). Further, the physiological role of ATP release during Shigella infection is not understood, but it is known that extracellular ATP - acting as danger signal - is a potent immunostimulatory mediator (Bours et al., 2006).

Our worked focussed on the effect of the T3S effector IpgD on hemichannel-dependent ATP release. IpgD is a InsPtd(4,5)P2 phosphatase that specifically yields the rare and poorly characterised lipid InsPtd(5)P (Niebuhr et al., 2002).

We found that infection of both unpolarised and polarised epithelial cells with an ipgD-deficient strain resulted in higher amounts of ATP in the extracellular medium as compared to wild-type, non-invasive or ipgD-complemented Shigella. Blockage of hemichannels dampened ATP release during infection.

Transient transfection of IpgD in epithelial cells abolished low extracellular Ca2+-induced hemichannel opening as determined by the uptake of a hemichannel-permeable dye from the extracellular medium. Interestingly, transfection of an InsPtd(4,5)P2 phosphatase yielding InsPtd(4)P instead of InsPtd(5)P had no effect on hemichannel opening, indicating that hemichannel closure is not a consequence of InsPtd(4,5)P2 degradation but rather of production of InsPtd(5)P. In accordance with this finding, treatment of epithelial cells with InsPtd(5)P inhibited ATP release induced by low extracellular Ca2+. Similarly, treatment of cells with InsPtd(5)P before infection with ipgD-deficient Shigella dampened ATP release. Also, when hemichannel opening was induced by low extracellular Ca2+ cellular InsPtd(5)P levels decreased, in keeping with a function in hemichannel regulation.

To understand the consequence of ATP release for infection the rabbit ileal loop model was used. The ipgD-deficient strain caused by far more severe destruction of the intestinal mucosa than wild-type and ipgD-complemented Shigella and extensive production of pro-inflammatory cytokines as assessed by rtPCR. Similarly to the ipgD-deficient mutant and in accordance with a pro-inflammatory function of extracellular ATP, injection of non-hydrolyzable ATP along with wild-type Shigella led to massive tissue destruction. To the contrary, injection of a suramin, an inhibitor of purinergic receptors, along with ipgD-deficient Shigella decreased its inflammatory phenotype.

Taken together our data indicate that IpgD, through production of InsPtd(5)P, dampens the appearance of extracellular ATP in order to avoid excessive activation of the immune system and tissue destruction. To our knowledge, this is the first report of a bacterial protein manipulating the cellular release of a danger signal. Further, we identified InsPtd(5)P as a key player in hemichannel regulation. As a consequence, our data assign a prominent role to InsPtd(5)P in the regulation of inflammation in the gut. The detailed molecular mechanism linking InsPtd(5)P levels to hemichannel activity are currently under investigation.

Our work opens new avenues in understanding infectious and inflammatory diseases and in the identification of new anti-inflammatory drug targets.