A major role for extracellular (eATP) in infectious diseases was not recognised until very recently. We have shown that intestinal epithelial cells infected with bacterial pathogens such as Shigella flexneri, Salmonella enterica serovar Typhimurium or enteropathogenic Escherichia coli (EPEC) secrete ATP across connexin-hemichannels. We have found that epithelial ATP secretion is an early alert response to infection, acting upstream of classical pro-inflammatory mediators (e.g. cytokines, chemokines and interferons) and provoking strong inflammation of the bowels (1). Further, we have shown that Shigella escapes this immune reaction: using a syringe-like protein complex (2), Shigella injects the enzyme IpgD directly into host cells. IpgD hydrolyses PtdIns(4,5)P2 to the poorly characterised lipid PtdIns5P (3). We have shown in vitro and in vivo that IpgD and PtdIns5P quickly induce hemichannel closure to stop ATP secretion (1). These data bridge the molecular mechanism regulating sterile inflammation and inflammation during infection. While our previous work has highlighted the fact that eukaryotic cells can respond to eATP by up-regulating inflammatory functions, it remains unknown whether bacteria can sense and respond to elevated eATP, e.g. to adapt to an inflammatory environment. We have shown that during acute inflammation in Shigella infection the luminal eATP concentration in the gut increases (1). In addition, we found that in patients suffering from certain types of inflammatory diseases of the bowels (IBD) and acute Clostridium difficile infection eATP concentrations in the gut lumen were elevated. These findings were confirmed using corresponding murine models (Groner et al., in preparation). Together, these data indicate that intestinal bacteria are exposed to eATP. Because the gut is highly compartmentalised and intestinal bacteria inhabit specific niches, it is reasonable to assume that microbes from distinct niches are exposed to distinct levels of eATP. For example, pathogens such as Shigella and EPEC inducing ATP secretion from the tissue are likely exposed to high concentrations of ATP, while members of the microbiota found in the centre of the lumen are likely exposed to lower eATP levels.
We have tested whether Shigella can sense and respond to elevated eATP by performing RNA-Seq on bacteria exposed to added ATP in vitro and have validated the hits by qRT-PCR (Tronnet, Cervantes Rivera and Puhar, unpublished results). We are extending the study to a number of E. coli strains with the aim of comparing the response in commensal, pathobiontic and pathogenic bacteria. To confirm the physiological relevance of in vitro findings and unravel the role of the response to eATP during disease, we will perform RNA-Seq on bacteria exposed to eATP in vivo using the rabbit ileal loop model for Shigella infection or murine models for E. coli infections.
1. A. Puhar, H. Tronchere, B. Payrastre, G. T. Nhieu, P. J. Sansonetti, Immunity 39, 1121-1131 (2013).
2. A. Puhar, P. J. Sansonetti, Curr. Biol. 24, pR784-R791 (2014).
3. K. Niebuhr et al., EMBO J. 21, 5069-5078 (2002).