Why Escherichia coli α‐hemolysin induces calcium oscillations in mammalian cells‐the pore is on its own

Abstract

Escherichia coli alpha-hemolysin (HlyA), archetype of a bacterial pore-forming toxin, has been reported to deregulate physiological Ca2+ channels, thus inducing periodic low-frequency Ca2+ oscillations that trigger transcriptional processes in mammalian cells. The present study was undertaken to delineate the mechanisms underlying the Ca2+ oscillations. Patch-clamp experiments were combined with single cell measurements of intracellular Ca2+ and with flowcytometric analyses. Application of HlyA at subcytocidal concentrations provoked Ca2+ oscillations in human renal and endothelial cells. However, contrary to the previous report, the phenomenon could not be inhibited by the Ca2+ channel blocker nifedipine and Ca2+ oscillations showed no constant periodicity at all. Ca2+ oscillations were dependent on the pore-forming activity of HlyA: application of a nonhemolytic but bindable toxin had no effect. Washout experiments revealed that Ca2+ oscillations could not be maintained in the absence of toxin in the medium. Analogously, propidium iodide flux into cells occurred in the presence of HlyA, but cells rapidly became impermeable toward the dye after toxin washout, indicating resealing or removal of the membrane lesions. Finally, patch-clamp experiments revealed temporal congruence between pore formation and Ca2+ influx. We conclude that the nonperiodic Ca2+ oscillations induced by HlyA are not due to deregulation of physiological Ca2+ channels but derive from pulsed influxes of Ca2+ as a consequence of formation and rapid closure of HlyA pores in mammalian cell membranes.

E. coli Alpha Hemolysin and Properties

“…In addition, sublytic concentrations of HlyA have been recently found to potently stimulate the inactivation of the serine/threonine protein kinase B (PKB), which enzyme plays a central role in host cellcycle progression, metabolism, vesicular trafficking, survival, and inflammatory-signaling pathways . These findings may help to explain previously published results implicating sublytic concentrations of HlyA in the inhibition of chemotaxis and in bacterial killing by phagocytes in addition to the HlyA-mediated stimulation of host apoptotic and inflammatory pathways (Cavalieri & Snyder, 1982), (Koschinski et al, 2006), (Mansson et al, 2007), (Tran Van Nhieu et al,2004), (Uhlen et al, 2000).…”

[Ca2+] Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia

“…This finding potentially argues for G␣ q activation in response to HlyA, which certainly comply with ATP-mediated P2Y receptor activation. It must, however, be noted that other groups support that the [Ca 2ϩ ] i events are caused by the HlyA-pore itself without any kind of subsequent signaling (36). To test the concept of HlyA-induced P2Y receptor activation, we used the same types of renal epithelia, in which we previously characterized the ATP and P2 receptor dependence of spontaneous [Ca 2ϩ ] i oscillations: MDCK cells and murine mTAL.…”

Ca2+/calmodulin-dependent invasion of microvascular endothelial cells of human brain by Escherichia coli K1

“…The E. coli K1 invasion of HBMEC is also blocked by U73122, a specific inhibitor of host PLC, implying an involvement of the IP 3 -sensitive intracellular Ca 2+ pool. An increased intracellular Ca 2+ has been implicated in mediating host cell invasion by several microorganisms, such as Salmonella typhimurium, Porphomonas gingivalis, enteropathogenic E. coli, Camphylobacter jejuni, Listeria monocytogenes, and Pseudomonas aeruginosa (Belton et al 2004;Bierne et al 2000;Dramsi and Cossart 2003;Hu et al 2005;Koschinski et al 2006;Pace et al 1993;Ratner et al 2001;Shiner et al 2006;Tran Van Nhieu et al 2004). In most cases, the [Ca 2+ ] i elevation has been attributed to the formation of calcium-permeable pores by bacterial toxins, such as hemolysin, vacuolating cytotoxin A, or listeriolysin, and the subsequent influx of extracellular Ca 2+ (Belton et al 2004;Dramsi and Cossart 2003;Koschinski et al 2006;Marlink et al 2003;Repp et al 2002;Soderblom et al 2005;Tran Van Nhieu et al 2004).…”