The Pore-forming Leukotoxins from <i>S. aureus</i> Involve Ca<sup>2+</sup> Release-Activated Ca<sup>2+</sup> Channels and Other Types of Ca<sup>2+</sup> Channels in Ca<sup>2+</sup> Entry into Neutrophils

Abstract

The pore-forming bi-component leukotoxins from <i>Staphylococcus aureus</i> induce two independent cellular events <i>1)</i> the formation of trans-membrane pores not permeable to divalent ions and <i>2)</i> the opening of pre-existing Ca²⁺ channels in human polymorphonuclear neutrophils (PMNs). The influx of Ca²⁺ and Mn²⁺ (Mn²⁺ was used as a Ca²⁺ surrogate) in Fura2-loaded human PMNs was determined by spectrofluorometry techniques. The present study showed that, in the presence of extracellular Ca²⁺, the staphylococcal HlgA/HlgB γ-hemolysin induced a rapid Ca²⁺ release from internal Ca²⁺ stores before the onset of a Mn²⁺ (Ca²⁺) influx. The sustained increase of Ca²⁺ and Mn²⁺ influx was partially inhibited by the ionic blockers of Ca²⁺ Release-Activated Ca²⁺ (CRAC) channels, La³⁺ and Ni²⁺. Furthermore, the incubation of human PMNs with either TMB8 or thapsigargin did inhibit significantly the Ca²⁺ release mediated by leukotoxins simultanously to a clear decrease of Ca²⁺ and Mn²⁺ influx. The internal Ca²⁺ release induced by γ-hemolysin was also inhibited by PMNs pretreatment with a pertussis toxin, NaF, caffeine, ryanodine, cinnarizine and flunarizine and consequently, the Mn²⁺ (Ca²⁺) influx was significantly reduced. Moreover, different Ca²⁺ signaling pathways blockers such as U73122, staurosporine, thyrphostin A9 and okadaic acid were tested on the leukotoxins activity. Taken together, this work provided evidence that, in the presence of extracellular Ca²⁺, bi-component staphylococcal leukotoxins provoked in human PMNs after a specific binding to their membrane receptors, a rapid depletion of internal Ca²⁺ stores mediating a CRAC channels activation. This Ca²⁺-dependent mechanism seems likely to be associated to the heterotrimeric G-proteins activation. Interestingly, in the absence of extracellular Ca²⁺, the staphylococcal leukotoxins tested induced the opening of an important divalent ions (Ca²⁺, Mn²⁺, Ni²⁺) pathway not sensitive to CRAC channels blockers. Consequently, we strongly suggested that other types of Ca²⁺ channels might be involved in bi-component leukotoxins activity, including Ca²⁺ channels dependent on the protein kinase C activation.