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 Ca<sup>2+</sup> channels in human polymorphonuclear neutrophils (PMNs). The influx of Ca<sup>2+</sup> and Mn<sup>2+</sup> (Mn<sup>2+</sup> was used as a Ca<sup>2+</sup> surrogate) in Fura2-loaded human PMNs was determined by spectrofluorometry techniques. The present study showed that, in the presence of extracellular Ca<sup>2+</sup>, the staphylococcal HlgA/HlgB γ-hemolysin induced a rapid Ca<sup>2+</sup> release from internal Ca<sup>2+</sup> stores before the onset of a Mn<sup>2+</sup> (Ca<sup>2+</sup>) influx. The sustained increase of Ca<sup>2+</sup> and Mn<sup>2+</sup> influx was partially inhibited by the ionic blockers of Ca<sup>2+</sup> Release-Activated Ca<sup>2+</sup> (CRAC) channels, La<sup>3+</sup> and Ni<sup>2+</sup>. Furthermore, the incubation of human PMNs with either TMB8 or thapsigargin did inhibit significantly the Ca<sup>2+</sup> release mediated by leukotoxins simultanously to a clear decrease of Ca<sup>2+</sup> and Mn<sup>2+</sup> influx. The internal Ca<sup>2+</sup> release induced by γ-hemolysin was also inhibited by PMNs pretreatment with a pertussis toxin, NaF, caffeine, ryanodine, cinnarizine and flunarizine and consequently, the Mn<sup>2+</sup> (Ca<sup>2+</sup>) influx was significantly reduced. Moreover, different Ca<sup>2+</sup> 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 Ca<sup>2+</sup>, bi-component staphylococcal leukotoxins provoked in human PMNs after a specific binding to their membrane receptors, a rapid depletion of internal Ca<sup>2+</sup> stores mediating a CRAC channels activation. This Ca<sup>2+</sup>-dependent mechanism seems likely to be associated to the heterotrimeric G-proteins activation. Interestingly, in the absence of extracellular Ca<sup>2+</sup>, the staphylococcal leukotoxins tested induced the opening of an important divalent ions (Ca<sup>2+</sup>, Mn<sup>2+</sup>, Ni<sup>2+</sup>) pathway not sensitive to CRAC channels blockers. Consequently, we strongly suggested that other types of Ca<sup>2+</sup> channels might be involved in bi-component leukotoxins activity, including Ca<sup>2+</sup> channels dependent on the protein kinase C activation.
Highlights
Pore-forming toxins (PFTs) are key virulence factors produced by a variety of important human bacterial pathogens, including Staphylococcus aureus (S. aureus), Group A and B streptococci, Streptococcus pneumoniae, Vibrio cholerae, Clostridium septicum, Escherichia coli and Mycobacterium tuberculosis [1,2]
We have reported that the HlgA/HlgB γ-hemolysin pair did provoke, in the absence or in the presence of 1 mM extracellular free Ca2+, the opening of pre-existing Ca2+ channels involving divalent ions influx through the plasma membrane of human polymorphonuclear neutrophils (PMNs) [15]
The leukotoxin induced a decrease in fluorescence intensity of Fura2-loaded neutrophils indicating an influx of Mn2+ into PMNs (Figure 2a)
Summary
Pore-forming toxins (PFTs) are key virulence factors produced by a variety of important human bacterial pathogens, including Staphylococcus aureus (S. aureus), Group A and B streptococci, Streptococcus pneumoniae, Vibrio cholerae, Clostridium septicum, Escherichia coli and Mycobacterium tuberculosis [1,2]. For numerous human pathogenic bacteria, PFTs expressed during infections contributed significantly to the virulence in vivo Among these cytotoxic proteins, the bi-component γ-hemolysin from S. aureus was first described by Smith & Price in 1938 [3] who isolated it from α, β, δ hemolysins and was purified during the 70s by different authors [4,5]. It is well known that γ-hemolysin is composed of three proteins: HlgA, HlgB and HlgC [7,8,9] which form two active pairs HlgA/HlgB and HlgC/HlgB constituting with the Panton-Valentin leukocidin (PVL) [10] a family of leukotoxins presenting comparable activities [11] It was first, suggested that the PVL created aspecific pores through the membrane of human polymorphonuclear neutrophils (PMNs) whose conformation did change into ion-sized pores in the presence of extracellular
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