Abstract
The bi-component leukotoxins; γ-hemolysin and Panton and Valentin Leukocidin (PVL) from Staphylococcus aureus induce two independent cellular events 1) the formation of trans-membrane pores not permeable to chloride (Cl-) ions and 2) the activation of at least, two modes of chloride fluxes (efflux/influx), including pre-existing Ca2+-activated Cl- channels (CaCC) in human polymorphonuclear neutrophils (PMNs). This was investigated by using spectrofluorometry techniques and the chloride-sensitive quencher fluorescent indicator, MQAE (N-(6-methoxyquinolyl) acetoacethyl ester). The ethidium bromide was used as an indicator for the trans-membrane pores formation by staphylococcal leukotoxins. In the absence of extracellular Ca2+, HlgA/HlgB, HlgC/HlgB and LukS-PV/LukF-PV leukotoxins from S. aureus induced a massive efflux of chloride (Cl-) ions. Interestingly, in the presence of extracellular Ca2+, the HlgA/HlgB γ-hemolysin provoked a biphasic response of Cl- movements (efflux/influx). Conversely to HlgA/HlgB and LukS-PV/LukF-PV, HlgC/HlgB leukotoxins did not induce any Cl- movement under this condition (e.g. in the presence of extracellular Ca2+). The potent Cl- channel inhibitor, DIDS, did inhibit significantly the Cl- fluxes caused by all pairs of staphylococcal leukotoxins tested in both conditions. In the present study, we found that the inhibitory effect of flufenamic acid, known as a Cl- channel inhibitor, was restricted only to the Ca2+-dependent Cl- influx triggered only by HlgA/HlgB and LukS-PV/LukF-PV leukotoxins. These findings might suggest that, Cl- fluxes in human neutrophils did involve at least, two different types of Cl- pathways, depending on the absence or presence of extracellular Ca2+. Both Cl- channels blockers, DIDS and flufenamic acid did not alter the pores formation by staphylococcal leukotoxins. Furthermore, under conditions when the membrane pores formation was blocked by divalent ions (Ca2+ and/or Zn2+), Cl- ions movements were still observed. Taken together, our results strongly provide an evidence that: i) trans-membrane pores formed by staphylococcal leukotoxins: HlgA/HlgB, HlgC/HlgB (γ-hemolysin) and LukS-PV/LukF-PV (PVL) do not drive Cl- ions fluxes ii) at least, two different types of Cl- ions pathways are activated, depending on the absence or presence of extracellular Ca2+, including Ca2+-activated Cl- channels (CaCC) and, iii) Ca2+-activated Cl- channels are mediated only by HlgA/HlgB and LukS-PV/LukF-PV leukotoxins.
Highlights
S. aureus secretes a family of bi-component pore-forming leukotoxins whose components belong either to class S or classs F [1]
We were interested in the present study to investigate an eventual activation of chloride channels by staphylococcal leukotoxins in human neutrophils, as it has previously demonstrated for other agonists [21,22]
The chloride ions efflux is an early event, that occurred following the human polymorphonuclear neutrophils (PMNs) activation by several physiological soluble agonists such as, tumor necrosis factor-α (TNF-α) [15, 21] or non-physiological soluble agonists like fMLP, PMA [16], and ionomycin with the concomitant decrease of the intracellular chloride content, we were interested in the present study, to investigate the Cl- ions movement in staphylococcal bi-component leukotoxins-activated human neutrophils
Summary
S. aureus secretes a family of bi-component pore-forming leukotoxins whose components belong either to class S or classs F [1]. The Panton-Valentine Leukocidin (class S: LukS-PV; class F: LukF-PV) is secreted by clinical strains associated with furuncles [2,3], and forms one toxic pair LukS-PV/LukF-PV. Fluxes in Human Neutrophils: Opening of Ca2+-activated Cl- Channels shown that, the class S component binding was a pre-requisite to binding of the class F component [5,6]. Both components (S/F) synergistically induce the opening of pre-existing Ca2+. Channels [7] and form pores through the membrane of human neutrophils [8]. It has been demonstrated that, these membrane pores were not permeable to divalent ions (Ca2+, Mn2+, Zn2+) but might be pathways for Na+ and K+ ions [7, 11]
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