Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding
Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding
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DOI：
10.1128/iai.00402-08
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发表时间：
2009-01-01
影响因子：
3.1
通讯作者：
Colin, Didier A.
中科院分区：
文献类型：
--
作者：
Meyer, Florent;Girardot, Raymonde;Colin, Didier A.
在这项研究中，结合的葡萄球菌双组分白细胞毒素Panton-Valentine杀白细胞素（LukF-PV）和γ-溶血素（HlgB）的F组分的多形核白细胞（PMN），单核细胞和淋巴细胞使用标记的突变体和流式细胞仪测定。通过使用荧光分光光度法或流式细胞术测量Ca 2+进入或孔形成来评价白细胞毒素活性。尽管HlgB在不存在S组分的情况下对细胞没有亲和力，但LukF-PV对PMN（解离常数[K-d]，6.2 +/- 1.9 nM; n = 8）、单核细胞（K-d，2.8 +/- 0.8 nM; n = 7）和淋巴细胞（K-d，1.2 +/- 0.2 nM; n = 7）具有高亲和力。仅在添加LukS-PV后，在PMN（K-d，1.1 +/- 0.2 nM; n = 4）和单核细胞（K-d，0.84 +/- 0.31 nM; n = 4）上或在添加HlgC后，在PMN、单核细胞和淋巴细胞上观察到HlgB的特异性结合。加入LukS-PV或HlgC可诱导LukF-PV与PMN的第二次特异性结合。HlgB和LukD仅与加入LukS-PV后结合的LukF-PV分子竞争。LukF-PV和LukD在LukS-PV存在下与HlgB在PMN和单核细胞上竞争。抗体的使用以及结合和活性时间过程之间的比较显示，在添加LukS-PV之前结合至靶细胞的LukF-PV分子是负责Ca 2+进入和孔形成的唯一LukF-PV分子。相反，活性HlgB分子是在添加LukS-PV后结合的HlgB分子。总之，LukF-PV必须连接到LukS-PV和膜的结合位点以具有毒素活性。
In this study, the binding of F components of the staphylococcal bicomponent leukotoxins Panton-Valentine leucocidin (LukF-PV) and gamma-hemolysin (HlgB) on polymorphonuclear neutrophils (PMNs), monocytes, and lymphocytes was determined using labeled mutants and flow cytometry. Leukotoxin activity was evaluated by measuring Ca2+ entry or pore formation using spectrofluorometry or flow cytometry. Although HlgB had no affinity for cells in the absence of an S component, LukF-PV had high affinity for PMNs (dissociation constant [K-d], 6.2 +/- 1.9 nM; n = 8), monocytes (K-d, 2.8 +/- 0.8 nM; n = 7), and lymphocytes (K-d, 1.2 +/- 0.2 nM; n = 7). Specific binding of HlgB was observed only after addition of LukS-PV on PMNs (K-d, 1.1 +/- 0.2 nM; n = 4) and monocytes (K-d, 0.84 +/- 0.31 nM; n = 4) or after addition of HlgC on PMNs, monocytes, and lymphocytes. Addition of LukS-PV or HlgC induced a second specific binding of LukF-PV on PMNs. HlgB and LukD competed only with LukF-PV molecules bound after addition of LukS-PV. LukF-PV and LukD competed with HlgB in the presence of LukS-PV on PMNs and monocytes. Use of antibodies and comparisons between binding and activity time courses showed that the LukF-PV molecules that bound to target cells before addition of LukS-PV were the only LukF-PV molecules responsible for Ca2+ entry and pore formation. In contrast, the active HlgB molecules were the HlgB molecules bound after addition of LukS-PV. In conclusion, LukF-PV must be linked to LukS-PV and to a binding site of the membrane to have toxin activity.