PDF(Pubmed)
Abstract：
An essential fact underlying the severity of Staphylococcus aureus (S. aureus) infection is the bicomponent leukocidins released by the pathogen to target and lyse host phagocytes through specific binding cell membrane receptors. However, little is known about the impact of post-transcriptional modification of receptors on the leukocidin binding.
In this study, we used small interfering RNA library (Horizon/Dharmacon) to screen potential genes that affect leukocidin binding on receptors. The cell permeability was investigated through flow cytometry measuring the internalization of 4\',6-diamidino-2-phenylindole. Expression of C5a anaphylatoxin chemotactic receptor 1 (C5aR1), sulfated C5aR1 in, and binding of 6x-His-tagged Hemolysin C (HlgC) and Panton-Valentine leukocidin (PVL) slow-component to THP-1 cell lines was detected and analyzed via flow cytometry. Bacterial burden and Survival analysis experiment was conducted in WT and myeloid TPST-cko C57BL/6N mice.
After short hairpin RNA (shRNA) knockdown of TPST2 gene in THP-1, HL-60, and RAW264.7, the cytotoxicity of HlgAB, HlgCB, and Panton-Valentine leukocidin on THP-1 or HL-60 cells was decreased significantly, and the cytotoxicity of HlgAB on RAW264.7 cells was also decreased significantly. Knockdown of TPST2 did not affect the C5aR1 expression but downregulated cell surface C5aR1 tyrosine sulfation on THP-1. In addition, we found that the binding of HlgC and LukS-PV on cell surface receptor C5aR1 was impaired in C5aR1+TPST2- and C5aR1-TPST2- cells. Phagocyte knockout of TPST2 protects mice from S. aureus infection and improves the survival of mice infected with S. aureus.
These results indicate that phagocyte TPST2 mediates the bicomponent leukocidin cytotoxicity by promoting cell membrane receptor sulfation modification that facilitates its binding to leukocidin S component.
In this study, we used small interfering RNA library (Horizon/Dharmacon) to screen potential genes that affect leukocidin binding on receptors. The cell permeability was investigated through flow cytometry measuring the internalization of 4\',6-diamidino-2-phenylindole. Expression of C5a anaphylatoxin chemotactic receptor 1 (C5aR1), sulfated C5aR1 in, and binding of 6x-His-tagged Hemolysin C (HlgC) and Panton-Valentine leukocidin (PVL) slow-component to THP-1 cell lines was detected and analyzed via flow cytometry. Bacterial burden and Survival analysis experiment was conducted in WT and myeloid TPST-cko C57BL/6N mice.
After short hairpin RNA (shRNA) knockdown of TPST2 gene in THP-1, HL-60, and RAW264.7, the cytotoxicity of HlgAB, HlgCB, and Panton-Valentine leukocidin on THP-1 or HL-60 cells was decreased significantly, and the cytotoxicity of HlgAB on RAW264.7 cells was also decreased significantly. Knockdown of TPST2 did not affect the C5aR1 expression but downregulated cell surface C5aR1 tyrosine sulfation on THP-1. In addition, we found that the binding of HlgC and LukS-PV on cell surface receptor C5aR1 was impaired in C5aR1+TPST2- and C5aR1-TPST2- cells. Phagocyte knockout of TPST2 protects mice from S. aureus infection and improves the survival of mice infected with S. aureus.
These results indicate that phagocyte TPST2 mediates the bicomponent leukocidin cytotoxicity by promoting cell membrane receptor sulfation modification that facilitates its binding to leukocidin S component.
摘要：
■金黄色葡萄球菌严重程度的基本事实(S.金黄色葡萄球菌）感染是病原体释放的双组分杀白细胞素，通过特异性结合细胞膜受体靶向和裂解宿主吞噬细胞。然而,关于受体转录后修饰对杀白细胞素结合的影响知之甚少。
■在这项研究中，我们使用小干扰RNA文库(Horizon/Dharmacon)筛选影响杀白细胞素与受体结合的潜在基因.通过流式细胞术测量4'的内在化来研究细胞通透性，6-二氨基-2-苯基吲哚。C5a过敏毒素趋化受体1(C5aR1)的表达,硫酸化C5aR1中，并通过流式细胞术检测并分析了6x-His标记的溶血素C（HlgC）和Panton-Valentine杀白细胞素（PVL）慢成分与THP-1细胞系的结合。在WT和髓样TPST-ckoC57BL/6N小鼠中进行细菌负荷和存活分析实验。
■THP-1，HL-60和RAW264.7中TPST2基因的短发夹RNA（shRNA）敲低后，HlgAB的细胞毒性，HlgCB,Panton-Valentine杀白细胞素对THP-1或HL-60细胞的作用明显减少，HlgAB对RAW264.7细胞的细胞毒性也显著降低。TPST2的敲低不影响C5aR1表达,但下调THP-1上的细胞表面C5aR1酪氨酸硫酸化。此外,我们发现，在C5aR1TPST2-和C5aR1-TPST2-细胞中，HlgC和LukS-PV在细胞表面受体C5aR1上的结合受损。TPST2的吞噬细胞敲除保护小鼠免受金黄色葡萄球菌感染并改善感染金黄色葡萄球菌的小鼠的存活率。
■这些结果表明，吞噬细胞TPST2通过促进细胞膜受体硫酸化修饰来介导双组分杀白细胞素的细胞毒性，从而促进其与杀白细胞素S组分的结合。
■在这项研究中，我们使用小干扰RNA文库(Horizon/Dharmacon)筛选影响杀白细胞素与受体结合的潜在基因.通过流式细胞术测量4'的内在化来研究细胞通透性，6-二氨基-2-苯基吲哚。C5a过敏毒素趋化受体1(C5aR1)的表达,硫酸化C5aR1中，并通过流式细胞术检测并分析了6x-His标记的溶血素C（HlgC）和Panton-Valentine杀白细胞素（PVL）慢成分与THP-1细胞系的结合。在WT和髓样TPST-ckoC57BL/6N小鼠中进行细菌负荷和存活分析实验。
■THP-1，HL-60和RAW264.7中TPST2基因的短发夹RNA（shRNA）敲低后，HlgAB的细胞毒性，HlgCB,Panton-Valentine杀白细胞素对THP-1或HL-60细胞的作用明显减少，HlgAB对RAW264.7细胞的细胞毒性也显著降低。TPST2的敲低不影响C5aR1表达,但下调THP-1上的细胞表面C5aR1酪氨酸硫酸化。此外,我们发现，在C5aR1TPST2-和C5aR1-TPST2-细胞中，HlgC和LukS-PV在细胞表面受体C5aR1上的结合受损。TPST2的吞噬细胞敲除保护小鼠免受金黄色葡萄球菌感染并改善感染金黄色葡萄球菌的小鼠的存活率。
■这些结果表明，吞噬细胞TPST2通过促进细胞膜受体硫酸化修饰来介导双组分杀白细胞素的细胞毒性，从而促进其与杀白细胞素S组分的结合。
