Staphylococcus aureus Susceptibility to Innate Antimicrobial Peptides, (cid:2) -Defensins and CAP18, Expressed by Human Keratinocytes
Staphylococcus aureus Susceptibility to Innate Antimicrobial Peptides, (cid:2) -Defensins and CAP18, Expressed by Human Keratinocytes
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发表时间：
2003
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通讯作者：
K. Midorikawa;K. Ouhara;H. Komatsuzawa;T. Kawai;S. Yamada;T. Fujiwara;Kenshi Yamazaki;K. Sayama;M. Taubman;H. Kurihara;K. Hashimoto;M. Sugai
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作者：
K. Midorikawa;K. Ouhara;H. Komatsuzawa;T. Kawai;S. Yamada;T. Fujiwara;Kenshi Yamazaki;K. Sayama;M. Taubman;H. Kurihara;K. Hashimoto;M. Sugai
角化细胞表达的抗菌肽人(cid:1) -防御素-1 （hBD1）、hBD2、hBD3和CAP18参与介导对细菌感染的先天防御。为了深入了解金黄色葡萄球菌感染，研究了金黄色葡萄球菌（包括耐甲氧西林金黄色葡萄球菌（MRSA））对这些抗菌肽的敏感性。基于定量PCR，与金黄色葡萄球菌接触可显著诱导人角质形成细胞表达hBD2 mRNA， hBD3和CAP18半胱氨酸残基表达形成3 (cid:2) -sheet和1 (cid:4) -helix（17,18）。我们的分子质量数据表明，每个合成(cid:2) -防御蛋白有三个二硫键。将10微升细菌悬浮液（10 5个细胞）接种到200 (cid:5) l的细菌中，加入或不加入不同浓度的抗菌肽，在37°C下孵育2小时。将适当稀释的反应混合物（100 (cid:5) l）涂于Trypticase大豆琼脂上，然后在37°C下孵育过夜。测定每个平板上菌落的数量。通过测定细胞存活率与总细胞数的比较来评价抗菌效果。同样的实验进行了三次。为评价NaCl的抑菌效果，采用10 mM NaP i （pH值为10 ~ 300 mM）进行上述抑菌试验。采用薄层电镜观察各抗菌肽对金黄色葡萄球菌的影响。S. 2PF-18在一系列乙醇浓度下最终g/ml为a缺陷，然后包埋在Spurr 's Epon中。薄片用金刚石刀用超微切片机切割，用JOEL JEM- 2000 EX II型电子显微镜在80千伏下检查。抗菌肽联合抗生素对金黄色葡萄球菌细胞的影响。由于化疗有时使用两种以上的抗生素，我们评估了抗菌肽与抗生素联合使用的效果。采用了以下两种方法。
The antimicrobial peptides human (cid:1) -defensin-1 (hBD1), hBD2, hBD3, and CAP18 expressed by keratinocytes have been implicated in mediation of the innate defense against bacterial infection. To gain insight into Staphylococcus aureus infection, the susceptibility of S. aureus , including methicillin-resistant S. aureus (MRSA), to these antimicrobial peptides was examined. Based on quantitative PCR, expression of hBD2 mRNA by human keratinocytes was significantly induced by contact with S. aureus , and expression of hBD3 and CAP18 cysteine residues and form three (cid:2) -sheets and one (cid:4) -helix (17, 18). Our molec- ular mass data suggested that each synthetic (cid:2) -defensin had three disulfide bonds. S. were and in mM sodium phosphate (NaP (pH was subjected to mild sonication to disperse clumping cells and to a concentration of 7 with (pH Ten microliters of a bacterial suspension (10 5 cells) was inoculated into 200 (cid:5) l of i with or without various concentrations of the antibacterial peptides and incubated fo r 2 h at 37°C. An appropriate dilution of the reaction mixture (100 (cid:5) l) was plated on Trypticase soy agar and then incubated at 37°C overnight. The of was determined the number of colonies on each plate. The antibacterial effect was estimated by determining the rate of survival of cells in comparison with the total number cells. The same experiment was carried out three times. To evaluate the effect of NaCl, 10 mM NaP i (pH containing various concentrations of NaCl (10 to 300 mM) was used in the antibacterial assay described above. Thin-section electron microscopy was performed to ob-serve the influence of each antimicrobial peptide on S. aureus Over- night of S. 2PF-18, was a A-deficient at a final g/ml in a series of ethanol con- centrations and then embedded in Spurr’s Epon. Thin sections were cut with an ultramicrotome with a diamond knife and were examined with a JOEL JEM- 2000 EX II electron microscope at 80 kV. Effects of combinations of antibacterial peptides with antibiotics on S. aureus cells. Since more than two antibiotics are sometimes used for chemotherapy, we evaluated the effects of combinations of antimicrobial peptides with antibiotics. The following two methods were employed.