Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Streptococcus pneumoniae D39
Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Streptococcus pneumoniae D39
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DOI：
10.1128/aac.05142-14
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发表时间：
2015-06-01
影响因子：
4.9
通讯作者：
Carlson, Erin E.
中科院分区：
文献类型：
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作者：
Kocaoglu, Ozden;Tsui, Ho-Ching T.;Carlson, Erin E.
选择性荧光β -内酰胺化学探针能够可视化青霉素结合蛋白（PBPs）在细菌细胞分裂不同阶段的转肽酶活性。为了促进PBP成像新荧光探针的开发，我们评估了20种市售的β -内酰胺对肺炎链球菌D39菌株未封装衍生物的PBP选择性抑制作用。活细胞用不同浓度的β -内酰胺类抗生素处理，随后用Bocillin FL （Boc-FL；荧光青霉素）孵育，使不受抑制的PBPs饱和。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和荧光扫描对荧光团标记的PBPs进行可视化。在所检测的20种化合物中，碳青霉烯类化合物（多利培南和美罗培南）对PBP1a、PBP2x和PBP3具有共选择性，而9种青霉素化合物中有6种对PBP2x和PBP3具有共选择性。相比之下，测试的七种头孢菌素化合物在其pbp结合谱中显示出可变性。三种头孢菌素化合物（头孢西丁、头孢氨苄和头孢素）和单巴坦氮曲南对PBP3有选择性，而只有头孢呋辛（一种头孢菌素）对PBP2x有选择性。用亚致死浓度的头孢呋辛处理肺炎链球菌培养物，抑制60%的PBP2x活性和低于20%的其他PBPs活性，导致形成细长细胞。相比之下，用氮曲南和头孢西丁浓度抑制高达70%的PBP3活性和低于30%的其他PBPs活性的肺炎链球菌培养物处理，没有明显的形态学变化。此外，除法罗培南、氨苄西林（美西林）和6-APA外，每种PBP的MIC和IC(50)s的相关性表明，肺炎球菌生长抑制主要是由于PBP2x的抑制。
Selective fluorescent beta-lactam chemical probes enable the visualization of the transpeptidase activity of penicillin-binding proteins (PBPs) at different stages of bacterial cell division. To facilitate the development of new fluorescent probes for PBP imaging, we evaluated 20 commercially available beta-lactams for selective PBP inhibition in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae. Live cells were treated with beta-lactam antibiotics at different concentrations and subsequently incubated with Bocillin FL (Boc-FL; fluorescent penicillin) to saturate uninhibited PBPs. Fluorophore-labeled PBPs were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence scanning. Among 20 compounds tested, carbapenems (doripenem and meropenem) were coselective for PBP1a, PBP2x, and PBP3, while six of the nine penicillin compounds were coselective for PBP2x and PBP3. In contrast, the seven cephalosporin compounds tested display variability in their PBP-binding profiles. Three cephalosporin compounds (cefoxitin, cephalexin, and cefsulodin) and the monobactam aztreonam exhibited selectivity for PBP3, while only cefuroxime (a cephalosporin) was selective for PBP2x. Treatment of S. pneumoniae cultures with a sublethal concentration of cefuroxime that inhibited 60% of PBP2x activity and less than 20% of the activity of other PBPs resulted in formation of elongated cells. In contrast, treatment of S. pneumoniae cultures with concentrations of aztreonam and cefoxitin that inhibited up to 70% of PBP3 activity and less than 30% of other PBPs resulted in no discernible morphological changes. Additionally, correlation of the MIC and IC(50)s for each PBP, with the exception of faropenem, amdinocillin (mecillinam), and 6-APA, suggests that pneumococcal growth inhibition is primarily due to the inhibition of PBP2x.