Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis.
Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis.
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DOI：
10.1021/acschembio.9b00977
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发表时间：
2020-05-15
影响因子：
4
通讯作者：
Carlson EE
中科院分区：
文献类型：
--
作者：
Sharifzadeh S;Dempwolff F;Kearns DB;Carlson EE
选择性化学探针能够对青霉素结合蛋白（PBP）进行单独研究，并提供有关其酶活性的空间和时间分辨率的关键信息。为了鉴定用于研究枯草芽孢杆菌中肽聚糖生物合成的新型探针的支架，我们使用基于荧光的测定来评估来自不同结构亚类的21种β-内酰胺抗生素的PBP抑制谱。大多数化合物易于标记PBP 1、PBP 2a、PBP 2b或PBP 4。几乎所有的青霉素支架都对PBP 2a、2b和4的全部或组合具有共选择性。另一方面，头孢菌素对PBP 1单独或与PBP 4（头孢曲松、头孢西丁）和2b（头孢噻肟）或2a、2b和4（头孢噻吩）沿着给药具有最低的IC 50值。总体而言，在B中，PBP 1的5种选择性抑制剂（氨曲南、法罗培南、哌拉西林、头孢呋辛和头孢磺啶）、PBP 5的1种选择性抑制剂（6-氨基青霉烷酸）和其它PBPs的多种共选择性抑制剂。枯草杆菌被发现。令人惊讶的是，碳青霉烯类强烈抑制PBP 3，以前显示对β-内酰胺类具有低亲和力，并推测参与B中的β-内酰胺耐药性。枯草杆菌。为了研究PBP 3的特定作用，我们开发了基于美罗培南核心的活性探针，并利用它们来监测PBP 3在活细胞中的活性。我们发现PBP 3活性定位为单个细胞中的补丁，并在细胞生长周期期间在隔膜和分裂部位集中为环。我们的基于活性的方法使空间分辨率的转肽活性的个别PBPs在这个模型微生物，这是不可能与以前的化学和生物学方法。
Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b, or PBP4. Almost all penicillin scaffolds were coselective for all or combinations of PBP2a, 2b, and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin) and 2b (cefotaxime) or 2a, 2b, and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime, and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid), and various coselective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in β-lactam resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.
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DOI：
10.1002/anie.201206749
发表时间：
2012-12-07
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Kuru E;Hughes HV;Brown PJ;Hall E;Tekkam S;Cava F;de Pedro MA;Brun YV;VanNieuwenhze MS
通讯作者：
VanNieuwenhze MS
DOI：
10.1073/pnas.73.6.1816
发表时间：
1976-01-01
影响因子：
11.1
作者：
BUCHANAN, CE;STROMINGER, JL
通讯作者：
STROMINGER, JL
影响因子：
3.2
作者：
Dougherty, TJ;Kennedy, K;Pucci, MJ
通讯作者：
Pucci, MJ
影响因子：
3
作者：
Horii, T;Ichiyama, S;Kobayashi, M
通讯作者：
Kobayashi, M
影响因子：
3.4
作者：
Loskill, Peter;Pereira, Pedro M.;Jacobs, Karin
通讯作者：
Jacobs, Karin