Vemula Harika, Ayon Navid J, Burton Alloch, Gutheil William G
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, USA.
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, USA
Antimicrob Agents Chemother. 2017 May 24;61(6). doi: 10.1128/AAC.02253-16. Print 2017 Jun.
Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway. Upstream inhibitors (fosfomycin, d-cycloserine, or d-boroalanine) reduced UDP-MurNAc-pentapeptide levels by more than fourfold. Alanine branch inhibitors (d-cycloserine and d-boroalanine) reduced d-Ala-d-Ala levels only modestly (up to 4-fold) but increased UDP-MurNAc-tripeptide levels up to 3,000-fold. Downstream pathway inhibitors (vancomycin, bacitracin, moenomycin, and oxacillin) increased UDP-MurNAc-pentapeptide levels up to 350-fold and UDP-MurNAc-l-Ala levels up to 80-fold, suggesting reduced MurD activity by downstream inhibitor action. Sub-MIC exposures demonstrated effects even at 1/8× MIC which strongly paralleled acute exposure changes. Time course data demonstrated that UDP-linked intermediate levels respond rapidly to vancomycin exposure, with several intermediates increasing three- to sixfold within minutes. UDP-linked intermediate level changes were also multiphasic, with some increasing, some decreasing, and some increasing and then decreasing. The total (summed) UDP-linked intermediate pool increased by 1,475 μM/min during the first 10 min after vancomycin exposure, providing a revised estimate of flux in this pathway during logarithmic growth. These observations outline the complexity of PG precursor response to antibiotic exposure in MRSA and indicate likely sites of regulation (entry and MurD).
在耐甲氧西林金黄色葡萄球菌（MRSA）中，测定了其暴露于几种细胞壁靶向抗生素后细胞质中肽聚糖（PG）前体的水平。进行了三项实验：（i）暴露于4倍最低抑菌浓度（MIC）水平（急性）；（ii）暴露于亚MIC水平（亚急性）；（iii）万古霉素作用的时间进程实验。在急性暴露实验中，正如预期的那样，磷霉素增加了UDP-GlcNAc，并导致相对于其他途径抑制剂，总UDP连接代谢物积累水平显著降低，表明进入该途径的量减少。上游抑制剂（磷霉素、d-环丝氨酸或d-硼丙氨酸）使UDP-MurNAc-五肽水平降低了四倍以上。丙氨酸分支抑制剂（d-环丝氨酸和d-硼丙氨酸）仅适度降低d-Ala-d-Ala水平（高达4倍），但使UDP-MurNAc-三肽水平增加了高达3000倍。下游途径抑制剂（万古霉素、杆菌肽、莫能菌素和苯唑西林）使UDP-MurNAc-五肽水平增加了高达350倍，UDP-MurNAc-L-Ala水平增加了高达80倍，表明下游抑制剂作用导致MurD活性降低。亚MIC暴露即使在1/8×MIC时也显示出与急性暴露变化强烈平行的效应。时间进程数据表明，UDP连接的中间产物水平对万古霉素暴露反应迅速，几种中间产物在几分钟内增加了三到六倍。UDP连接的中间产物水平变化也是多相的，一些增加，一些减少，一些先增加后减少。万古霉素暴露后的前10分钟内，总（汇总）UDP连接的中间产物库以1475μM/分钟的速度增加，这为对数生长期该途径的通量提供了一个修订后的估计值。这些观察结果概述了MRSA中PG前体对抗生素暴露反应的复杂性，并指出了可能的调控位点（进入和MurD）。
