Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli.
Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli.
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DOI：
10.1007/s00216-017-0610-5
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发表时间：
2017-12
影响因子：
4.3
通讯作者：
Östman C
中科院分区：
文献类型：
--
作者：
Fyrestam J;Östman C
对现代医学成就的主要威胁之一是抗菌素耐药性。血红素的细菌获取机制的分子靶向已被认为是一种替代抗生素。本研究建立了液相色谱-质谱联用液-液萃取简易清理法同时测定微生物中血红素和卟啉血红素前体的方法。通过实验设计对提取工艺参数进行优化，得到了回收率高、基质效应小、精密度高的方法。以大肠杆菌为模型微生物，研究了培养基中添加剂对血红素生物合成的影响。5-氨基乙酰丙酸和血红素分别使大肠杆菌中血红素浓度增加1.5倍和4.5倍。5-氨基乙酰丙酸的加入绕过了大肠杆菌血红素生物合成的负反馈控制，导致细胞内卟啉含量高。当血红素用作培养添加剂时获得的高浓度血红素表明大肠杆菌从其周围环境中摄取血红素。相比之下，在生长培养基中添加原卟啉钴IX减少了大肠杆菌中血红素的数量，这表明该化合物具有模拟真正的血红素并抑制血红素获取机制的能力。本文的在线版本（10.1007/s00216-017-0610-5）包含补充材料，仅供授权用户使用。
One of the main threats to the achievements in modern medicine is antimicrobial resistance. Molecular targeting of bacterial acquisition mechanisms of heme has been suggested to be an alternative to antibiotics. In the present study, HPLC-MS/MS combined with a simple clean-up based on liquid-liquid extraction has been developed and evaluated for simultaneous determination of heme and porphyrin heme precursors in microorganisms. Experimental design was used to optimize the extraction parameters, to obtain a method with high recovery, low matrix effects, and high precision. The effects of additives in the culture medium on the biosynthesis of heme were studied using Escherichia coli as a model microorganism. 5-Aminolaevulinic acid and hemin increased the heme concentration in E. coli by a factor of 1.5 and 4.5, respectively. Addition of 5-aminolaevulinic acid bypassed the E. coli negative feedback control of heme biosynthesis, which led to high amounts of intracellular porphyrins. The high heme concentration obtained when hemin was used as a culture additive shows that E. coli has an uptake of heme from its surroundings. In contrast, addition of cobalt protoporphyrin IX to the growth medium reduced the amount of heme in E. coli, demonstrating this compound’s ability to mimic real heme and inhibit the heme acquisition mechanisms. The online version of this article (10.1007/s00216-017-0610-5) contains supplementary material, which is available to authorized users.
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影响因子：
7.4
作者：
SCHNEEGURT, MA;BEALE, SI
通讯作者：
BEALE, SI
影响因子：
4.1
作者：
BROWN, SB;SHILLCOCK, M;JONES, P
通讯作者：
JONES, P
影响因子：
3
作者：
KJELDSTAD, B;JOHNSSON, A;SANDBERG, S
通讯作者：
SANDBERG, S
DOI：
10.1016/s1570-0232(02)00914-5
发表时间：
2003-03-05
影响因子：
3
作者：
Polson, C;Sarkar, P;Grant, R
通讯作者：
Grant, R
影响因子：
6.7
作者：
Alteri CJ;Hagan EC;Sivick KE;Smith SN;Mobley HL
通讯作者：
Mobley HL