Screening system for xenosiderophores as potential drug delivery agents in mycobacteria
Screening system for xenosiderophores as potential drug delivery agents in mycobacteria
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DOI：
10.1128/aac.45.5.1317-1322.2001
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发表时间：
2001-05-01
影响因子：
4.9
通讯作者：
Möllmann, U
中科院分区：
文献类型：
--
作者：
Schumann, G;Möllmann, U
为了建立可被分枝杆菌利用的异种铁载体的筛选系统，我们产生了一组耻垢分枝杆菌的突变体，其在公知的铁获取系统的不同步骤中被阻断。通过化学诱变，从菌株mc（2）155产生了一种具有分枝杆菌素生物合成阻断的突变体。外螯素生物合成基因fxbA和铁外螯素摄取基因fxuA，先前由菲斯等人鉴定（E，H，菲斯，S，Yu，和W，R，Jacobs，Jr，，Mel. Microbiol，14：557-559，1994）。将相邻的染色体片段用于同源重组，以便用来自转座子Tn 903的卡那霉素抗性基因替换野生型基因。通过PCR证实基因替换。分离的突变体显示出预期的表型：fxbA突变体在外螯素生物合成中有缺陷，而fxuA突变体分泌的外螯素的量显著大于亲本菌株分泌的量，这是由于它们在铁外螯铁蛋白摄取方面的缺陷，如在生长促进测定中所证明的。这组新的突变体允许通过与外螯素或分枝杆菌素的配体交换、通过使用单独的铁载体摄取途径或通过使用外螯素通透酶来区分为分枝杆菌提供铁的铁载体。所有这些类型的铁摄取途径都是用25种外源性铁载体作为测试物质来鉴定的。铁载体的作用没有配体交换是潜在的候选人作为药物载体，可用于克服渗透性介导的阻力。
In order to establish a screening system for xenosiderophores which can be utilized by mycobacteria, we generated a set of mutants of Mycobacterium smegmatis that are blocked in different steps of the well-known iron acquisition system. One mutant with a block in mycobactin biosynthesis was generated from strain mc(2)155 by chemical mutagenesis, The exochelin biosynthesis gene fxbA and the ferric exochelin uptake gene fxuA, previously identified by Fiss et al, (E, H, Fiss, S, Yu, and W, R, Jacobs, Jr,, Mel. Microbiol, 14:557-559, 1994), were knocked out by gene replacement. Adjacent chromosomal fragments were used for homologous recombination in order to replace wild-type genes by the kanamycin resistance gene from transposon Tn903, Gene replacement was confirmed by PCR, The isolated mutants show the expected phenotype: fxbA mutants are defective in exochelin biosynthesis, whereas fxuA mutants excrete a significantly larger amount of exochelin compared to the amount excreted by the parent strain, This is due to their defectiveness in ferriexochelin uptake, as demonstrated in growth promotion assays. This new set of mutants allows differentiation of siderophores that supply mycobacteria with iron by ligand exchange with exochelin or mycobactin, by the use of separate siderophore uptake routes, or by the use of the exochelin permease, All these types of iron uptake routes were identified with 25 exogenous siderophores as test substances. Siderophores that act without ligand exchange are potential candidates as drug vectors that can be used to overcome permeability-mediated resistance.