Peptide deformylase in Staphylococcus aureus:: Resistance to inhibition is mediated by mutations in the formyltransferase gene
Peptide deformylase in Staphylococcus aureus:: Resistance to inhibition is mediated by mutations in the formyltransferase gene
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DOI：
10.1128/aac.44.7.1825-1831.2000
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发表时间：
2000-07-01
影响因子：
4.9
通讯作者：
Trias, J
中科院分区：
文献类型：
--
作者：
Margolis, PS;Hackbarth, CJ;Trias, J
肽去甲酰基酶是一种细菌酶，代表了抗生素发现的新靶点。在金黄色葡萄球菌中鉴定出两种去甲酰基酶同源物 defA 和 defB。位于转化酶基因上游的 defA 同源物通过基因组分析进行鉴定，并通过 PCR 从染色体 DNA 中克隆，不同的同源物 defB 是通过与在阿拉伯糖限制条件下生长的 P-BAD-def 大肠杆菌菌株的阿拉伯糖依赖性表型互补从金黄色葡萄球菌基因组文库中克隆的。 defB（而非 defA）在大肠杆菌中的过表达与去甲酰基酶活性的增加和对去甲酰基酶特异性抑制剂放线菌素（actinonin）的敏感性降低相关。 defB 基因在野生型金黄色葡萄球菌中不能被破坏，这表明该编码功能性去甲酰基酶的基因是必需的。相反，defA 基因可能会失活；该基因的功能尚不清楚。放线菌素耐药突变体在体外生长缓慢，并且未表现出与其他类别抗生素的交叉耐药性。与亲本相比，放线菌素抗性菌株在小鼠脓肿模型中产生了减毒感染，表明该菌株在体内也具有生长劣势。对放线菌素抗性突变体的序列分析表明，每个突变体都含有 fmt 基因的功能丧失突变。当野生型 fmt 基因被引入这些突变菌株时，对放线菌素的敏感性得到恢复。金黄色葡萄球菌 Delta fmt 菌株也对放线菌素具有抗性，表明缺乏甲酰基转移酶活性的菌株不需要功能性去甲酰基酶活性。因此，fmt 突变体中的 defB 基因可能被破坏。
Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB, were identified in Staphylococcus aureus. The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR, ri distinct homolog, defB, was cloned from an S, aureus genomic library by complementation of the arabinose-dependent phenotype of a P-BAD-def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E, coli of defB, but not defA, correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. The defB gene could not be disrupted in wild-type S, aureus suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, the defA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not show cross-resistance to other classes of antibiotics. When compared to the parent, an actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the fmt gene. Susceptibility to actinonin was restored when the wild-type fmt gene was introduced into these mutant strains. An S, aureus Delta fmt strain was also resistant to actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, the defB gene could be disrupted in an fmt mutant.