宫颈霉素A-D是由肌腱链霉菌HKI0179产生的双糖基化聚酮类抗生素,具有对革兰氏阳性细菌的杀菌活性。在这项研究中,宫颈霉素C(CmC)处理在枯草芽孢杆菌168中引起意大利面样表型,具有细长的弯曲细胞,细胞分裂后保持连接,表现出染色体分离缺陷,导致没有DNA的鬼细胞。CmC处理的金黄色葡萄球菌的电子显微镜(3×MIC)显示肿胀的细胞,畸形的隔垫,细胞壁增厚,和粗糙的细胞壁表面。在枯草芽孢杆菌中的掺入试验表明在高的宫颈霉素浓度下对DNA生物合成有影响。的确,DNA促旋酶亚基B基因(gyrB)的人工下调增加了琼脂扩散试验中的宫颈霉素活性,and,在高浓度下(从62.5×MIC开始),该抗生素在体外抑制金黄色葡萄球菌DNA促旋酶超螺旋活性。为了更全面地了解CmC的作用方式,进行了宫颈霉素处理的与未处理的金黄色葡萄球菌细胞的转录组学和蛋白质组学。有趣的是,3×MIC的宫颈霉素没有引起特征性反应,这表明体内DNA促旋酶活性受到干扰。相反,宫颈霉素诱导CtsR/HrcA热休克操纵子的表达和自溶素的表达,与核糖体靶向抗生素庆大霉素相似。总之,我们确定了DNA促旋酶为靶标,但是在低浓度下,电子显微镜和组学数据揭示了宫颈霉素的更复杂的作用模式,包括热休克反应的感应,表明细胞中的蛋白质应激。重要抗生素革兰氏阳性菌的耐药性是现代医学中的一个新兴问题,迫切需要具有新型作用方式的新型抗生素。来自链霉菌的次生代谢产物是抗生素的重要来源,如腱链霉菌HKI0179产生的子宫颈霉素复合物。枯草芽孢杆菌和金黄色葡萄球菌对宫颈霉素C的表型反应表明染色体分离和隔膜形成缺陷。这种作用首先归因于宫颈霉素C和DNA促旋酶之间的相互作用。然而,cervimin处理与未处理的金黄色葡萄球菌细胞的组学数据表明了不同的作用模式,因为应激反应不包括SOS反应,但类似于对诱导误译或过早链终止并引起蛋白质应激的抗生素的反应。总之,这些结果指出了一种可能的新机制,该机制在细胞中产生蛋白质应激,并随后导致细胞和染色体分离的缺陷。
Cervimycins A-D are bis-glycosylated
polyketide antibiotics produced by Streptomyces tendae HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in Bacillus subtilis 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of CmC-treated Staphylococcus aureus (3 × MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in B. subtilis indicated an effect on DNA biosynthesis at high cervimycin concentrations. Indeed, artificial downregulation of the DNA gyrase subunit B gene (gyrB) increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 × MIC), the antibiotic inhibited S. aureus DNA gyrase supercoiling activity in vitro. To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated S. aureus cells were performed. Interestingly, 3 × MIC of cervimycin did not induce characteristic responses, which would indicate disturbance of the DNA gyrase activity in vivo. Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting antibiotic gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations, electron microscopy and omics data revealed a more complex mode of action of cervimycin, which comprised induction of the heat shock response, indicating protein stress in the cell.IMPORTANCEAntibiotic resistance of Gram-positive bacteria is an emerging problem in modern medicine, and new antibiotics with novel modes of action are urgently needed. Secondary metabolites from Streptomyces species are an important source of antibiotics, like the cervimycin complex produced by Streptomyces tendae HKI 0179. The phenotypic response of Bacillus subtilis and Staphylococcus aureus toward cervimycin C indicated a chromosome segregation and septum formation defect. This effect was at first attributed to an interaction between cervimycin C and the DNA gyrase. However, omics data of cervimycin treated versus untreated S. aureus cells indicated a different mode of action, because the stress response did not include the SOS response but resembled the response toward antibiotics that induce mistranslation or premature chain termination and cause protein stress. In summary, these results point toward a possibly novel mechanism that generates protein stress in the cells and subsequently leads to defects in cell and chromosome segregation.