Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants
Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants
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DOI：
10.1016/j.mimet.2010.09.008
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发表时间：
2010-11-01
影响因子：
2.2
通讯作者：
Brueggemann, Holger
中科院分区：
文献类型：
--
作者：
Soerensen, Meike;Mak, Tim N.;Brueggemann, Holger
痤疮丙酸杆菌是一种经常与炎症性疾病如寻常痤疮相关的皮肤病。尽管基因组序列的可用性痤疮丙酸杆菌的功能研究是稀缺的，由于缺乏这种细菌的遗传操作的方法。在这里，我们提出了一种插入诱变的方法，用于特定的痤疮丙酸杆菌基因的失活。通过采用同源重组，目的基因可以被破坏并用红霉素抗性盒替换。我们使用这种方法产生camp 2（PPA 0687）和camp 4（PPA 1231）的敲除突变体，编码具有预测的共溶血活性的CAMP因子同源物。使用特异性抗CAMP 2/CAMP 4血清通过PCR和Western印迹实验证实了这两个基因的成功失活。Delta camp 2而不是Delta camp 4突变体在与绵羊红细胞的CAMP反应中表现出降低的溶血活性，表明CAMP 2是痤疮丙酸杆菌的主要活性共溶血因子。CAMP因子的生物学相关性仍然不清楚，因为camp 2或camp 4的破坏没有显著改变HaCaT细胞对痤疮丙酸杆菌的转录组应答。本文提出的插入突变方法将有助于痤疮丙酸杆菌的进一步研究。(C)2010 Elsevier B. V.保留所有权利。
P. acnes is a skin commensal that is frequently associated with inflammatory diseases such as acne vulgaris. Despite the availability of the genome sequence functional studies on P. acnes are scarce due to a lack of methods for genetic manipulation of this bacterium. Here we present an insertional mutagenesis approach for the inactivation of specific P. acnes genes. The gene of interest can be disrupted and replaced with an erythromycin-resistance cassette by employing homologous recombination. We used this method to generate knock-out mutants of camp2 (PPA0687) and camp4 (PPA1231), encoding CAMP factor homologs with predicted co-hemolytic activities. The successful inactivation of the two genes was confirmed by PCR and Western blotting experiments using specific anti-CAMP2/CAMP4 sera. The Delta camp2 but not the Delta camp4 mutant exhibited reduced hemolytic activity in the CAMP reaction with sheep erythrocytes, indicating that CAMP2 is the major active co-hemolytic factor of P. acnes. The biological relevance of the CAMP factors remains unclear as disruption of camp2 or camp4 did not significantly alter the transcriptome response of HaCaT cells to P. acnes. The here presented insertional mutagenesis approach will facilitate future studies on P. acnes. (C) 2010 Elsevier B.V. All rights reserved.