The Panton-Valentine leukocidin is a virulence factor in a murine model of necrotizing pneumonia.
The Panton-Valentine leukocidin is a virulence factor in a murine model of necrotizing pneumonia.
复制标题
Panton-Valentine 杀白细胞素是坏死性肺炎小鼠模型中的毒力因子。
DOI：
10.1086/651026
复制
发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Bowden,MGabriela
中科院分区：
文献类型：
--
作者：
Vandenesch,François;Couzon,Florence;Boisset,Sandrine;Benito,Yvonne;Brown,EricL;Lina,Gerard;Etienne,Jerome;Bowden,MGabriela
致编辑：我们饶有兴趣地阅读了Villaruz等人关于金黄色葡萄球菌agr位点点突变导致表型的文章，我们和其他研究者将其归因于pton - valentine leukocidin (PVL)[1]。在这篇文章中，作者指出，在我们的一项研究中使用的一种菌株b[2]“在agr中含有意想不到的突变，这极大地改变了基因表达。”作者还指出，“Labandeira-Rey等人描述的同一菌株的毒力表型受agr突变而不是PVL的影响。”Villaruz等人的结果促使我们在自己的菌株库中检查agr位点的完整性。如图1A所示，菌株RN6390、LUG855 （RN6390的Fpvl溶原）、LUG776 （LUG855 Dpvl）、LUG862 （LUG776中的pvl互补质粒）和LUG1564 （LUG776中的空载体）在血琼脂培养基中接种后显示正常的b溶血，而RN6911 （RN6390 Dagr衍生物）没有溶血。正如作者所描述的那样，溶血是“agr功能的一种常见而简单的读数”[1]。因此，与Villaruz等人描述的结果相反，Labandeira-Rey等人研究中使用的LUG855菌株显示出正常的agr表型。此外，我们对RN6390、LUG776和LUG855的agr P2-P3区域（核苷酸1724-1458）进行了测序，发现该序列与RN6390亲本株NCTC8325-4的序列相同。我们进行Northern blot分析，检测RNAIII转录本的表达，作为agr活性的直接读数（图1B）。正如预期的那样，RNAIII转录本在大多数测试菌株中以相似的水平产生，包括LUG855，但它们不存在于Dagr RN6911或其Fpvl溶原LUG856中。利用免疫印迹法对蛋白A的表达分析证实了我们之前发表的结果，即菌株LUG855与其等基因Dpvl衍生物（LUG776）及其亲本菌株（RN6390）相比，蛋白A的产生水平有所增加（图1C）。在含有编码PVL的质粒的Dpvl菌株（LUG862）中也观察到spa的过表达，但在纯载体对照（LUG1564）中未见过表达。关于PVL的产生，Villaruz等人认为“PVL在LUG855中的表达非常低，很可能是由于lukSF-PV具有很强的agr控制，而LUG855中存在缺陷。”使用特异性酶联免疫吸附试验[3]，我们测量了从选定菌株培养的废培养基中产生的lukS-PV水平（图1D）。我们的研究结果表明，LUG855在早期固定阶段产生了~ 500 ng/mL的培养基，而质粒补充菌株LUG862产生了高达5倍的培养基，可能是因为该菌株中使用的表达载体的多拷贝性质。这些水平的PVL产生与功能agr位点一致。相比之下，Dagr， Fpvl溶原菌株LUG856的PVL产量降低。因此，我们同意作者的说法，即lukSF-PV可能处于监管控制之下。
To the Editor—We read with great interest the article by Villaruz et al about a point mutation in the agr locus of Staphylococcus aureus causing phenotypes that we and other investigators had attributed to Panton-Valentine leukocidin (PVL)[1]. In this article, the authors stated that a strain used in one of our studies [2]“contained an unintended mutation in agr, which dramatically changed gene expression.” The authors also stated that “the virulence phenotype described by Labandeira-Rey et al for the same strain was influenced by the agr mutation and not PVL.” The results obtained by Villaruz et al prompted us to check the integrity of the agr locus in our own strain stocks. As shown in Figure 1A, strains RN6390, LUG855 (Fpvl lysogen of RN6390), LUG776 (LUG855 Dpvl), LUG862 (plasmid pvl complementation in LUG776), and LUG1564 (empty vector in LUG776) show normal b-hemolysis when plated in blood-agar medium, whereas RN6911 (the RN6390 Dagr derivative) showed no hemolysis. As described by the authors, bhemolysis is “a common and simple readout for agr functionality”[1]. Therefore, contrary to the results described by Villaruz et al, the LUG855 strain used in the studies of Labandeira-Rey et al displays a normal agr phenotype. In addition, we have sequenced the agr P2-P3 region (nucleotides 1724–1458) in RN6390, LUG776, and LUG855 and found that the sequences were identical to those reported for the RN6390 parental strain, NCTC8325-4. We performed Northern blot analyses to detect the expression of RNAIII transcripts as a direct readout for agr activity (Figure 1B). As expected, RNAIII transcripts were generated at similar levels in most strains tested, including LUG855, but they were not present in the Dagr RN6911 or its Fpvl lysogen, LUG856. Expression analyses of protein A by use of immunoblot assays confirmed our previously published results, in which the strain LUG855 showed an increased level of protein A production compared with its isogenic Dpvl derivative (LUG776) and its parental strain (RN6390)(Figure 1C). Overexpression of spa is also seen in the Dpvl strain complemented by a plasmid encoding PVL (LUG862), but it is not seen in the vector-only control (LUG1564). Regarding the PVL production, Villaruz et al claim that “the expression of PVL in LUG855 was very low, most likely owing to strong agr control of lukSF-PV, which was defective in LUG855.” Using a specific enzyme-linked immunosorbent assay [3], we have measured the level of lukS-PV production in the spent medium from cultures of selected strains (Figure 1D). Our results show that LUG855 produced∼ 500 ng/mL culture medium during early stationary phase, whereas the plasmid-complemented strain LUG862 produced up to 5-fold more, presumably because of the multicopy nature of the expression vector used in this strain. These levels of PVL production are consistent with a functioning agr locus. By comparison, the Dagr, Fpvl lysogen strain LUG856 showed a reduced level of PVL production. Therefore, we agree with the authors’ statement that lukSF-PV may be under agr regulatory control.