Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation
Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation
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DOI：
10.1074/jbc.m114.583989
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发表时间：
2014-08-08
影响因子：
4.8
通讯作者：
Federle, Michael J.
中科院分区：
文献类型：
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作者：
Aggarwal, Chaitanya;Jimenez, Juan Cristobal;Federle, Michael J.
化脓性链球菌是一种人类限制性病原体，在世界范围内造成与感染相关的大量死亡。最近的研究表明，链球菌产生并响应于几种分泌的肽信号分子（信息素），包括那些被称为短疏水肽（SHP），通过群体感应机制调节基因表达。在转运到细菌细胞中后，信息素结合到属于转录因子的Rgg家族的受体蛋白并调节其活性。以前，我们报道了生物膜调节的Rgg 2/3群体感应电路在S。化脓本研究的目的是确定无细胞培养上清液中促进生物膜形成的成熟信息素的组成。采用生物发光报告检测活性信息素培养上清液通过反相色谱分离，并使用质谱法来表征其特性。令人惊讶的是，检测到多种长度不同的SHP。使用生物发光报告子和生物膜生长测定法单独测试每个变体的合成肽，并且尽管组中的活性差异很大，但包含全长天然肽的C-末端8个氨基酸的肽是最有活性的。直接Rgg/SHP相互作用，使用荧光偏振测定法，利用FITC标记的肽配体。肽受体亲和力被认为是低至500 nM，其结合亲和力直接与观察到的生物活性。揭示天然产生的信息素沿着测定它们对同源受体的亲和力是设计化合物的重要步骤，所述化合物的目的定位于旨在通过干扰细菌通讯来治疗感染的未来疗法。
Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nM and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing com-pounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication.