Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics
Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics
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DOI：
10.1046/j.1462-2920.2003.00532.x
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发表时间：
2003-12-01
影响因子：
5.1
通讯作者：
Eberl, L
中科院分区：
文献类型：
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作者：
Arevalo-Ferro, C;Hentzer, M;Eberl, L
革兰氏阴性细菌铜绿假单胞菌是一种机会性人类病原体，其导致免疫功能低下患者的严重医院感染，并且是囊性纤维化的主要病原体。该细菌利用两个相互关联的群体感应（QS）系统，这依赖于N-酰基高丝氨酸内酯（阿勒）信号分子，控制毒力因子的表达和生物膜的发展。在这项研究中，我们比较了PAO 1亲本菌株的细胞内，细胞外和表面蛋白组分的蛋白质模式的同基因lasI rhlI双突变体通过二维凝胶电泳（2-DE）。该分析表明，在所有检测到的蛋白质点中，23.7%的蛋白质点的强度在两个菌株之间差异超过2.5倍。我们只考虑那些真正QS调节的蛋白质点，这些蛋白质点在没有信号分子的情况下在突变体中发生变化，但在培养基中补充AHL时被拯救到野生型情况。通过MALDI-TOF肽图对这些蛋白质点进行表征。27个蛋白质被确定为以前报道的阿勒控制，其中几个良好的特点毒力因子。对于鉴定的蛋白质之一，丝氨酸蛋白酶PrpL，建立生物化学测定以验证该因子的表达确实是QS调节的。此外，它表明，群体感应阻断剂C-30特异性地干扰表面组分的67%的AHL控制的蛋白质点的表达，证实了该化合物的高特异性。重要的是，20个新的QS调节蛋白被确定，其中许多涉及铁的利用，这表明群体感应和铁调节系统之间的联系。这些蛋白质中的两种，BXR和HasAp，是铜绿假单胞菌中存在的两种不同的血液摄取系统的组分。在协议的发现，这两种蛋白质的QS级联正调控，我们表明，lasI rhlI双突变体生长不良，血红蛋白作为唯一的铁源相比，野生型。这些结果将血红蛋白利用添加到铜绿假单胞菌中通过QS控制的表型列表中。AHL调节蛋白相对于调节基因数量的惊人高数量表明群体感应控制也通过转录后机制起作用。为了加强这一假设，我们研究了群体感应的作用，在翻译后修饰的HasAp，细胞外蛋白所需的自由和血红蛋白结合血红素的摄取。
The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem.