A new look at the biosynthesis of 4-hydroxy-2-alkylquinolines in Pseudomonas aeruginosa

这项研究计划旨在破译4-羟基-2-烷基喹啉(HAQs)的生物合成，这是一类参与细菌之间的交流方式--“群体感应”的化合物。HAQ是由铜绿假单胞菌产生的，它是一种利用群体感应来控制毒力因子表达的机会性病原体。抑制该细菌中HAQS的产生会导致其毒力的显著降低。然而，Haqs的生物合成还没有完全阐明，为了有效地对抗这些信号化合物的产生，重要的是要找到参与其生物合成的酶和中间体的作用。虽然我们知道pqsABCDE操纵子的基因是合成HAQ所必需的，但它们编码的酶的作用只有部分知道。自从20世纪50年代中期HAQs被发现以来，人们一直认为3-酮脂肪酸是HAQs生物合成的中间产物。在初步实验中，我们发现参与其中的中间体不是3-酮脂肪酸，而是脂肪酸。这一范式的改变使人们对这一生物合成途径中涉及的一些酶的作用有了新的认识。因此，我们现在认为HAQ的生物合成是一个两步过程，其中PqsD负责将邻氨基苯甲酸与丙二酸衍生物偶联生成2-氨基苯甲酰乙酸，该中间体与脂肪酸反应生成HAQS。我们将通过纯化我们假设参与第二步的酶PqsB和PqsC来证实这一假设。我们之前还发现了一系列似乎专门抑制PqsC和/或PqsB的化合物。一旦了解了这两种酶的具体作用，我们将研究它们对这些化合物的抑制作用。我们将寻找这些化合物的类似物，它们可能比我们已经拥有的那些更有活性。我们的长期目标是开发一种新的方法，通过靶向铜绿假单胞菌的群体感应来抑制感染，这将是抗生素使用的替代方法，这种方法会导致耐药性的发展。

This research program is aimed at deciphering the biosynthesis of 4-hydroxy-2-alkylquinolines (HAQs), which are a class of compounds that are involved in "Quorum Sensing", a mode of communication between bacteria. HAQs are produced by Pseudomonas aeruginosa, an opportunistic pathogen that uses Quorum Sensing to control the expression of virulence factors. Inhibiting the production of HAQs in this bacterium leads to a considerable decrease of its virulence. However, the biosynthesis of HAQs is not fully elucidated and in order to efficiently counter the production of these signaling compounds, it is important to find the role of the enzymes and intermediates involved in their biosynthesis. Although we know that the genes from the pqsABCDE operon are required for HAQ synthesis, the role of the enzymes that they encode is only partially known. Since the discovery of HAQs in the mid 1950s, it was believed that 3-ketofatty acids are intermediates in the biosynthesis of HAQs. In preliminary experiments, we discovered that instead of 3-ketofatty acids, fatty acids were the intermediates involved. This paradigm change brings new light on the role of some of the enzymes implicated in this biosynthesis pathway. Thus we now think that HAQ biosynthesis is a two-step process in which PqsD is responsible for the coupling of anthranilic acid with a malonate derivative to produce 2-amino-benzoylacetic acid and that this intermediate reacts with a fatty acid to produce HAQs. We will confirm this hypothesis by purifying the enzymes PqsB and PqsC that we hypothesize are involved in this second step. We also previously found a series of compounds that seem to inhibit specifically PqsC and/or PqsB. Once the specific role these two enzymes will be understood, we will study their inhibition by these compounds. We will look for analogs of these compounds that might be more active than those we already have. Our long-term goal is to develop a new way to inhibit infections by P. aeruginosa by targeting their quorum sensing, which would be an alternative to the use of antibiotics, an approach which leads to the development of resistance.