Active Efflux and Diffusion Are Involved in Transport of Pseudomonas aeruginosa Cell-to-Cell Signals

Many gram-negative bacteria communicate by N-acyl homoserine lactone signals called autoinducers (AIs). In Pseudomonas aeruginosa, cell-to-cell signaling controls expression of extracellular virulence factors, the type II secretion apparatus, a stationary-phase sigma factor (ς(s)), and biofilm differentiation. The fact that a similar signal, N-(3-oxohexanoyl) homoserine lactone, freely diffuses through Vibrio fischeri and Escherichia coli cells has led to the assumption that all AIs are freely diffusible. In this work, transport of the two P. aeruginosa AIs, N-(3-oxododecanoyl) homoserine lactone (3OC(12)-HSL) (formerly called PAI-1) and N-butyryl homoserine lactone (C(4)-HSL) (formerly called PAI-2), was studied by using tritium-labeled signals. When [(3)H]C(4)-HSL was added to cell suspensions of P. aeruginosa, the cellular concentration reached a steady state in less than 30 s and was nearly equal to the external concentration, as expected for a freely diffusible compound. In contrast, [(3)H]3OC(12)-HSL required about 5 min to reach a steady state, and the cellular concentration was 3 times higher than the external level. Addition of inhibitors of the cytoplasmic membrane proton gradient, such as azide, led to a strong increase in cellular accumulation of [(3)H]3OC(12)-HSL, suggesting the involvement of active efflux. A defined mutant lacking the mexA-mexB-oprM-encoded active-efflux pump accumulated [(3)H]3OC(12)-HSL to levels similar to those in the azide-treated wild-type cells. Efflux experiments confirmed these observations. Our results show that in contrast to the case for C(4)-HSL, P. aeruginosa cells are not freely permeable to 3OC(12)-HSL. Instead, the mexA-mexB-oprM-encoded efflux pump is involved in active efflux of 3OC(12)-HSL. Apparently the length and/or degree of substitution of the N-acyl side chain determines whether an AI is freely diffusible or is subject to active efflux by P. aeruginosa.

许多革兰氏阴性菌（Gram-negative bacteria）通过被称为自动诱导物（autoinducers，AIs）的N-酰基高丝氨酸内酯（N-acyl homoserine lactone）信号进行细胞间通讯。在铜绿假单胞菌（Pseudomonas aeruginosa）中，细胞间信号传导调控细胞外毒力因子、II型分泌装置（type II secretion apparatus）、稳定期σ因子（ς(s)）以及生物被膜分化的表达。此前研究发现，类似信号分子N-(3-氧己酰基)高丝氨酸内酯（N-(3-oxohexanoyl) homoserine lactone）可自由穿过费氏弧菌（Vibrio fischeri）与大肠杆菌（Escherichia coli）的细胞，这使得学界曾普遍认为所有自动诱导物均为可自由扩散的分子。本研究以氚标记的信号分子为工具，探究了铜绿假单胞菌的两种自动诱导物——N-(3-氧十二烷酰基)高丝氨酸内酯（N-(3-oxododecanoyl) homoserine lactone，3OC(12)-HSL，旧称PAI-1）与N-丁酰基高丝氨酸内酯（N-butyryl homoserine lactone，C(4)-HSL，旧称PAI-2）的跨膜转运过程。当将[³H]C(4)-HSL加入铜绿假单胞菌的细胞悬浮液后，细胞内该分子的浓度在30秒内即达到稳态，且与胞外浓度近乎相等，这与可自由扩散分子的特性相符。与之形成对比的是，[³H]3OC(12)-HSL则需要约5分钟才能达到稳态，且细胞内浓度为胞外水平的3倍。向体系中加入细胞质膜质子梯度抑制剂（如叠氮钠）后，细胞内[³H]3OC(12)-HSL的积累量显著升高，这提示该分子的转运涉及主动外排过程。一株缺失mexA-mexB-oprM编码的主动外排泵的工程突变株，其细胞内[³H]3OC(12)-HSL的积累水平与经叠氮钠处理的野生型细胞相近。外排相关实验进一步验证了这一观测结果。本研究结果表明，与C(4)-HSL不同，铜绿假单胞菌的细胞对3OC(12)-HSL不具备自由通透性。取而代之的是，mexA-mexB-oprM编码的外排泵介导了3OC(12)-HSL的主动外排过程。显然，N-酰基侧链的长度和/或取代程度决定了自动诱导物是可自由扩散，还是需要通过铜绿假单胞菌的主动外排系统进行转运。