Reduced Intracellular c-di-GMP Content Increases Expression of Quorum Sensing-Regulated Genes in Pseudomonas aeruginosa

Song Lin Chua, Yang Liu, Yingying Li, Hui Jun Ting, Gurjeet S. Kohli, Zhao Cai, Pipob Suwanchaikasem, Kelvin Kau Kit Goh, Sean Pin Ng, Tim Tolker-Nielsen, Liang Yang, Michael Givskov

Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP content compared with cells with higher c-di-GMP content. The induced expression of pqs and rhl QS required a functional PqsR, the transcriptional regulator of pqs QS. Next, we observed increased production of pqs and rhl-regulated virulence factors, such as pyocyanin and rhamnolipids, in P. aeruginosa cells with low c-di-GMP levels, conferring them with increased intracellular survival rates and cytotoxicity against murine macrophages. Hence, our data suggested that low intracellular c-di-GMP levels in bacteria could induce QS-regulated virulence, in particular rhamnolipids that cripple the cellular components of the innate immune system.