Quorum sensing and c-di-GMP-dependent alterations in gene transcripts and virulence-associated phenotypes in a clinical isolate of Aeromonas hydrophila

Recently, we demonstrated that the LuxS-based quorum sensing (QS) system (AI-2) negatively regulated the virulence of a diarrheal isolate SSU of Aeromonas hydrophila, while the ahyRI-based (AI-1) N-acyl-homoserine lactone system was a positive regulator of bacterial virulence. Thus, these QS systems had opposing effects on modulating biofilm formation and bacterial motility in vitro models and in vivo virulence in a speticemic mouse model of infection. In this study, we linked these two QS systems with the bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) in the regulation of virulence in A. hydrophila SSU. To accomplish this, we examined the effect of overproducing a protein with GGDEF domain, which increases c-di-GMP levels in bacteria, on the phenotype and transcriptional profiling of genes involved in biofilm formation and bacterial motility in wild-type (WT) versus its QS null mutants. We provided evidence that c-di-GMP overproduction dramatically enhanced biofilm formation and reduced motility of the WT A. hydrophila SSU, which was equitable with that of the ΔluxS mutant. On the contrary, the ∆ahyRI mutant exhibited only a marginal increase in the biofilm formation with no effect on motility when c-di-GMP was overproduced. Overall, our data indicated that c-di-GMP overproduction modulated transcriptional levels of genes involved in biofilm formation and motility phenotype in A. hydrophila SSU in a QS-dependent manner, involving both AI-1 and AI-2 systems.