Pseudomonas aeruginosa PAO1 Raw sequence reads

Current investigation on bacterial chronic adaptation is focused on the examination of antibiotic resistance and virulence markers. However, recent advances in next generation sequencing (NGS) shed light on adaptive evolution of the pathogens to the host environment during long-term colonization. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve the current treatment strategies against the chronic infections. Here, we employed an adaptive experimental evolution strategy to investigate the defence mechanism by the opportunistic pathogen Pseudomonas aeruginosa to reactive oxygen species (ROS), which are commonly produced by the host immune cells. Evolution of P. aeruginosa in the sub-lethal concentration H2O2 led to the emergence of “cystic fibrosis-associated” rough small colony variants (RSCV), while an antioxidant, glutathione reduced the occurrence of the RSCVs. NGS analysis showed that point mutations including in the wspF gene, which encoded a repressor of the WspR diguanylate cyclase (DGC), was responsible for the occurrence of RSCVs. The evolved RSCVs had increased intracellular cyclic-di-GMP (c-di-GMP) content, which correlated to higher resistance to phagocytosis and severity in a Caenorhabditis elegans infection model, as compared to the ancestral PAO1 strain. This was also a universal phenomenon across pathogenic species, as increasing intracellular c-di-GMP content of Burkholderia cenocepacia and Klebsiella pneumoniae by over expression of a DGC enhanced their ROS resistance. Our study suggested that ROS from the host immune cells is an essential driving force for selecting hyper-biofilm forming pathogenic variants.