A bistable type III secretion system is primed and loaded in Pseudomonas aeruginosa hypervirulent isolates with positively selected codon changes in exsA or exsD

Abstract

Pseudomonas aeruginosa has multiple virulence factors that promote airway infections in people with immune deficiencies. Most clinical isolates from human infections have functional type III secretion systems (T3SSs) that are subject to bistable regulation and secrete the cytotoxic effector ExoS into neutrophils. During airway infection in people with cystic fibrosis (CF), P. aeruginosa maintains bistable regulation or genetically adapts by inactivating or hyperactivating T3SS expression. CF clinical isolates with hyperactive T3SSs have codon changes in the transcriptional regulators, exsA or exsD. It is not known how these mutations: 1) arise, 2) promote hypervirulence, or 3) impact bistable T3SS regulation. To understand how these mutants arise, ~24,000 genomes in the NCBI Pathogens database were searched for positively selected mutations in exs genes based on nonsynonomous to synonymous codon change ratios. Missense and nonsense mutations were detected in exsA (47) or exsD (45) the majority of which are predicted to be positively selected. To determine how one of these codon changes promotes hypervirulence, an exsAT48I isolate was used to establish lung infections in mice engineered with a CF genotype. The exsAT48I mutant and an exsA+ control had similar lung burdens, but the former caused exacerbated weight loss and lung inflammation, suggesting immunopathology from phagocyte death as the cause of hypervirulence. Two additional hyperactive mutants were identified by screening a small collection of isolates from children with CF. Immunoblotting and fluorescence microscopy showed that hyperactive exsA or exsD mutants increased production of T3SS injectisomes and effectors, indicating they have locked on (primed and loaded) phenotypes. ExsA dimer modeling revealed that codon changes like T48I in the {beta}-barrel domain cause a conformational change that hyperactivates the transcription factor. These results define that hyperactive mutants are positively selected, hypervirulent due to lung inflammation, and have primed and loaded T3SSs.