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Bacterial type II toxin-antitoxin (TA) systems are abundant genetic elements and are involved in a diverse array of physiological processes. These systems encode an antitoxin protein that directly binds and effectively neutralizes the protein toxin. Recent studies have highlighted the key roles of type II TA modules in bacterial virulence and pathogenesis, but the underlying mechanisms remain unclear. Here, we investigated the antitoxin HigA in Pseudomonas aeruginosa infection. Proteomic analysis of the higA deletion strain revealed an enhanced expression of pathogenic proteins. We further verified that HigA negatively controlled T3SS and T6SS expression by directly interacting with the promoter regions of the regulators amrZ and exsA , respectively. In other words, the reversal of HigA-mediated transcriptional inhibition on stress stimulation could induce virulence genes. These findings confirm the crucial roles of the type II antitoxin in bacterial infection, which highlights the potential of the HigBA TA system as an antibacterial treatment target.

Type II toxin-antitoxin (TA) systems modulate many essential cellular processes in prokaryotic organisms. Recent studies indicate certain type II antitoxins also transcriptionally regulate other genes, besides neutralizing toxin activity. Herein, we investigated the diverse transcriptional repression properties of type II TA antitoxin PaHigA from Pseudomonas aeruginosa. Biochemical and functional analyses showed that PaHigA recognized variable pseudopalindromic DNA sequences and repressed expression of multiple genes. Furthermore, we presented high resolution structures of apo-PaHigA, PaHigA-P higBA and PaHigA-P pa2440 complex, describing how the rearrangements of the HTH domain accounted for the different DNA-binding patterns among HigA homologues. Moreover, we demonstrated that the N-terminal loop motion of PaHigA was associated with its apo and DNA-bound states, reflecting a switch mechanism regulating HigA antitoxin function. Collectively, this work extends our understanding of how the PaHigB/HigA system regulates multiple metabolic pathways to balance the growth and stress response in P. aeruginosa and could guide further development of anti-TA oriented strategies for pathogen treatment., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)