Primers used for RT-qPCR.

<div><p>The opportunistic bacterial pathogen <i>Pseudomonas aeruginosa</i> causes a wide range of infections that are difficult to treat, largely because of the spread of antibiotic-resistant isolates. Antivirulence therapy, <i>í</i>.<i>e</i>. the use of drugs that inhibit the expression or activity of virulence factors, is currently considered an attractive strategy to reduce <i>P</i>. <i>aeruginosa</i> pathogenicity and complement antibiotic treatments. Because of the multifactorial nature of <i>P</i>. <i>aeruginosa</i> virulence and the broad arsenal of virulence factors this bacterium can produce, the regulatory networks that control the expression of multiple virulence traits have been extensively explored as potential targets for antivirulence drug development. The intracellular signaling molecule diadenosine tetraphosphate (Ap4A) has been reported to control stress resistance and virulence-related traits in some bacteria, but its role has not been investigated in <i>P</i>. <i>aeruginosa</i> so far. To fill this gap, we generated a mutant of the reference strain <i>P</i>. <i>aeruginosa</i> PAO1 that lacks the Ap4A-hydrolysing enzyme ApaH and, consequently, accumulates high intracellular levels of Ap4A. Phenotypic and transcriptomic analyses revealed that the lack of ApaH causes a drastic reduction in the expression of several virulence factors, including extracellular proteases, elastases, siderophores, and quorum sensing signal molecules. Accordingly, infection assays in plant and animal models demonstrated that ApaH-deficient cells are significantly impaired in infectivity and persistence in different hosts, including mice. Finally, deletion of <i>apaH</i> in <i>P</i>. <i>aeruginosa</i> clinical isolates demonstrated that the positive effect of ApaH on the production of virulence-related traits and on infectivity is conserved in <i>P</i>. <i>aeruginosa</i>. This study provides the first evidence that the Ap4A-hydrolysing enzyme ApaH is important for <i>P</i>. <i>aeruginosa</i> virulence, highlighting this protein as a novel potential target for antivirulence therapies against <i>P</i>. <i>aeruginosa</i>.</p></div>