Pseudomonas aeruginosa H3-T6SS Combats H 2 O 2 Stress by Diminishing the Amount of Intracellular Unincorporated Iron in a Dps-Dependent Manner and Inhibiting the Synthesis of PQS.

The type VI secretion system (T6SS), a protein translocation nanomachine, is widely distributed in Gram-negative bacteria and delivers effectors directly into target cells or the extracellular environment to help the bacteria gain a competitive fitness advantage and promote bacterial survival in harmful environments. In this study, we demonstrated that the synthesis of the Pseudomonas quinolone signal (PQS) in Pseudomonas aeruginosa PAO1 was inhibited by the H3-T6SS gene cluster under iron-rich conditions, and that this inhibition was relieved under iron starvation conditions. Conversely, PQS differentially regulated the expression of the H3-T6SS structural genes and the effector protein gene tseF . The expression of tseF was inhibited by PQS, while the expressions of the H3-T6SS structural genes were positively regulated by PQS. Further studies showed that the H3-T6SS was involved in the resistance of P. aeruginosa to oxidative stress caused by hydrogen peroxide (H 2 O 2 ). Interestingly, H3-T6SS expression was neither induced by H 2 O 2 stress nor regulated by OxyR (a global anti-oxidative transcriptional regulator) but was positively regulated by RpoS (a major transcription regulator of the stress response). In addition, we found that the clpV3 (a structural gene of H3-T6SS) mutation resulted in upregulation of two proteins related to PQS synthesis and many proteins related to oxidative stress resistance, while the expression of some iron storage proteins, especially Dps, were significantly downregulated. Furthermore, the clpV3 mutation led to an increase in the intracellular free Fe 2+ content of P. aeruginosa . Further studies showed that both the PQS deficient mutation and overexpression of dps effectively restored the H 2 O 2 sensitive phenotype of the H3-T6SS mutant. Finally, we proposed the following model of H3-T6SS-mediated resistance to H 2 O 2 stress in P. aeruginosa . H3-T6SS not only reduces the intracellular free Fe 2+ level by upregulating the expression of ferritin Dps, but also inhibits the synthesis of PQS to mediate the resistance of P. aeruginosa to H 2 O 2 stress. This study highlights the important role of H3-T6SS in the ability of P. aeruginosa to combat H 2 O 2 stress and provides a perspective for understanding the stress response mechanism of bacteria.