Inhibition of the Antiviral Response by P. aeruginosa Depends on LasR and AprA
Since the previous data implied an involvement of the quorum sensing protein LasR, we again used further clinical CF isolates (CFI-V) and their counterparts with a targeted LasR mutation. In line with the reported importance of LasR in the expression of proteases (6) all LasR deficient CF isolates displayed significantly lower protease activity in CM (Figure 5A). In parallel, all CF isolates with functional LasR were able to suppress the induction of MX1 and OAS1 after RSV infection whereas their LasR deficient counterparts were not (Figure 5B). The most important proteases regulated by LasR are AprA, LasA, LasB, and PrpL. In order to investigate which LasR dependent protease is involved in the modulation of the antiviral response, we used strain PA14 and transposon mutants of PA14 with specific protease deficiency. We observed that even though all protease deficient mutants showed a decrease in total protease activity in CM (Figure 5C), only AprA-deficient PA14 was not able anymore to suppress the induction of antiviral genes MX1 and OAS1 (Figure 5D). These observations indicate that the LasR-regulated protease AprA degrades IFNλ, thereby blocking the induction of the antiviral response upon RSV infection.
Figure 5  Inhibition of antiviral response by P. aeruginosa depends on quorum sensing and the secreted protease AprA. (A) Protease activity of P. aeruginosa CF isolates and corresponding LasR deficient strains was assessed using a skim milk agar assay. (B) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa from (A) or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. (C) Protease activity of P. aeruginosa strain PA14 and corresponding protease deficient strains was assessed using skim milk agar. (D) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa from (C) or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. All experiments n = 4–5, ANOVA and Bonferroni post-test was used for statistical analysis. (D) Statistical analysis was done in comparison to the control (ctr) or to treatment with PA14 (indicated by prefix #). Significant differences were considered at *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to the control condition. n.s., not significant.