P. aeruginosa PAO1 Degrades IFNλ by Secretion of Proteases
Since P. aeruginosa is known to secret various proteases, we next tested whether CM of P. aeruginosa is able to degrade recombinant IFNλ directly (Figure 3A). CM of both strains, PAO1 and Boston, degraded recombinant IFNλ but PAO1 was much more efficient with complete destruction of added IFNλ even after 1h of incubation. To elucidate the mechanism of action, we heat treated (10 min, 95°C) or filtered (molecular weight cut-off 30 kDa) the conditioned medium from both P. aeruginosa strains (Figures 3B,C). Both treatments were able to restore MX1 and OAS1 induction by RSV in PAO1-conditioned cells indicating that the inhibitory factors in PAO1-CM might be proteins of <30 kDA. We next analyzed the general secretion of proteases by both strains using casein degradation (Figure 3D) or zymography (Figure 3E). Although both strains showed protease secretion, protease activity was much higher in PAO1 (Figure 3D). Zymography also showed that PAO1 had higher AprA and LasA/B activity (as determined by size) whereas the Boston strain only expressed low amounts of AprA (Figure 3E).
Figure 3  P. aeruginosa secretes a protease degrading directly IFNλ. (A) Recombinant IFNλ was incubated in conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium at 37°C for 1, 2, or 4 h. Subsequently, levels of IFNλ were analyzed by western blot. (B) Conditioned media was heated for 10 min at 95°C. Subsequently BEAS2B cells were pretreated with heat treated or non-treated conditioned medium of P. aeruginosa (PAO1 and Boston) or with control medium and subsequently infected with RSV. Induction of MX1 mRNA was analyzed by qRT-PCR after 14 h post-infection. (C) Conditioned media was filtered using a cutoff of 30 kDa. Subsequently BEAS2B cells were pretreated with the fraction larger than 30 kDa, smaller than 30 kDa, non-filtered conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium and subsequently infected with RSV. Induction of MX1 mRNA was analyzed by qRT-PCR after 14 h post-infection. (D) Protease activity in conditioned medium of P. aeruginosa (PAO1 and Boston) was assessed using skim-milk agar. (E) Elastase activity in conditioned medium of P. aeruginosa (PAO1 and Boston) was assessed by zymography. All experiments n = 3–4, ANOVA and Bonferroni post-test was used for statistical analysis. 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.