Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression

Abstract

Insights into the host factors and mechanisms mediating the primary host responses after pathogen presentation remain limited, due in part to the complexity and genetic intractability of host systems. Here, we employ the model Drosophila melanogaster to dissect and identify early host responses that function in the initiation and progression of Pseudomonas aeruginosa pathogenesis. First, we use immune potentiation and genetic studies to demonstrate that flies mount a heightened defense against the highly virulent P. aeruginosa strain PA14 when first inoculated with strain CF5, which is avirulent in flies; this effect is mediated via the Imd and Toll signaling pathways. Second, we use whole-genome expression profiling to assess and compare the Drosophila early defense responses triggered by the PA14 vs. CF5 strains to identify genes whose expression patterns are different in susceptible vs. resistant host-pathogen interactions, respectively. Our results identify pathogenesis- and defense-specific genes and uncover a previously undescribed mechanism used by P. aeruginosa in the initial stages of its host interaction: suppression of Drosophila defense responses by limiting antimicrobial peptide gene expression. These results provide insights into the genetic factors that mediate or restrict pathogenesis during the early stages of the bacterial-host interaction to advance our understanding of P. aeruginosa-human infections.

Fig. 1.

Immune potentiation elicited by initial inoculation with CF5 cells protects flies from subsequent infection with virulent PA14 cells. Infection with CF5 at 3, 6, 12, or 24 h before PA14 infection delays mortality and increases fly survival. This protection maximizes at 6 h after primary inoculation and lasts for >24 h; P < 0.0001 (Table 4).

Fig. 2.

P. aeruginosa-triggered immune potentiation requires both the Imd and Toll signaling pathways. Fly survival after preinoculation with CF5 of rel^- (A) and spz^- (B) mutant flies and of a imd^-;spz^- double mutant (C). P < 0.0001 for A and B and P > 0.2 for C (Table 4).

Fig. 3.

Relative AMP levels induced in PA14- and CF5-infected flies over time. Values for selected AMPs correspond to PA14 vs. mock injury (black bars) and CF5 vs. mock injury (gray bars) as percent activation of expression levels at 1, 6, and 12 h after inoculation. Several differences between PA14 and CF5 infection are seen: Attacin A (AttA) and Cecropin A1 (CecA1) show prominent differences at 1 and 6 h; Diptericin (Dipt) and Drosomycin (Drom) at 1 h; Drosocin (Drc) at all three time points; and Defensin (Def) at 1 and 12 h. Note the negative percent change in expression levels of Drc and Drom at 1 h after PA14 infection, suggesting they are suppressed by PA14.

Fig. 4.

Hierarchical cluster analysis of the 241 differentially expressed DGs and PGs. Each gene is clustered across naive, sterile-injury, PA14-infection, and CF5-infection conditions and is presented colorwise in a single row. The darker the color, the lower (blue) or the higher (red) the expression vs. the mean expression level. Groups of similarly affected genes are defined by each cluster. DG clusters A–C (DGA–C) predominantly contain immunity genes (green bars), the DGD cluster contains predominantly tissue reconstruction genes (blue bar), and the DGE cluster (red bar) contains stress proteins. The black bar marks the PGs. The complete list of gene identities is presented in Table 3.

Fig. 5.

Highly virulent PA14 cells suppress AMP expression. (A) Fly survival after infection with PA14 and PA14-isogenic mutant D12. (B) Relative expression of 14 AMP genes in flies infected with PA14 vs. avirulent PA14-isogenic mutant D12 cells. Cecropin C′ refers to the splice variant of the gene. Color representations are the same as in Fig. 4. (C) Overexpression of the AMP genes AttA, CecA, Def, and Drc, but not Dipt or Drom, renders flies significantly less susceptible to PA14 infection (P < 0.0001; Table 4).