Human host defense peptide LL-37 stimulates virulence factor production and adaptive resistance in Pseudomonas aeruginosa

Abstract

A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor.

Figure 1. Summarized microarray data of dysregulated P. aeruginosa genes in response to LL-37.

Mid-log phase cultures of P. aeruginosa PAO1 were grown in MH broth containing either 20 µg/ml LL-37 or no LL-37 for 2 h at 37°C following RNA extraction and microarray analysis. The graph shows functions of more than 1.5-fold up- or downregulated genes according to the Pseudomonas Genome Database . Hypothetical genes are not shown.

Figure 2. Time-killing of P. aeruginosa PAO1 by antibiotics ciprofloxacin (A) or gentamicin (B) in the absence or presence of LL-37.

Mid-log phase bacterial cultures were incubated with either 20 µg/ml LL-37 (filled circles) or without LL-37 (open squares) for 2 h. Following dilution of bacterial cultures to 10⁷ cells/ml and addition of 3-fold MIC concentrations of antibiotics ciprofloxacin (0.18 µg/ml) or gentamicin (1.5 µg/ml), colony forming units at indicated time points were determined using the optimized drop plate method . Experiments were performed in triplicate. The figure shows representative results of one experiment. Error bars indicate standard deviations of 10 spots per sample plated out on two different agar plates (n = 10).

Figure 3. Quantification of metabolites elastase (A), pyocyanin (B) and PQS (C) in PAO1 WT supernatants after 21 h incubation without or with LL-37.

Mid-log phase cultures of PAO1 WT were grown in MH broth containing either 20 µg/ml LL-37 or no LL-37 (control) for 21 h at 37°C. OD₆₀₀ values after 21 h were comparable in treated samples and controls, indicating no growth inhibition by LL-37. Elastase activity (A) and pyocyanin concentration (B) in bacterial supernatants were determined photometrically. PQS levels (C) were quantified by LC-MS/MS. Boxes include median (black line), 25^th and 75^th percentiles of normalized data (n≥6). Statistical significance was calculated by Mann-Whitney-Test (**: p≤0.01, ***: p≤0.001).