Human Host Defense Peptide LL-37 Stimulates Virulence Factor Production and Adaptive Resistance in Pseudomonas aeruginosa

Nikola Strempel,Olivier Lesouhaitier,Julien Vieillard,Gerald Brenner-Weiss,Robert Geffers,Anke Neidig,Joerg Overhage,Michael Nusser,Suzanne Fleiszig

doi:10.1371/journal.pone.0082240

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.

Highlights

Pseudomonas aeruginosa is a widespread Gram-negative water and soil bacterium, which is, in addition, one of the most important opportunistic human pathogens causing severe infections in immunocompromised persons, such as burn wound, catheter and urinary tract infections or chronic pneumonia in cystic fibrosis (CF) patients [1]
Determined OD600 values were comparable in treated P. aeruginosa samples and control cultures, ranging between 0.7 and 1.2 after the incubation time of 2 h, which confirmed that growth was not inhibited by applied LL-37 concentrations
We demonstrate that both, virulence factor production as well as the adaptive resistance against fluoroquinolone and aminoglycoside antibiotics, are considerably stimulated by the host defense peptide LL-37, when applied at concentrations that are comparable to the high LL-37 levels found in body fluids at sites of inflammation

Summary

Introduction

Pseudomonas aeruginosa is a widespread Gram-negative water and soil bacterium, which is, in addition, one of the most important opportunistic human pathogens causing severe infections in immunocompromised persons, such as burn wound, catheter and urinary tract infections or chronic pneumonia in cystic fibrosis (CF) patients [1]. Multiple virulence factors have been identified to affect the pathogenicity of P. aeruginosa These factors comprise the expression of extracellular appendices flagella, type IV pili and type III secretion systems, the production of alginate and lipopolysaccharide (LPS) and the synthesis of secreted exocompounds such as proteases (e.g. elastase) and other enzymes, toxins, phenazines, rhamnolipids, hydrogen cyanide (HCN) and quorum sensing molecules (e.g. 4-quinolone PQS) [4,5]. P. aeruginosa virulence is controlled by a highly complex and in large parts not fully understood signaling network including the Las, Rhl and PQS quorum sensing systems which induce the expression of various virulence factors e.g. in response to high cell densities or other external stimuli like iron limitation [6]. Activation of mentioned two-component systems induces the expression of the LPS modifying operon arnBCADTEFugd, leading to a reduced net charge of LPS due to the addition of 4-aminoarabinose to lipid A, which impairs the self-promoted uptake of cationic compounds across the outer membrane and thereby enhances tolerance to these compounds [4,14]

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