Synthetic Peptides to Target Stringent Response-Controlled Virulence in a Pseudomonas aeruginosa Murine Cutaneous Infection Model

Abstract

Microorganisms continuously monitor their surroundings and adaptively respond to environmental cues. One way to cope with various stress-related situations is through the activation of the stringent stress response pathway. In <i>Pseudomonas aeruginosa</i> this pathway is controlled and coordinated by the activity of the RelA and SpoT enzymes that metabolize the small nucleotide secondary messenger molecule (p)ppGpp. Intracellular ppGpp concentrations are crucial in mediating adaptive responses and virulence. Targeting this cellular stress response has recently been the focus of an alternative approach to fight antibiotic resistant bacteria. Here, we examined the role of the stringent response in the virulence of <i>P. aeruginosa</i> PAO1 and the Liverpool epidemic strain LESB58. A Δ<i>relA</i>/Δ<i>spoT</i> double mutant showed decreased cytotoxicity toward human epithelial cells, exhibited reduced hemolytic activity, and caused down-regulation of the expression of the alkaline protease <i>aprA</i> gene in stringent response mutants grown on blood agar plates. Promoter fusions of <i>relA</i> or <i>spoT</i> to a bioluminescence reporter gene revealed that both genes were expressed during the formation of cutaneous abscesses in mice. Intriguingly, virulence was attenuated <i>in vivo</i> by the Δ<i>relA</i>/Δ<i>spoT</i> double mutant, but not the <i>relA</i> mutant nor the Δ<i>relA</i>/Δ<i>spoT</i> complemented with either gene. Treatment of a cutaneous <i>P. aeruginosa</i> PAO1 infection with anti-biofilm peptides increased animal welfare, decreased dermonecrotic lesion sizes, and reduced bacterial numbers recovered from abscesses, resembling the phenotype of the Δ<i>relA</i>/Δ<i>spoT</i> infection. It was previously demonstrated by our lab that ppGpp could be targeted by synthetic peptides; here we demonstrated that <i>spoT</i> promoter activity was suppressed during cutaneous abscess formation by treatment with peptides DJK-5 and 1018, and that a peptide-treated <i>relA</i> complemented stringent response double mutant strain exhibited reduced peptide susceptibility. Overall these data strongly indicated that synthetic peptides target the <i>P. aeruginosa</i> stringent response <i>in vivo</i> and thus offer a promising novel therapeutic approach.