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 Pseudomonas aeruginosa 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 P. aeruginosa PAO1 and the Liverpool epidemic strain LESB58. A ΔrelA/ΔspoT double mutant showed decreased cytotoxicity toward human epithelial cells, exhibited reduced hemolytic activity, and caused down-regulation of the expression of the alkaline protease aprA gene in stringent response mutants grown on blood agar plates. Promoter fusions of relA or spoT to a bioluminescence reporter gene revealed that both genes were expressed during the formation of cutaneous abscesses in mice. Intriguingly, virulence was attenuated in vivo by the ΔrelA/ΔspoT double mutant, but not the relA mutant nor the ΔrelA/ΔspoT complemented with either gene. Treatment of a cutaneous P. aeruginosa 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 ΔrelA/ΔspoT infection. It was previously demonstrated by our lab that ppGpp could be targeted by synthetic peptides; here we demonstrated that spoT promoter activity was suppressed during cutaneous abscess formation by treatment with peptides DJK-5 and 1018, and that a peptide-treated relA complemented stringent response double mutant strain exhibited reduced peptide susceptibility. Overall these data strongly indicated that synthetic peptides target the P. aeruginosa stringent response in vivo and thus offer a promising novel therapeutic approach.
Highlights
Antibiotics are arguably the most successful medical intervention in human history
Stringent response mutants often exhibit attenuated virulence and persistence as well as enhanced survival rates in a variety of Gram-positive (Kazmierczak et al, 2009; Frank et al, 2014; Mansour et al, 2016; Zhu et al, 2016), mycobacterial (Klinkenberg et al, 2010; Weiss and Stallings, 2013), and Gram-negative bacterial infections (Haralalka et al, 2003; Dean et al, 2009; Vogt et al, 2011; Xu et al, 2016). These animal models provided evidence that the stringent response is crucial during infection, and we recently demonstrated that the stringent response of Gram-positive S. aureus can be effectively directly targeted in vivo [15] by small synthetic cationic antibiofilm peptides (Pletzer et al, 2016)
All organisms were cultured at 37◦C in LB (Thermo Scientific), double Yeast Tryptone, King’s B (KB), or modified synthetic cystic fibrosis medium (MSCFM) (Palmer et al, 2007; Yeung et al, 2012)
Summary
The lack of effective antibiotics would have devastating effects in medicine such as causing death after major surgeries and even minor injuries. Resistance to most antibiotics has become a global threat to public health and strategies to overcome this danger are urgently needed. Life-threatening situations with pathogens that are resistant to several classes of antibiotics are on the rise and the widespread distribution of those organisms is of great concern (Ventola, 2015). The stringent stress response pathway is a ubiquitous process among bacteria and, importantly, mammalian hosts lack this pathway (Pollard et al, 1980), indicating that the stringent response is an excellent selective drug target in bacteria
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