Abstract
Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.
Highlights
Biofilms are structured multicellular communities of microorganisms associated with surfaces
Biofilms cause at least 65% of all human infections, being prevalent in device-related infections, infections on body surfaces and in chronic infections
At concentrations that had no effect on planktonic growth (Table 1), this peptide was able to potently prevent biofilm formation (Fig. 1, middle panels) and eradicate preformed (2-day old) biofilms (Fig. 1, right hand panels) formed by diverse species of Gram-negative bacteria and the Gram-positive bacterium Staphylococcus aureus
Summary
Biofilms are structured multicellular communities of microorganisms associated with surfaces They have been widely studied, in part because they cause at least 65% of all human infections, being prevalent in device-related infections, on body surfaces (skin and soft tissue, lung, bladder, endocarditis, etc.) and in chronic infections [1,2]. The cell synthesizes two small signaling nucleotides, guanosine 59-diphosphate 39-diphosphate (ppGpp) and guanosine 59-triphosphate 39-diphosphate (pppGpp), collectively denoted (p)ppGpp [3] These serve as a second messenger response that is induced by a variety of stress conditions, is highly conserved in both Gram-negative and Gram-positive species [3,4], regulates the expression of a plethora of genes [3], and is known to play a role in biofilm formation in certain species [5,6,7,8,9,10,11], some variability has been observed [6,7,9,12]
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