A broad-spectrum anti-biofilm peptide that targets a cellular stress response

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate increased adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. In this work, I first identified peptide 1037, which inhibited biofilm formation in a broad-spectrum manner and proposed that this activity might be due to the effect of the peptide on biofilm-associated processes. However, these processes are not conserved in bacteria and therefore did not explain the broad-spectrum activity of the peptide. Additional screens identified 1018 as a potent anti-biofilm peptide that prevented biofilm formation and led to the eradication of mature biofilms in both Gram-negative and Gram-positive bacteria. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. To explain the broad-spectrum activity of the peptide, I hypothesized that it acted to inhibit a common stress response, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this notion, increasing (p)ppGpp synthesis 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 reduced biofilm formation. Eliminating (p)ppGpp expression after 2 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, and in vitro directly interacted with ppGpp. These results indicate that 1018 targets (p)ppGpp and marks it for degradation, thus providing an explanation for the broad-spectrum activity of the peptide. Further, the peptide was found to be synergistic with different classes of antibiotics to prevent and eradicate bacterial biofilms. Thus the peptide represents a novel strategy to potentiate antibiotic activity against biofilms. Further studies identified even more potent D-enantiomeric anti-biofilm peptides DJK-5 and DJK-6 that also prevented (p)ppGpp accumulation, were highly synergistic with conventional antibiotics and exhibited in vivo activity. Targeting biofilms represents a novel approach against drug-resistant bacterial infections.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat