No abstract

Thanatin targets protein-protein interactions between LptA and LptD in the periplasmic bridge required for LPS transport.

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel ␤-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The discovery of novel antibiotics with new mechanisms of action is an important goal in antibiotic research to combat infections caused by multidrug-resistant bacteria, in particular Gram-negative microorganisms with their unique asymmetric outer membrane (OM) 3 (1). Naturally occurring cationic host defense peptides that form part of the innate immunity in many organisms have recently attracted great interest in the search for new clinically useful antibiotics (2). We explore here an approach to antibiotic discovery based upon host defense peptide-inspired macrocyclic peptidomimetics as a potential source of antibiotics displaying target selectivity and potency not seen with naturally occurring host defense peptides. In previous work (3), we described a family of macrocyclic ␤-hairpin peptidomimetics with potent and selective antimicrobial activity against Pseudomonas spp., which were shown to have a novel mechanism of action targeting the ␤-barrel outer membrane protein LptD in Pseudomonas aeruginosa and inhibiting its key lipopolysaccharide (LPS) transport function in OM biogenesis (4). We report here the discovery of a new conformationally constrained ␤-hairpin peptidomimetic (called JB-95) having potent antimicrobial activity against a panel of Grampositive and Gram-negative bacteria and, in particular, against Escherichia coli. JB-95 shows minimal inhibitory concentrations (MICs) of ϳ0.25 g/ml against E. coli, including many multidrug-resistant clinical strains. We report the solution structure of JB-95 and investigations into its mechanism of action against E. coli. Experimental ProceduresPeptide Synthesis-The methods for synthesis and characterization of all peptides have been described previously (5). JB-95 was of Ͼ95% purity by analytical reverse phase HPLC ( Antibacterial Assays-MICs were determined in microtiter plates in Mueller-Hinton-I (MH-I) medium using the broth

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections. Due to its high intrinsic and adaptive resistance to antibiotics, infections caused by this organism are difficult to treat and new therapeutic options are urgently needed. Novel peptidomimetic antibiotics that target outer membrane (OM) proteins have shown great promise for the treatment of P. aeruginosa infections. Here, we have performed genome-wide mutant fitness profiling using transposon sequencing (Tn-Seq) to identify resistance determinants against the recently described peptidomimetics L27-11, compounds 3 and 4, as well as polymyxin B2 (PMB) and colistin (COL). We identified a set of 13 core genes that affected resistance to all tested antibiotics, many of which encode enzymes involved in the modification of the lipopolysaccharide (LPS) or control their expression. We also identified fitness determinants that are specific for antibiotics with similar structures that may indicate differences in their modes of action. These results provide new insights into resistance mechanisms against these peptide antibiotics, which will be important for future clinical development and efforts to further improve their potency.

In Fig. 1 of this Article, the label for residue 24 in peptide 4 should have been 'Thr' instead of 'Dab', and in peptide 5, the labels for the 'N' and 'C' termini should have been reversed (with the C terminus next to Dab, and the N terminus next to Trp). This figure has been corrected online.