Bacterial Resistance to Antisense Peptide Phosphorodiamidate Morpholino Oligomers

Abstract

cPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF) 3 RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is ␤-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF) 3 RXB-AcpP was 2.5 M (14 g/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF) 3 RXB-AcpP was 4 ؋ 10 ؊7 mutations/cell division. A spontaneous (RFF) 3 RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF) 3 RXB-AcpP was 40 M (224 g/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF) 3 RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF) 3 RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF) 3 RXB-AcpP. Deletion of sbmA caused resistance to (RFF) 3 RXB-AcpP. We conclude that resistance to (RFF) 3 RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF) 3 R-XB PPMOs may be transported across the plasma membrane by SbmA.

Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

“…MccJ25 is a 21-amino-acid posttranslationally modified peptide that adopts a lasso structure, whereas MccB17 and bleomycin contain thiazole heterocycles (MccB17 also contains oxazoles). Moreover, recent findings suggest the involvement of SbmA also in the internalization of antisense peptide phosphorodiamidate morpholino oligomers and PNAs (13,14), expanding the spectrum of substrates transported by this protein. More importantly, a high level of promiscuity to export structurally unrelated peptides is a feature typical of multidrug transporters (28,29).…”

“…SbmA is involved in the uptake of the lasso peptide microcin J25 (4) and is also required for the direct uptake of the eukaryotic proline-rich antimicrobial peptides Bac7 (5) and PR-39 (12). Mutants of SbmA were recently found to be resistant to the antisense peptide phosphorodiamidate morpholino oligomers, suggesting that these molecules may be transported across the plasma membrane by SbmA (13). In addition, a role of this protein in the uptake of peptide nucleic acid (PNA) conjugates in E. coli has been proposed, indicating SbmA as the first protein identified to recognize PNA (14).…”

Mechanism of Escherichia coli Resistance to Pyrrhocoricin

“…The natural function of SbmA is known to be hijacked by different classes of exogenous antimicrobial peptides, including mi- crocins B17 and B25 secreted by some enteric bacteria (31,44), the glycopeptide bleomycin (45), antisense peptide phosphorodiamidate morpholino oligomers (46), and peptide nucleic acids (47). The peptides described above kill bacteria without membrane lysis; once inside the cytoplasm, they act on one or more intracellular targets.…”

Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers

“…The only example of resistance to PPMOs thus far was shown to be due to the transporter SbmA in E. coli. Interestingly, SbmA mutants were resistant to RFF-linked PMOs but retained activity to the same PMO linked to an RXR-based peptide (38). In addition, PNAs have been shown to be transported by SbmA in E. coli; however, certain peptide-conjugated PNAs, including (RXR) 4 XB, enter through an SbmA-independent mechanism (39).…”