Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene
Gram-negative bacteria (GNB) cause common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance (MDR) and mortality. The emergence and spread of MDR-GNB strains limit therapeutic options and highlight the need to develop new therapeutic strategies. In this study, the peptide (RXR)4XB- and (KFF)3K-conjugated peptide nucleic acids (PPNAs) were developed to target rpoD, which encodes an RNA polymerase primary σ70 that is thought to be essential for bacterial growth. Their antimicrobial activities were tested against different clinical isolates of MDR-GNB in vitro and in infection models. The (RXR)4XB- and (KFF)3K- conjugated PNAs were bactericidal against different strains of MDR-GNB in concentration-dependent and sequence-selective manner, whereas a PPNA with a scrambled base sequence had no effect on growth. Among tested PPNAs, (RXR)4XB conjugate PPNA06 showed more potent and broad spectrum inhibition in multidrug-resistant Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, and Shigella flexneri in vitro and in vivo. The results were associated with suppression of rpoD mRNA and σ70 expression, as well as σ70 downstream regulated genes including ftsZ, mazF, prfB, rpoS, seqA, turfB and ygjD. The treatment of PPNA06 on mono- or multiple MDR-GBN infected human gastric mucosal epithelial cells demonstrated the complete inhibition on bacterial growth and no influence on morphology and growth of human cells. Also, PPNA06 did not show the induction of antibiotic resistance as compared with classical antibiotics in GNB. These findings firstly demonstrate that rpoD is potential target for developing antisense antibiotics, and indicate that peptide conjugates of anti-rpoD PNA are active against GNBs in vitro and in vivo. Our results offer a feasible strategy for treating MDR-GNB infections.
Journal: Biomaterials - Volume 33, Issue 2, January 2012, Pages 659–667