title: Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene.
abstract: 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)(4)XB- and (KFF)(3)K-conjugated peptide nucleic acids (PPNAs) were developed to target rpoD, which encodes an RNA polymerase primary σ(70) 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)(4)XB- and (KFF)(3)K- 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)(4)XB 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 σ(70) expression, as well as σ(70) 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.
authors: Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xian 710032, PR China.
authors: Hui
authors: H
authors: Bai
authors: Yu
authors: Y
authors: You
authors: Hua
authors: H
authors: Yan
authors: Jingru
authors: J
authors: Meng
authors: Xiaoyan
authors: X
authors: Xue
authors: Zheng
authors: Z
authors: Hou
authors: Ying
authors: Y
authors: Zhou
authors: Xue
authors: X
authors: Ma
authors: Guojun
authors: G
authors: Sang
authors: Xiaoxing
authors: X
authors: Luo
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D057846:Cell-Penetrating Peptides / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics; D008826:Microbial Sensitivity Tests; D020135:Peptide Nucleic Acids / Q000494:pharmacology*; D016372:RNA, Antisense / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000187:drug effects / Q000235:genetics; D012763:Shigella flexneri / Q000187:drug effects / Q000235:genetics; D012808:Sigma Factor / Q000037:antagonists & inhibitors*
chemical_list: D000900:Anti-Bacterial Agents; D057846:Cell-Penetrating Peptides; D020135:Peptide Nucleic Acids; D016372:RNA, Antisense; D012333:RNA, Messenger; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases