Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids

Abstract

Gene function studies in bacteria lag behind progress in genome sequencing, in part because current reverse genetics technology based on genome disruption does not allow subtle control of gene expression for all genes in a range of species. Essential genes and clustered regions are particularly problematic. Antisense technology offers an attractive alternative for microbial genomics. Unfortunately, bacteria lack RNAi mechanisms and conventional oligonucleotides are not taken up efficiently. However, in Escherichia coli, efficient and gene-specific antisense knock down is possible using antisense peptide nucleic acids (PNAs) attached to carrier peptides (KFFKFFKFFK). Carrier peptides can enter a range of microbial species, and in this study we asked whether peptide-PNAs could mediate antisense effects in Staphylococcus aureus. Using low micromolar concentrations we observed dose- and sequence-dependent inhibition of the reporter gene gfp and endogenous gene phoB. Also, antisense peptide-PNAs targeted to the essential genes fmhB, gyrA, and hmrB were growth inhibitory. Control peptide-PNAs were much less effective, and sequence alterations within the PNA and target mRNA sequences reduced or eliminated inhibition. Further development is needed to raise the antibacterial potential of PNAs, but the present results show that the approach can be used to study gene function and requirement in this important pathogen.

Novel Anion Liposome-Encapsulated Antisense Oligonucleotide Restores Susceptibility of Methicillin-ResistantStaphylococcus aureusand Rescues Mice from Lethal Sepsis by TargetingmecA

“…The loss of effectiveness of commonly used antibacterial antibiotics such as ␤-lactam drugs further adds to the dilemma, calling for the immediate need to develop a new strategy and agents that can specifically combat MRSA infection. The concept of using antisense antibiotics is innovative, and antisense antibiotics have been proposed as a new hope for bacterial infection therapy through the targeting of specific genes in bacteria (10,29).…”

Peptide Nucleic Acid Antisense Oligomer as a Therapeutic Strategy against Bacterial Infection: Proof of Principle Using Mouse Intraperitoneal Infection

“…Methods have been recently developed to overcome this membrane barrier resistance. For example, some cell-permeabilizing peptides, when covalently attached to ODNs, enable the resulting peptide-ODN conjugate to traverse the outer membrane and have been shown to greatly improve antisense activity (10,13,23). Negatively charged liposomes have been successfully used to encapsulate and deliver ODNs into bacterial cells (7).…”

Antisense effect of pyrrolidine-based oxy-peptide nucleic acids in Escherichia coli

“…To facilitate internalization by an E. coli cell, the POPNA was conjugated to the C-terminus of a cellpenetrating peptide (N′-KFFKFFKFFK-C′) [10][11][12] . We performed the LacZ reporter assay 13,14 to examine the antisense effect of the conjugated POPNA.…”