Inhibition of Gene Expression and Growth by Antisense Peptide Nucleic Acids in a Multiresistant β-Lactamase-Producing Klebsiella pneumoniae Strain

Abstract

Klebsiella pneumoniae causes common and severe hospital-and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of ␤-lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF) 3 K peptide and investigated whether they could mediate gene-specific antisense effects in K. pneumoniae. No outer membrane permeabilization was observed with antisense PNAs when used alone. Antisense peptide-PNAs targeted at two essential genes, gyrA and ompA, were found to be growth inhibitory at concentrations of 20 M and 40 M, respectively. Mismatched antisense peptide-PNAs with sequence variations of the gyrA and ompA genes when used as controls were not growth inhibitory. Bactericidal effects exerted by peptide-anti-gyrA PNA and peptide-anti-ompA PNA on cells were observed within 6 h of treatment. The antisense peptide-PNAs specifically inhibited expression of DNA gyrase subunit A and OmpA from the respective targeted genes in a dose-dependent manner. Both antisense peptide-PNAs cured IMR90 cell cultures that were infected with K. pneumoniae (10 4 CFU) in a dose-dependent manner without any noticeable toxicity to the human cells.

Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency against Escherichia coli In Vitro and In Vivo

“…When antisense oligomers are added to pure bacterial cultures, they have been shown to decrease the levels of expression of reporter genes such as luciferase (5,7), activate endogenous genes such as ␤-galactosidase (7), or inhibit growth by targeting essential genes or RNA (9,10,11,14,23,28). In addition, antisense oligomers reduce infection and increase the survival of mice infected with Escherichia coli (8,21,22).…”

Inhibition of aac(6′)-Ib -mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria

“…Several groups have experimented with a variety of strategies to induce uptake of oligonucleotides and analogs into the cytosol. These strategies included liposome encapsulation, modification of the oligonucleotide by introduction of structures like hairpins, or attachment of cell-permeabilizing peptides (18,(24)(25)(26)(27)(28)(29)(30). We are presently carrying out assays using these approaches to induce penetration of LNA/DNA cooligomers into E. coli and other bacteria.…”

Antisense Antibacterials: From Proof-Of-Concept to Therapeutic Perspectives

“…Large amount of data concerning toxicity of CPP-PNA conjugates have been collected from ex vivo studies (Alksne & Projan, 2000;Kurupati & et al, 2007), in which antisense peptidePNAs cured cell cultures that were infected with bacteria in a dose-dependent manner without any noticeable toxicity to the human cells. In vivo inhibition of gene expression and growth have been observed for anti-acpP CPP-PNA conjugates in mouse intraperitoneal E. coli infection.…”

“…Validated essential genes among different bacterial species include fbpA/ fbpB/fbpC (Harth & et al, 2002(Harth & et al, , 2007 and glnA1 (Harth & et al, 2000) in Mycobacterium tuberculosis, gyrA/ompA in Klebsiella pneumonia (Kurupati & et al, 2007), inhA in Mycobacterium smegmatis (Kulyte & et al, 2005), oxyR/ahpC in Mycobacterium avium (Shimizu & et al, 2003), NPT/EhErd2 in Entamoeba histolytica (Stock & et al, 2000(Stock & et al, , 2001, gtfB in Streptococcus mutans (Guo & et al, 2006), fmhB/ gyrA/hmrB (Nekhotiaeva & et al, 2004a) and fabI (Ji & et al, 2004) in Staphylococcus aureus, 23S rRNA (Xue-Wen & et al, 2007), 16S rRNA plus lacZ/bla (Good & Nielsen, 1998), and RNAse P (Gruegelsiepe & et al, 2006) in Escherichia coli, and acpP in Burkholderia cepacia (Greenberg & et al, 2010), Escherichia coli (Deere & et al, 2005b;Geller & et al, 2003aGeller & et al, , 2003bGeller & et al, , 2005Mellbye & et al, 2009Mellbye & et al, , 2010Tan & et al, 2005;Tilley & et al, 2007) as well as Salmonella enterica serovar Typhimurium (Mitev & et al, 2009;Tilley & et al, 2006). Mellbye et al, First proof-of-principle evidence was given by White et al in 1997 for successful increasing the bactericidal activity of norfloxacin by antisense inhibiting the marRAB operon in Escherichia coli (White & et al, 1997).…”