Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneumoniae strain.
Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of beta-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 muM and 40 muM, 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.
Kurupati
Prathiba
P
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore 117597, Singapore.
Tan
Kevin Shyong Wei
KS
Kumarasinghe
Gamini
G
Poh
Chit Laa
CL
D016375:Antisense Elements (Genetics) / Q000494:pharmacology*; D002478:Cells, Cultured; D002511:Cephalosporins; D002876:Chromosomes, Bacterial / Q000187:drug effects / Q000201:enzymology; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D020868:Gene Silencing / Q000187:drug effects*; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D007763:Lac Operon / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000494:pharmacology*; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D016375:Antisense Elements (Genetics); D002511:Cephalosporins; D004269:DNA, Bacterial; D007202:Indicators and Reagents; D020135:Peptide Nucleic Acids; D012329:RNA, Bacterial; D012333:RNA, Messenger; D001618:beta-Lactamases; C021720:nitrocefin