title: Inhibition of Mycobacterium smegmatis gene expression and growth using antisense peptide nucleic acids.
abstract: Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria.
authors.affiliation: Center for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden. agne.kulyte@sh.se
authors.forename: Agné
authors.initials: A
authors.lastname: Kulyté
authors.forename: Natalia
authors.initials: N
authors.lastname: Nekhotiaeva
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Awasthi
authors.forename: Liam
authors.initials: L
authors.lastname: Good
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D016375:Antisense Elements (Genetics) / Q000235:genetics / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000187:drug effects; D015536:Down-Regulation / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D010088:Oxidoreductases / Q000187:drug effects; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D000900:Anti-Bacterial Agents; D016375:Antisense Elements (Genetics); D001426:Bacterial Proteins; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium