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- Kulyte, A, et al.
(författare)
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Inhibition of Mycobacterium smegmatis gene expression and growth using antisense peptide nucleic acids
- 2005
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Ingår i: Journal of molecular microbiology and biotechnology. - : S. Karger AG. - 1464-1801. ; 9:2, s. 101-109
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Tidskriftsartikel (refereegranskat)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 <i>Escherichia coli </i>and<i> Staphylococcus aureus</i>. Here we asked whether peptide-PNAs could mediate antisense effects in <i>Mycobacterium smegmatis</i>. We first targeted the <i>gfp</i> 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-<i>gfp</i> effects. Two peptide-PNAs targeted to the essential gene <i>inhA</i> 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.
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- Kuttuva, Gunaratna R., et al.
(författare)
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Peptide-mediated delivery of green fluorescent protein into yeasts and bacteria
- 2002
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 215:2, s. 267-272
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Tidskriftsartikel (refereegranskat)abstract
- Stringent microbial cell barriers limit the application of many substances in research and therapeutics. Carrier peptides that penetrate or translocate across cell membranes may help overcome this problem. To assess peptide-mediated delivery into two yeast and three bacterial species, a range of cell penetrating and signal peptide sequences were fused to green fluorescent protein (GFP), expressed in Escherichia coli, partially purified and incubated with growing cells. Fluorescence microscopy indicated several peptides that mediated delivery. In particular, VLTNENPFSDP efficiently delivered GFP into Candida albicans and Staphylococcus aureus, while YKKSNNPFSD was most efficient for Bacillus subtilis and CFFKDEL for Escherichia coli. Carrier peptides may improve delivery of certain large molecular mass molecules into microorganisms for research and therapeutic applications.
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- Kuttuva, Gunaratna R., et al.
(författare)
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The signal peptide NPFSD fused to ricin A chain enhances cell uptake and cytotoxicity in Candida albicans
- 2003
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 301:2, s. 529-534
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Tidskriftsartikel (refereegranskat)abstract
- Microorganisms possess stringent cell membranes which limit the cellular uptake of antimicrobials. One strategy to overcome these barriers is to attach drugs or research reagents to carrier peptides that enter cells by passive permeation or active uptake. Here the short endocytosis signal peptide NPFSD was found to efficiently deliver both FITC and GFP,into Saccharomyces cerevisiae and Candida albicans with uptake into the majority of cells in a population. The NPFSD signal is itself non-toxic, but when fused to the ricin A chain toxin (RTA) the peptide enhanced both cell uptake and toxicity against C albicans, which like other yeasts is resistant to naked RTA. Cell entry required at least 1 h incubation, temperatures above 4degreesC, and an energy source, and uptake was outcompeted with free peptide. Therefore, the NPFSD peptide can carry a range of compounds into yeasts and this delivery route holds promise to enhance the activity of antifungals.
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- Nekhotiaeva, N., et al.
(författare)
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Cell entry and antimicrobial properties of eukaryotic cell-penetrating peptides
- 2003
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 17:15, s. 394-
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Tidskriftsartikel (refereegranskat)abstract
- Antimicrobial drug action is limited by both microbial and host cell membranes. Microbes stringently exclude the entry of most drugs, and mammalian membranes limit drug distribution and access to intracellular pathogens. Recently, cell-penetrating peptides (CPPs) have been developed as carriers to improve mammalian cell uptake. Given that CPP's are cationic and often amphipathic, similar to membrane active antimicrobial peptides, it may be possible to use CPP activity to improve drug delivery to microbes. Here, two CPPs, TP10 and pVEC, were found to enter a range of bacteria and fungi. The uptake route involves rapid surface accumulation within minutes followed by cell entry. TP10 inhibited Candida albicans and Staphylococcus aureus growth, and pVEC inhibited Mycobacterium smegmatis growth at low micromolar doses, below the levels that harmed human HeLa cells. Therefore, although TP10 and pVEC entered all cell types tested, they preferentially damage microbes, and this effect was sufficient to clear HeLa cell cultures from noninvasive S. aureus infection. Also, conversion of the cytotoxicity indicator dye SYTOX Green showed that TP10 causes rapid and lethal permeabilization of S. aureus and pVEC permeabilizes M. smegmatis, but not HeLa cells. Therefore, TP10 and pVEC can enter both mammalian and microbial cells and preferentially permeabilize and kill microbes.
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