Intracellular Toxicity of Proline-Rich Antimicrobial Peptides Shuttled into Mammalian Cells by the Cell-Penetrating Peptide Penetratin

Hansen, Anders Kragh; Schäfer, Ingo; Knappe, Daniel; Seibel, Peter; Hoffmann, Ralf

doi:10.1128/aac.00585-12

Cited by 63 publications

(56 citation statements)

References 37 publications

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“…With a view toward augmenting innate immunity, HBD-2 mRNA transfection into monocyte-derived macrophages was shown to be effective against Mycobacterium tuberculosis, where the AMP and bacterium inexplicably colocalized in phagosomes (37). Similarly, insect-derived short proline-rich antimicrobial peptides (48) linked to a cell-penetrating peptide, penetratin, entered the cytoplasm of two mammalian cell lines without marked toxicity; intracellular resistance was increased against Micrococcus luteus but not against Escherichia coli (48). The insectderived peptides show homology to mammalian heat shock proteins, but concern about potential cross-reactivity with host proteins and an autoimmune response may limit their clinical utility.…”

Section: Discussionmentioning

confidence: 99%

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

et al. 2013

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bTo protect against invading bacteria, oral epithelial cells appear to use two effector antimicrobial peptides (AMPs): calprotectin (S100A8-S100A9 heterodimer [S100A8/A9]) in the cytosol and cathelicidin antimicrobial protein (CAMP) in endosomes. We sought to learn whether innate immunity might be augmented benignly to increase resistance against invasive bacteria. Epithelial cells were transiently transfected with mRNA constructs containing either the CAMP, S100A8, and S100A9 open reading frames, A8-IRES-A9 (fusion sequence), or A8-nIRES-A9 (fusion with native internal ribosome entry site [IRES] sequence). CAMP, S100A8, and S100A9 protein levels generally peaked between 16 and 44 h after mRNA transfection, depending on the construct; CAMP was processed to LL-37 over time. Following transfection with the respective mRNAs, CAMP and S100A8/A9 each independently increased resistance of epithelial cells to invasion by Listeria and Salmonella for up to 48 h; tandem S100A8/A9 constructs were also effective. Cotransfection to express S100A8/A9 and CAMP together augmented resistance, but synergy was not seen. Independent of the new proteins produced, transfection reduced cell viability after 48 h by 20%, with only 2% attributable to apoptosis. Taken together, these results suggest that epithelial cell resistance to invasive pathogens can be augmented by transient transfection of antimicrobial mRNAs into epithelial cells.

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Section: Discussionmentioning

confidence: 99%

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

et al. 2013

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“…Thus, the relative peak areas should reflect the peptide molar ratios very well. In contrast, the signal corresponding to [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Api88 increased continuously over the observation period, but its peak area was still below 10% after 2 h (Fig. 1A).…”

Section: Resultsmentioning

confidence: 82%

“…S1), which points to the C-terminally truncated peptides [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] Api88 and 1-16 Api88, respectively. The peak area of [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] Api88 increased very rapidly during the first 30 min to 70% of the initial peak area of Api88 and then showed a further slight increase to 80% within the next 30 min, before decreasing slowly afterwards (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Both classes act by the same mechanism, i.e., they freely diffuse through the outer membrane, invade the periplasmic space, enter the cytoplasm via an irreversible permease/transporter-mediated uptake (e.g., SbmA transporter), and ultimately inhibit their bacterial targets (e.g., chaperone DnaK) (11)(12)(13)(14). This mechanism is highly specific for bacteria, as PrAMPs do not enter mammalian cells (except for some cells of the immune system) and even when artificially introduced are only slightly toxic (15,16).…”

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confidence: 99%

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Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

Berthold

Czihal

Fritsche

et al. 2013

Antimicrob Agents Chemother

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dProline-rich antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their pharmaceutical potential in treating systemic bacterial infections. Besides the native peptides, several shortened, modified, or even artificial sequences were highly effective in different murine infection models. Most recently, we showed that the 18-residue-long peptide Api88, an optimized version of apidaecin 1b, was efficient in two different animal infection models using the pathogenic Escherichia coli strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal leucine residue. To improve its in vitro characteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 with Lornithine or L-homoarginine increased the serum stabilities by more than 20-fold (half-life, ϳ4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is N,N,N=,N=-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine and D-amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an E. coli strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient in vivo than Api88.

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“…Inside the cell, the AMPs (pyrrhocoricin and drosocin) interact with the target, which is mainly the 70 kDa heat shock protein DnaK. They can also interfere with DNA and RNA synthesis by binding to nucleic acids (Kragol et al, 2002;Li et al, 2006;Nicolas, 2009) and have been shown to target E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii (Mattiuzzo et al, 2007;Hansen et al, 2012).…”

Section: Working Mechanism Of Ampsmentioning

confidence: 99%

Insect proteins as a potential source of antimicrobial peptides in livestock production. A review

2017

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Intracellular Toxicity of Proline-Rich Antimicrobial Peptides Shuttled into Mammalian Cells by the Cell-Penetrating Peptide Penetratin

Cited by 63 publications

References 37 publications

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

Insect proteins as a potential source of antimicrobial peptides in livestock production. A review

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