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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 4, 2003 as doi:10.1096/fj.03-0449fje. |
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Center for Genomics and Bioinformatics, Karolinska Institutet, 17177 Stockholm, Sweden; and
* Department of Neurochemistry and Neurotoxicology, Stockholm University, SE-106 91, Stockholm, Sweden
2 Correspondence: Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, 17177 Stockholm, Sweden. E-mail: liam.good{at}cgb.ki.se
SPECIFIC AIM
Cell-penetrating peptides (CPPs) are membrane active sequences that translocate into mammalian cells and can be used to improve the cell uptake and biodistribution of large molecular weight drugs. CPPs are structurally similar to membrane active antimicrobial peptides involved in host innate immunity, and here we aimed to determine whether two examples of CPPs, pVEC and TP10, can enter microorganisms.
PRINCIPAL FINDINGS
1. Cell-penetrating peptides enter a range of microbial species
Two well-characterized cell-penetrating peptides, TP10 and pVEC, with attached fluorescein tags were added to cultures of growing microorganisms and the accumulation of intracellular fluorescence showed that both CPPs entered gram positive and gram negative bacteria and fungi (Fig. 1
). It is unclear how CPPs enter mammalian cells, and more than one mechanism may be involved. It is too early to comment on the details of microbial cell entry; however, the entry route is clearly peptide dependent and involves rapid surface accumulation within minutes, followed by cell entry (Fig. 1)
.
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2. Cell-penetrating peptides show potent antimicrobial properties
Most CPPs enter mammalian cells without associated membrane damage. In the case of microbial cells, which differ greatly in membrane composition, peptide entry could cause cell leakage and death. To test this possibility, TP10 and pVEC were added to growing microorganisms in standard serial broth dilution and zone of inhibition assays. TP10 was growth inhibitory against Candida albicans and Staphylococcus aureus and pVEC was growth inhibitory against Mycobacterium smegmatis at low micromolar doses. TP10 showed potent growth inhibition against S. aureus with a sharp toxicity threshold.
3. Cell-penetrating peptides can inhibit the growth of microorganisms without apparent damage to human cells
The antimicrobial properties of TP10 and pVEC suggest that these peptides, which can deliver cargo molecules into mammalian cells, may be able to kill bacteria in the presence of human cells. To test this, we first determined the hemolytic activity of the peptides against rat erythrocytes and found little evidence of cell lysis. Next, HeLa cell cultures were infected with 105 CFU/mL of noninvasive S. aureus, and TP10 was added immediately postinfection (Fig. 2
). This system can be viewed as a simple model for the growth of an extracellular pathogen in the presence of host cells. TP10 added at 6 µM prevented S. aureus growth with no apparent affects on HeLa cell growth. At higher concentrations, TP10 further reduced or eliminated S. aureus colony-forming units; at 15 µM TP10 eliminated all CFUs, confirming bactericidal effects. Therefore, TP10 can kill S. aureus at doses that do not appear to harm human cells.
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4. Cell-penetrating peptides preferentially permeabilize bacteria
Bactericidal effects of TP10 against S. aureus indicate that TP10 damages bacterial cell membranes during cell entry, but not mammalian cells. To further investigate the level of cell membrane damage caused by TP10 during cell entry, the peptide was added to S. aureus and HeLa cell cultures containing SYTOX Green, a high-affinity nucleic acid stain excluded from healthy cells but one that can enter at points of membrane damage. TP10 added at low micromolar concentrations caused a rapid fluorescence increase in S. aureus cultures but no fluorescence increase in HeLa cells cultures. pVEC permeabilized M. smegmatis, consistent with its antimicrobial effects. Therefore, while TP10 and pVEC enter microbial and mammalian cells, they permeabilize and kill microbes at concentrations that show no damage to HeLa cells.
CONCLUSIONS AND SIGNIFICANCE
Cell-penetrating peptides enter mammalian cells and distribute in vivo with surprising efficiency. Despite a structural similarly to antimicrobial peptides and their potential to improve the distribution of a variety of drugs, CPPs had not been evaluated for their ability to enter microbes, although there are examples of model CPPs that damage microbial membranes. Here we assessed the microbial cell uptake and antimicrobial properties of two well-characterized CPPs: pVEC and TP10. When present at low micromolar concentrations these peptides enter all species of microbial cells tested through rapid surface accumulation, followed by internalization (Fig. 1)
. pVEC is growth inhibitory against M. smegmatis and TP10 is growth inhibitory against S. aureus and C. albicans. Cell entry of peptides into M. smegmatis is fascinating in that mycobacteria possess the thickest biomembranes hitherto known in nature and extremely low permeability.
CPPs are a loosely defined class of peptides that display striking mammalian cell uptake properties. The uptake mechanism(s) for CPPs are unclear, and there is evidence for both energy-independent and -dependent cell translocation. Interest in CPPs stems from their capacity to improve the mammalian cell uptake and biodistribution of large molecular weight therapeutics, including other peptides and proteins. Many antimicrobials suffer from poor distribution or cell uptake. Here we show that CPPs can enter microbial cells and are antimicrobial. A combination of mammalian cell penetration without associated cell damage and antimicrobial activity is appealing for antimicrobial drug development. Although we have only begun to pursue this line of research, we used TP10 to clear a human cell culture of growing S. aureus (Fig. 2)
.
How can CPPs pass mammalian cells without causing membrane damage but kill microbial cells? It is too early to adequately address this question; however, cell entry may involve phase transitions within membranes that can be reversible or directional and dependent on peptide and membrane composition. This could explain how differences in membrane composition provide a basis for microbial killing on the one hand and eukaryotic cell passage on the other. Mammalian and microbial cell membranes differ, and cell entry and killing may involve dependent or independent mechanisms. Finally, cell entry and differential membrane damage may be a common feature of membrane active peptides (Fig. 3
).
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Antimicrobial drugs are limited by mammalian and microbial cell membranes; overcoming these barriers to infectious disease therapy is a challenge for biomedicine. Here we show that two CPPs known to efficiently enter mammalian cells—pVEC and TP10—enter microbial cells. Antimicrobial effects were observed against several pathogens, and the TP10 peptide was found to permeabilize and kill S. aureus cells without damaging human cells. These observations show that the mammalian cell penetration properties of CPPs and the well-known cell permeabilization properties of cationic antimicrobial peptides can coexist within a single cationic and amphipathic peptide. In the future it may be possible to combine the host distribution properties of CPPs with antimicrobial peptide activity to treat infections that are difficult to access with conventional strategies.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0449fje 
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) and 5 µM (•) and added to HeLa cells at 1 µM (
), 5 µM (•), and 10 µM (
). Increased fluorescence indicates cell permeabilization and death. Maximal permeabilization (
) was provided using lysostaphin (100 µg/mL) for S. aureus, 5% Triton X for M. smegmatis and 1% Triton X for HeLa cells. Background fluorescence is apparent in samples containing SYTOX Green but lacking added peptide (x).