An important part of the project is inhibition of growth or killing of bacteria with the use of antimicrobial peptides (AMPs). In order to get an idea of the toxicity of our peptides we conducted several minimal inhibiting concentration (MIC) experiments. These MIC measurements where done on E. coli, B. subtilis andS. delphini, with the first as representative of our Gram-negative expression host, the second for the Gram-positive targets and the last for our specific target. The AMPs Signiferin, maximin H5 and magainin were chosen from literature [1,2,3] as being active against staphylococcus species but not against E. coli in order to able to use the latter as the expression host. MICs were also determined for the novel peptides we designed as described ( here).
- Grow the appropriate strains overnight and make a 1/50 to 1/100 dilution the next morning and wait for the OD at 600nm to reach 0,4-0,6.
- Make a 100µM stock of the AMP in LB medium, this solution will serve as a 100 X stock, as the expected MIC range of our AMPs lays between 5 and 40 µM.
- Then make the combinations as shown in table 1 in a 96 well plate, for the plate reader to take readings using an plate reader capable of shaking and heating to 37˚C. Taking measurements every 10 minutes.
|No cells + 1mM||40µM||30µM||25µM||20µM||15µM||10µM||7.5µM||5.0µM||2.0µM||1.0µM||Cell + No AMP|
The MIC of signiferin on S. delphini, B. subtilis and E. coli were done according to the protocol described above. S. delphini is the most sensitive for signiferin, as the MIC was determined to be 1µM (Figure 1A), no growth is seen at this concentration. The MIC for B. subtilis was determined to be 10µM (Figure 1B).
Figure 1: MICs of signiferin. 1A: signiferin on S. delphini, 1B: signiferin on B. subtilus
The expected selectivity of the chosen peptides for Staphylococcus was confirmed by these experiments, as for all the peptides for which we could determine a MIC, that MIC was the lowest on S. delphini. Maximin H5 and magainin did not give a measurable reduction in growth below 40µM (Figure 2), making us decide not to proceed testing, as modeling showed it was not possible to reach these concentrations through expression in E.coli.
Figure 2: MICs of maximin-H5 and magainin. 2A: maximin-H5 on S. delphini, 2B: magainin on S.delphini
The fact that for none of the peptides a MIC could be determined for E. coli (>150µM) further confirms the expected selectivity towards Gram-positives (Figure 3).
Figure 3: MICs of Maximin-H5, Signiferin and Magainin on E. coli
Figure 4: MICs of peptidor. 4A: peptidor on S. delphini, 4B: peptidor on B. subtilus
Figure 5:MICs of staphycine 5A:staphycine on S. delphini, 5B: staphycine on B. subtilus
Figure 6: MICs of peptidor and staphycin. 6A peptidor on E. coli, 6B staphycine on E. coli
COS-1 toxicity determination
In order to determine the toxicity of the AMPs on mammalian cells we tested the most promising peptides on COS-1 cells. COS-1 cells were chosen as they, because of their simian nature, strongly resemble human cells with respect to membrane properties and crucial cell functions and mechanisms . COS-1 cells are originally derived from Green African Monkey kidney cells in 1964, and since then have been extensively used in laboratoria around the world in molecular and cellular research on mammalian cells . As signifirin, staphycine and peptidor gave the best results in the experiments described above they were chosen to be tested with concentrations up to 150µM on COS-1 cells. Normally healthy COS-1 cells attach to the bottom of the well, showing a clear fibroblastic morphology . When they start to die they will swell and eventually detach from the bottom forming spherical cells which upon lysis leave behind cell debris.
Signifirin showed to be toxic above 100µM, whereas staphycine and peptidor turned out not to be toxic up to concentrations of 150µM (Figure 7). Two controls are shown, as peptidor and signiferin are dissolved in water whereas staphycine is in 80% DMSO. All photos shown are taken 24 hours after induction, but are representative for the photos taken 4 hours after induction. In the 150µM signiferin experiment no live cells could be observed at both 4 hours and 24 hours after induction.
Figure 7: Toxicity test of AMPs on COS-1 cells
The MIC experiments show three peptides to be effective against S. delphini, while not being toxic for E.coli or COS-1 cells. Two of the three peptides found are novel peptides designed by our modeler team. These peptides could be used in further research towards novel AMPs, as they also show a lower toxicity towards mammalian cells than the signiferin that was found in literature. Interesting to see is also that a low (<10µM) was found for staphycine on B. subtilis, showing a low toxicity on mammalian cells does not necessarily mean lowering toxicity to bacterial species, what could be thought after comparing the MIC results with the toxicity tests on COS-1. We were not able to determine a MIC value for maximin, magainin and derpini, this could be due to multiple reasons. As derpini is a novel peptide, it could be assumed it is not designed properly and it is not toxic to one of the bacterial species tested on. Another reason, which would also go up for maximin and magainin, would be the possibility of cohesion of the peptides to the plastic tubes they were dissolved and kept in. A way to determine this could be to add a certain amount, 20-80% for example, of DMSO to the peptide solution. This could help the peptide to dissolve if cohesion to the plastic is the problem. The amount of DMSO would have to be adjusted for, but this should be no problem as this was also done for staphycine.
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