Team:TU-Delft/PeptideCharacterization

Peptide Characterization

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).

Procedure

1. 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.
2. 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.
3. 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.

Results

MIC determination

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 Maximim-H5

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 Maximim-H5 and Magainin

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 Maximim-H5, Signiferin and Magainin in E.coli

Of the novel peptides two, two showed to be active against S.delphini; staphycine and peptidor, with MICs of respectively 30 and 40µM (Figure 4 and Figure 5). The MIC of staphycine on B. subtilis however was determined to be <10µM (Figure 5B) lower than that for S. delphini. As for the peptides described above, no MIC could be determined for E.coli (Figure 6).

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 [4]. 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. 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 [5]. Normally healthy COS-1 cells attach to the bottom of the well, showing a clear fibroblastic morphology [6]. 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

References

1. V.M. Maselli, D. Bilusich, et al., Host-defence skin peptides of the Australian Streambank Froglet Crinia riparia: isolation and sequence determination by positive and negative ion electrospray mass spectrometry, Rapid Communications in Mass Spectrometry, Volume 20, Issue 5, pages 797–803, Mar 2006.
2. Lai R, Liu H, et al., An anionic antimicrobial peptide from toad Bombina maxima, Biochem Biophys Res Commun, 26;295(4):796-9, Jul 2002
3. M. Zasloff, Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor, Proc Natl Acad Sci U S A.;84(15):5449-53 Aug 1987.
4. J.F. Hancock, “COS Cell Expression”, Methods in Molecular Biology. Vol 8 pp 153-158, 1992.
5. Fred C. Jensen, Anthony J. Girardi, et al., “Infection of Human and Simian Tissue Cultures with Rous Sarcoma Virus”, Proc Natl Acad Sci U S A. 1964 July; 52(1): 53–59. Jul 1964.
6. P. Shashidharan, G.W. Huntley, et al., “Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody, Brain Research. Volume 773, Issues 1–2, Pages 139–148, Okt 1997.