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Cationic Antimicrobial <strong>Peptide</strong>s 133<br />
interaction of interest. Up to now, only the SPOT synthesis on cellulose (see also<br />
Chapter 4) has been utilized to screen large amounts of short peptides for antimicrobial<br />
activity against microbes of choice (31). We will outline in the following<br />
how the cellulose peptides were used to screen for antimicrobial activity and<br />
what results were gained from this approach.<br />
The peptides were synthesized as large spots (diameter about 0.6 cm) at high<br />
density, which can reach up to 1.9 �mol/cm 2 (32). About 1000 peptides were<br />
synthesized per 20 × 29 cm cellulose sheet. After the final side-chain deprotection<br />
step, the membrane was washed and the peptides were cleaved from<br />
the membrane (33). At this point, the peptides still remain adsorbed onto the<br />
membrane but will dissolve once a liquid is added. The peptides were then<br />
punched out with a normal single-hole puncher, and the spots were transferred<br />
into a 96-well microtiter plate. Distilled water was added in each well to dissolve<br />
the peptides.<br />
The next stage involved introducing bioluminescence into P. aeruginosa to<br />
increase the sensitivity and high throughput readout of the assay. Alternatively,<br />
unmodified microbes can be used as well (31). Bioluminescence was gained by<br />
cloning the luxCDABE in P. aeruginosa. The bioluminescence reaction involves<br />
oxidation of reduced riboflavin mononucleotide (FMNH2) and a long-chain fatty<br />
aldehyde. The genes luxCDE provide the synthesis of a fatty aldehyde and<br />
luxAB encode the luciferase subunits. In order to generate light, the bacterium<br />
must provide reduced flavin mononucleotide and molecular oxygen, and both<br />
are abundant as long as the bacteria are energized. Any event that changes<br />
the energy level of the bacteria, and therefore FMNH2 concentration, can be<br />
monitored by detection changes in light intensity. Luminescence was measured<br />
by a luminometer in a multiplex format (e.g., microtitre tray). P. aeruginosa<br />
isolate, strain H1001, with a Tn5-luxCDABE transposon inserted into the fliC<br />
(34) grows normally and expresses luminescence in an abundant and constitutive<br />
fashion. This strain, together with buffer and a carbon source, was transferred<br />
in each well of a 96-well microtiter plate, suitable for luminescence measurements.<br />
In each well of the first row of the microtiter plate, dissolved peptides<br />
(see previous paragraph) were added and a serial dilution was performed. After<br />
4 h of incubation time, the luminescence was measured, and the effect of each<br />
peptide at different concentration was evaluated. It was shown that the loss in<br />
luminescence parallels with killing of bacteria (35).<br />
Using the Spot synthesis strategy, a complete substitution analysis of a 12mer<br />
peptide was performed (35). Each amino acid of the original 12mer peptide<br />
Bac2A was substituted by all other 19 gene-encoded amino acids (Fig. 1 shows<br />
the principle of this approach). Substitution analysis is a powerful tool for investigating<br />
the importance of each amino acid for the interaction of interest, and<br />
it can help identify key amino acids or important patterns for the interaction.
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