Name: testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut)
Uploaded: 2010-12-29T17:30:00
Description: Watch this Scientific Journal Video about Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

We thank Dr. Allison Richard, former director of the LSU peptide facility for the fluorescent ligand synthesis, the Interdisciplinaray Center for Biotechnology Research, UF for the ligand-lytic peptide synthesis, and the Socolovsky Microscope facility for providing access to fluorescence microscopes. Funding was provided by the SERDP Exploratory Development Program (SEED) of the Department of Defense, Department of Energy and Environmental Protection Agency, the Biotechnology AgCenter Interdisciplinary Team Program and the state of Louisiana.

Experiment 1: Extraction of termite gut protozoa under anaerobic conditions
<ol>
	<li>Use a fan box (Coy Laboratories) in a glove box to constantly circulate air through a desiccant and type D catalyst Stak-Paks to control humidity and oxygen levels and eliminate uneven temperatures. Fill the glove box with a continuous stream of nitrogen for 20 to 30 min. Monitor oxygen levels with an oxygen sensor (C-squared, Inc.) for 1 h. Use nitrogen to reach and maintain anaerobic conditions when needed.</li>
	<li>Prepare Tra...

<ol>
	<li>Hansel, W., Leuschner, C., Enright, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugates+of+lytic+peptides+and+LHRH+or+&#946;CG+target+and+cause+necrosis+of+prostate+cancers+and+metastases.">Conjugates of lytic peptides and LHRH or &#946;CG target and cause necrosis of prostate cancers and metastases.</a> Mol. Cell. Endocrinol. 269, 26-33 (2007).</li><li>Husseneder, C., Collier, R. E., Bourtzis, K., Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paratransgenesis+for+termite+control.">Paratransgenesis for termite control.</a> Insect Symbiosis. 3, 361-376 (2009).</li><li>Husseneder, C., Grace, J. K., Oishi, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genetically+engineered+bacteria+(Escherichia+coli)+to+monitor+ingestion,+loss+and+transfer+of+bacteria+in+termites.">Use of genetically engineered bacteria (Escherichia coli) to monitor ingestion, loss and transfer of bacteria in termites.</a> Curr. Microbiol. 50, 119-123 (2005).</li><li>Husseneder, C., Grace, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+termite+gut+bacteria+deliver+and+spread+foreign+genes+in+termite+colonies.">Genetically engineered termite gut bacteria deliver and spread foreign genes in termite colonies.</a> Appl. Microbiol. Biotechnol. 68, 360-367 (2005).</li><li>Javadpour, M. M., Juban, M. M., Lo, W. C., Bishop, S. M., Alberty, J. B., Cowell, S. M., Becker, C. L., Mc Laughlin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+antimicrobial+peptides+with+low+mammalian+cell+toxicity.">De novo antimicrobial peptides with low mammalian cell toxicity.</a> J. Med. Chem. 39, 3107-3113 (1996).</li><li>Lai, P. Y., Tamashiro, M., Fuji, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+and+distribution+of+the+three+species+of+symbiotic+protozoa+in+the+hindgut+of+Coptotermes+formosanus+(Isoptera).">Abundance and distribution of the three species of symbiotic protozoa in the hindgut of Coptotermes formosanus (Isoptera).</a> Proc. Haw. Entomol. Soc. 24, 271-276 (1983).</li><li>Leuschner, C., Hansel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+disrupting+lytic+peptides+for+cancer+treatments.">Membrane disrupting lytic peptides for cancer treatments.</a> Curr. Pharm. Des. 10, 2299-2310 (2004).</li><li>Mutwiri, G. K., Henk, W. G., Enright, F. M., Corbeil, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+Antimicrobial+Peptide,+d-Hecate,+on+Trichomonads.">Effect of the Antimicrobial Peptide, d-Hecate, on Trichomonads.</a> J. Parasitol. 86, 1355-1359 (2000).</li><li>Trager, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+a+cellulose-digesting+flagellate,+Trichomonas+termopsidis,+and+of+certain+other+termite+protozoa.">The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other termite protozoa.</a> Biol. Bull. 66, 182-190 (1934).</li></ol>

Ligand-lytic peptides have been successfully used to effectively target and destroy cancer cells (Hansel and Leuschner 2004, Hansel et al. 2007). Based on this concept, we developed a heptapeptide ligand that binds to the surface of protozoa in the gut of Formosan subterranean termites and coupled it to a lytic peptide with the goal to destroy these obligate cellulose-digesting symbionts in the gut of termites to achieve termite control (Husseneder and Collier 2009).
We successfully ...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sigmacote</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>SL-2</td>
<td>&nbsp;</td>
<tr>
<td>EDANS</td>
<td>&nbsp;</td>
<td>Novabiochem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anaerobic glove box</td>
<td>&nbsp;</td>
<td>Coy Laboratories, Inc.</td>
<td>Custom made</td>
<td>&nbsp;</td>
<tr>
<td>Intellus environmental controller</td>
<td>&nbsp;</td>
<td>Percival</td>
<td>I36NL</td>
<td>&nbsp;</td>
<tr>
<td>PC-10 Glass micropipette puller</td>
<td>&nbsp;</td>
<td>Narishige Scientific Instrument Lab</td>
<td>PC-10</td>
<td>&nbsp;</td>
<tr>
<td>Glass needles (Model GD-1, 1 X 900 mm)</td>
<td>&nbsp;</td>
<td>Narishige Scientific Instrument Lab</td>
<td>GD-1</td>
<td>&nbsp;</td>
<tr>
<td>Leitz micromanipulators</td>
<td>&nbsp;</td>
<td>Vermont Optechs, Inc.</td>
<td>ACS01</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Tritech Research, Inc.</td>
<td>MINJ-1</td>
<td>&nbsp;</td>
<tr>
<td>Microcaps</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>1-000-0005</td>
<td>&nbsp;</td>
<tr>
<td>LEICA fluorescence imaging system</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMRxA2</td>
<td>&nbsp;</td>
<tr>
<td>LEICA dissecting scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>MZ16</td>
<td>&nbsp;</td>
<tr>
<td>LEICA microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMLB</td>
<td>&nbsp;</td>
<tr>
<td>Olympus dissecting scope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZ61</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).
In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni, Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.
In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as "Trojan Horses" to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.

Watch this Scientific Journal Video about Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean ...

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