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

Summary

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).

Abstract

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.

Protocol

Experiment 1: Extraction of termite gut protozoa under anaerobic conditions

1. 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.
2. Prepare Trager U media (Trager 1934) and adjust pH to 7.0. Sparge the filter sterilized media in the glove box with a mixture of 2.5% hydrogen, 5% carbon dioxide....

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sigmacote	Sigma-Aldrich	SL-2
EDANS	Novabiochem, EMD Millipore
Anaerobic glove box	Coy Laboratories, Inc.	Custom made
Intellus environmental controller	Percival Scientific, Inc.	I36NL
PC-10 Glass micropipette puller	Narishige International	PC-10
Glass needles (Model GD-1, 1 X 900 mm)	Narishige International	GD-1
Leitz micromanipulators	Vermont Optechs, Inc.	ACS01
Microinjector	Tritech Research, Inc.	MINJ-1
Microcaps	Drummond Scientific	1-000-0005
LEICA fluorescence imaging system	Leica Microsystems	DMRxA2
LEICA dissecting scope	Leica Microsystems	MZ16
LEICA microscope	Leica Microsystems	DMLB
Olympus dissecting scope	Olympus Corporation	SZ61