Тестирование Protozoacidal активность лиганд-литических пептиды против термитов Gut Protozoa В пробирке (Protozoa культуры) и В естественных условиях (Микроинъекция в Термит кишке)
Summary
Мы существующему порядку демонстрации того, что лиганды связываются с поверхностью мембраны целлюлозы переваривания простейших в кишечнике Формозы подземных термитов помощью флуоресцентной микроскопии и лигандов, что в сочетании с литических пептидов убивать этих простейших<em> В пробирке</em> (Анаэробных простейших культуры) и<em> В естественных условиях</em> (Инъекции в кишке термитов).
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
Discussion
Лиганд-литических пептидов, которые были успешно использованы для эффективного прицельное действие способствует разрушению раковых клеток (Гензель и Лейшнер 2004, Гензель и соавт. 2007). Основываясь на этой концепции, мы разработали гептапептида лиганд, который связывается с поверхн…
Declarações
The authors have nothing to disclose.
Acknowledgements
Мы благодарим доктор Элисон Ричард, бывший директор объекта ЛГУ пептид для флуоресцентной синтез лиганда, Interdisciplinaray Центра биотехнологических исследований, UF для лиганд-литического синтеза пептидов, а объект Socolovsky микроскоп для обеспечения доступа к флуоресцентных микроскопов. Финансирование было предоставлено развития SERDP поисковые программы (SEED) из Министерства обороны, Министерства энергетики и охране окружающей среды, биотехнологии AgCenter Междисциплинарная команда программы и штата Луизиана.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Sigmacote | Sigma Aldrich | SL-2 | ||
| EDANS | Novabiochem | |||
| Anaerobic glove box | Coy Laboratories, Inc. | Custom made | ||
| Intellus environmental controller | Percival | I36NL | ||
| PC-10 Glass micropipette puller | Narishige Scientific Instrument Lab | PC-10 | ||
| Glass needles (Model GD-1, 1 X 900 mm) | Narishige Scientific Instrument Lab | GD-1 | ||
| Leitz micromanipulators | Vermont Optechs, Inc. | ACS01 | ||
| Microinjector | Tritech Research, Inc. | MINJ-1 | ||
| Microcaps | Drummond Scientific Company | 1-000-0005 | ||
| LEICA fluorescence imaging system | Leica | DMRxA2 | ||
| LEICA dissecting scope | Leica | MZ16 | ||
| LEICA microscope | Leica | DMLB | ||
| Olympus dissecting scope | Olympus | SZ61 |
Referências
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