原虫シロアリ腸に対するリガンド - 溶解性ペプチドのProtozoacidal活性を試験する in vitroで（原生動物文化）と in vivoで（シロアリ後腸へのマイクロインジェクション）

The overall goal of this procedure is to demonstrate that a synthesized ligands specifically binds to the cellulose digesting protozoa in the gut of form mosen subterranean termites, and that the ligand coupled with lytic peptide kills these protozoa in vitro in anaerobic protozoa culture and in vivo by injection into the termite hind gut. This is accomplished by first extracting worker termite gut protozoa under anaerobic conditions. The second step of the procedure is to confirm binding of a fluorescent coupled ligand to termite gut protozoa in vitro.

The third step of the procedure is to show that ligand coupled litic peptide kills termite gut protozoa in vitro. The final step of the procedure is to inject fluorescent coupled ligand and ligand coupled litic peptide into the termite hind gut to confirm binding two and destruction of gut protozoa in vivo. Using protozoa culture injections into the termite hind gut and fluorescence microscopy, we show that a ligand coupled to lytic peptide binds two and efficiently kills termite gut protozoa in vitro and in vivo.

The attachment of the ligand to the lytic peptide increased the toxicity to protozoa compared to lytic peptide alone. While it reduced toxicity to non-target organisms such as e coli, most likely because the ligand did not bind the bacterial cell membrane, the loss of protozoa leads to the death of the termites in less than two weeks. The presenter technique of using LIG colitic peptides to kill the symbiotic protozoa in the gut of subterranean termites is part of the effort to develop alternative techniques for termite control.

With less reliance on chemicals without the symbions in their guts, termites die. We extract protozoa from the termite gut to demonstrate that the ligand binds to the protozoa and that the ligand lytic peptide kills the protozoa Wood particles inside the Protozoa show autofluorescence. So it's important to compare the results of protozoa treated with fluorescent ligand to the untreated protozoa.

To avoid any false positives, We inject peptides directly into the hind gut because if the peptides are fed to the termites, the protease is in the midgut and the for gut will digest them. Using farge display libraries from New England Bio Labs, we identified 19 hep peptide sequences that bind to protozoa. We chose one of the ligands with a peptide sequence that showed similarities to putative glycoproteins known from the troma brucei membrane.

After peptide synthesis, we coupled the synthesized peptide to a C terminal fluorescent probe by a solid state peptide synthesis using the E-D-A-N-S novatech resin to assess litic properties of peptide conjugates. In this procedure, we use a conjugate of the synthesized ligand and the litic peptide ate that was previously synthesized at the LSU protein facility one day prior to experimentation. Use sigma coat to ize all materials used and prevent absorption of protozoa or peptides to surfaces.

Apply an even layer and allow to dry overnight before use the following day. Prepare a fan box in a glove box to constantly circulate air at room temperature through a desiccant and type D catalyst stack packs to reduce the humidity and oxygen levels for over one hour. Fill the glove box with a continuous stream of nitrogen for 20 to 30 minutes and use additional nitrogen to maintain anaerobic conditions when needed.

Next, prepare Traeger U media and adjust the pH to seven spae the filter sterilized media in the glove box via a 200 microliter tip attached to a tube with a mixture of 2.5%hydrogen, 5%carbon dioxide, and 92.5%nitrogen for one hour to remove oxygen residues in the glove box. Once materials are ready, use forceps to obtain a termite worker from a Petri dish and submerge the whole body into a beaker filled with 70%ethanol gently S SW holding the termite with forceps for approximately 10 seconds. To remove surface contaminants, remove the worker from the ethanol and allow to dry on a clean Kim wipe for about 20 seconds.

In the glove box, prepare a siloized microscope slide with 100 microliters of Trager U media for collecting the termite gut and a tube of 900 microliters of Trager U media for collecting protozoa. Then using a dissecting microscope and a pair of sterile fine tipped forceps, hold the worker abdomen and grab the tip of the abdomen with another pair of forceps to gently pull the gut upward or downward In a 45 degree angle, try to collect four gut midgut and hind gut in one piece. Place tenter mic guts in a drop of 100 microliters of Traeger U media on a microscope.

Slide next, Pierce the hind guts with a pair of sterile fine dissecting probes to release the protozoa and gently transfer the gut contents with a 200 microliter pipette into a one milliliter micro centrifuge tube containing 900 microliters of Traeger edia. Allow five seconds for sedimentation of gut wall fragments and transfer 900 microliters of the supernatant to a fresh tube. Transfer te microliters of protozoa culture to a sigma coated microscope.

Slide and check the condition of protozoa under a microscope at 200 times magnification. Prepare control cultures of aerobic protozoa, tetra piriformis, amoeba, ug, gleaner, and paramecium, as well as an overnight culture of e coli in the culture media as recommended by the supplier immediately after obtaining protozoa, fixed termite gut protozoa any other protozoan controls and e coli with 10%formaldehyde at four degrees Celsius for 12 hours. Then centrifuge the protozoa solution at 30 times gravity for 10 minutes.

Discard the supinate and wash the pellet containing the fixed protozoa twice with one milliliter of Trager U media reus, bend the pellet in one milliliter of Traeger Edia incubate the fixed microorganisms for one to two hours with 100 microliters of a solution of synthesized ligand coupled to the fluorescent dye E-D-A-N-S. Using a final concentration of 50 micromolar ligand dissolved in water. Observe the microorganisms treated with ligands and untreated controls under a fluorescent microscope at 400 times magnification at an absorbance maximum at 341 nanometers and an emission in the blue region at 471 nanometers.

To assess the ligand conjugated litic peptide tic properties pipette six aliquots of 198 microliters of protozoa culture prepared as shown in section three into 0.5 milliliter einor tubes. Add two microliters of a 100 micromolar solution of a ligand litic peptide to half the aliquots of the termite gut protozoa culture. For a final concentration of one micromolar, add two microliters of sterile water as a control to the other half of the aliquots.

After one hour transfer 10 microliters of each protozoa culture to a slide. Compare the survival of ligand conjugated litic peptide treated protozoa to that of controls under a microscope at 200 times magnification pull needles using a NGI PC 10 micropipet puller with a dual stage heat level. To obtain a tip size of 20 to 30 microns, confirm the tip size by measuring it under a microscope.

Using a micrometer fill one needle with approximately 30 microliters of 50 micromolar fluorescently marked ligand suspended in water Using an attach syringe, fill another needle water for the control. Attach a micro pipette to a holder and a micro manipulator. Attach a needle to the injection system holder in a second micro manipulator.

Set initial injection parameters to approximately one second pulse length and 10 to 12 PSI injection. Then advance the needle slowly into the micro pipette and inject the solution using a foot pedal after injection. Remove the micro pipette and record the length of the injected solution.

Using a veer caliper, calculate the injected volume from the known parameters of the micro pipette and adjust the pressure and pulse length to expel 0.3 microliters of solution. Next, prepare 20 termites for each treatment for injection. By first squeezing the end of the abdomen with soft forceps to remove any excretion present in the rectum.

Then immobilize the termite workers by chilling on ice for five minutes while termites immobilize. Make receivers for holding termite workers by cutting off 100 microliter pipette tips. Using a scalpel blade, cut the tip to a length of 10 to 12 millimeters and use according to the size of termites and place them into a vial.

With a screw top, attach the receiver to the micro manipulator. Place a worker on a Petri dish on its dorsal side and aspirate the worker headfirst into the receiver using a nitrogen suction pump so that the terminus of the worker protrudes from the receiver holding the termite in the receiver carefully advance the filled needle using the micro manipulator to insert it into the worker anus. Inject 0.3 microliters of the fluorescently marked ligand or water control.

Place the workers injected with ligand or water into separate Petri dishes with damp filter paper and keep them overnight at 26 degrees Celsius with 78%relative humidity. After 24 hours, extubate guts from the injected termites and collect the protozoa as shown previously. Observe the ligand binding in protozoa from injected termites using fluorescence microscopy to assess lytic properties of ligand conjugated lytic peptides in living termites.

Prepare injection materials, equipment, and termites as previously done for injections and inject 0.3 microliters of 500 micromolar ligand lytic peptide solution or water as a control into the hind gut of 20 termite workers After 24 hours extra paid guts from several workers and observe gut contents under the microscope at 200 times magnification as soon as death of all protozoa in the treated termite gut is confirmed. Keep remaining treated termites and controls in Petri dishes with moist filter paper and observe mortality daily. We confirm that the ligand coupled to a fluorescent probe binds to all three species of protozoa, p grassi, h, heart, mani, and sla.

From the hind gut of four mosson subterranean termites, inde detectable densities, ligand binding occurs on the whole cell surface and is concentrated in the anterior region of the protozoa on the axis, still a sheet of microtubules and the nucleus MP grass eye. We also detected fluorescence in all tested free living aerobic protozoa species, which suggests that the ligand binds to structures generic to protozoa. While no ligand binding was observed for e coli, one micromolar of ligand litic peptide killed all three species of protozoa from the gut of omo and subterranean termite workers, and also the free living tea piriformis in vitro in less than 10 minutes while control stayed alive.

This image shows the progressive loss of membrane integrity of the termite gut protozoa treated with ligand litic peptide. No difference was observed in the number of e coli colonies between treatments of ligand lytic peptide, and water Litic peptide with no ligand, however, reduce the number of e coli colonies considerably. This suggests that attaching a ligand to the lytic peptide to some degree protects non-target microorganisms from lysis injection of 0.3 microliters of 500 micromolar ligand lytic peptide killed all three species of protozoa in the gut of foremost and subterranean termites within 24 hours, and termites died after 10 days when ligand coupled litic peptide was injected.

Protozoa and termites died three times sooner than when non ligand conjugated litic peptides were injected as shown in husen and Colia 2009, suggesting that the ligand increases protozoa cydal efficiency of litic peptides Under optimal culture conditions. All three proa species extracted from the gut of Omo. Subterranean termites should stay alive and healthy for at least three days.

However, if there are oxygen residues left in the media, then the movement of the protozoa stops immediately. If the wrong media is used and the osmotic pressure in the cells gets too high, then the membranes of the protozoa bul out and the protozoa rupture. If the osmotic pressure in the cells gets too low or the membranes are compromised, then the protozoa shrivel and shrink Fixing of protozoa makes it easier to detect the binding of fluorescent couple ligand to the protozoa.

It's difficult to detect fluorescence and discern it from autofluorescence of wood particles when the protozoa are freely moving around. Once this technique is mastered, approximately 30 termites per hour can be injected with the aid of a assistant. Though the ligand colitic peptide was developed for killing protosome symbiance in the gut of subterranean termites and thus for termite control, but it also could be useful for developing drugs against protosome parasites such as slash mania, ponoma, te, and odium, which cause many diseases in humans.

After watching this video, you should have a good understanding on how to obtain and maintain protozoa extracted from the termite gut in culture, how to show specific binding of a ligand to protozoa via fluorescence microscopy, how to demonstrate the proto suicide action of ligand colitic peptide in vitro. And last but not least, how to inject peptides into the tumor time gut for in vivo confirmation.