Antimicrobial peptides to kill gut symbionts

Lytic peptides, which are a ubiquitous part of the nonspecific immune system of eukary-otes, disrupt the membranes of bacteria and protozoa by forming channels that lead to cell death (Mutwiri et al , 2000; Boman, 1995, 2003) Because lytic peptides are largely inactive against the electrically neutral, cholesterol-containing cell membranes of higher eukary-otes (Javadpour et al ., 1996; Kamysz et al ., 2003; Boman, 2003), most lytic peptides have low or no toxicity to nontarget organisms such as beneficial insects, humans, and other mammals Because of their mode of action (membrane disruption), the development of resistance mechanisms to lytic peptides is less likely than to chemical insecticides The highly evolved natural lytic peptides, as well as synthetic derivatives, may provide an environmentally friendly alternative to chemical insecticides, because concentrations in the micromolar range are active against microorganisms (Wade et al , 1990) and do not leave toxic residues in the environment

Because it has been shown that antimicrobial peptides can have activity against insect transmitted parasites (Shahabuddin et al , 1998), several paratransgenic approaches to control vector borne diseases involve the use of lytic peptides to kill the protozoan disease agent in the insect host For example, Hu and Aksoy (2005) discussed a paratransgenic strategy of using Sodalis glossinidius, a midgut symbiont of the tsetse fly, to express lytic peptides that were characterized from the tsetse fly fat body to control trypanosome transmission by tsetse The proposed host bacterium S. glossinidius shows high level of resistance against lytic peptides that are part of the innate immune system of the tsetse fly (Hu and Aksoy, 2005). Durvasula et al . (1997) genetically engineered Rhodococcus rhodnii, the endosymbiont of the triatomid Rhodnius prolixus, to express Cecropin A, a lytic peptide lethal to the protozoan Trypanosoma cruzi, which causes Chagas disease . Stable expression of the lytic peptide from the endosymbiont into the bug's hindgut resulted in dramatic reduction of the number of parasites Although the use of lytic peptides in these projects aimed at eliminating disease-causing parasites from insects without toxicity to the host itself, the goal in termite control would be to destroy the symbiotic community in the worker gut and thus starve the termite colony

To be able to test the efficacy of lytic peptides against protozoa in the gut of the For-mosan subterranean termite in vitro, we isolated the three species of protozoa (Koidzumi, 1921) from the termite gut. Using sterile Trager U media at pH 7.0 (Trager 1934) sparged with 2 . 5% hydrogen, 5% carbon dioxide, and 92 .5% nitrogen, protozoa cultures could be maintained in an anaerobic glove-box for more than 24 hours outside the termite gut This technique enabled us to test the efficacy of lytic peptides against the termite gut protozoa in anaerobic conditions similar to the termites' hindgut environment Following exposure to concentrations of 50 ]M of L-Cecropin B, L-Melittin, L-Hecate, and D-Hecate (Hancock et al ., 1995; Mutwiri et al , 2000), protozoa were deformed and movement of the protozoa ceased All three protozoa species died within 5-10 minutes following lytic peptide application in culture, whereas untreated protozoa cultures stayed alive for at least 24 hours and longer However, when the natural L-enantiomers of the lytic peptides were fed to termite workers, no defaunation was observed (Figure 17. 1). The lytic peptides were possibly digested by general proteases while passing through the digestive tract To test this assumption, synthetic D-enantiomers of Hecate, which are resistant to enzymatic degradation and inac-tivation (Wade et al , 1990), were fed to termite workers When termites were fed a 500 ] M D-Hecate solution, vesicles formed inside the protozoa, protozoan membranes ruptured (Figure 17. 2a), and defaunation of the gut was complete within a few days (Figure 17. 2b) . Because D-peptides cannot be synthesized naturally by any living organisms, they are not useful in a paratransgenic system

The majority of symbionts, including the protozoa, live in the anaerobic pouches of the hindgut and are largely protected from digestion because the hindgut is not likely to contain general proteases (Fujita et al ., 2001) . Therefore, we tested whether L-forms of lytic peptides could stay active in the hindgut . Using microinjection, approximately 0 . 5 ]L of 10 mM Tris-HCl, pH 7.4 (control) or 500 ]M lytic peptide solution was injected into the hind-gut of termite workers Death of all protozoa in the guts was observed within 72 hours of lytic peptide treatment (Figure 17 3) Termites were defaunated and died within a range

Figure 17.1 (Color figure follows p. 238.) Healthy termite gut containing a dense protozoa population.
Figure 17.2a (Color figure follows p. 238.) Deterioration of protozoa in the hindgut after workers were fed D-Hecate . 1 = vesicles inside affected protozoan . 2 = dead protozoan .

Figure 17.2b (Color figure follows p. 238.) Defaunated hindgut .

of a few days (L-Melittin) to 6 weeks (L- and D-Hecate, Cecropin B) The latter is consistent with death by starvation, as 6 weeks is approximately the life span of workers that have been defaunated with the protozoacidal drug metronidazole (Raina et al , 2004) The bee component venom Melittin is a very potent antimicrobial peptide The accelerated death

Figure 17.3 (Color figure follows p. 238.) Defaunation of worker hindgut after injection of lytic peptides

after Melittin injection and observations of disintegration of the gut suggest that Melittin applied in concentrations of 500 ]M and higher attacks not only the protozoa but also the termite gut itself Therefore, the toxicity of Melittin against some eukaryotic cells (Boman and Hultmark, 1987) may prevent its application in a paratransgenic system Although the application of Melittin should lead to a quick demise of individual termite workers, such a fast-acting toxin might not spread sufficiently among colony members to guarantee eradication of the entire termite colony Slow-acting lytic peptides, such as Hecate and Cecropin, are therefore the toxins of choice to be delivered to and expressed in the hindgut by a paratransgenic microbial shuttle system

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