Expression Patterns And Recombinant Expression Of Antimicrobial Peptide Genes From Housefly (Musca Domestica)

The most of multicellular organisms live in surroundings laden with pathogens. The survival of them depends on host defense mechanisms involving various components in vivo. In high vertebrates such as mammalian, the host defense systems depend on two types of immune: the innate immunity and the acquired immunity. But the defenses of invertebrate, such as insects, only depend on innate immunity.A character of the innate immunity is that endogenous peptides are constitutively expressed or induced, which provide a fast and effective means of defence against pathogens. This group of peptides termed 'antimicrobial peptides' (AMPs). AMPs make up of a primitive immune mechanism and are found in a wide range of eukaryotic organisms, from humans to plants and insects. Some of them are found in microorganisms. Most of these peptides have small molecular weight, high isoelectric point, heat stability, overall net positive charge under the physiological condition, a broad antimicrobial spectrum and so on. They not only function on Gram-positive bacteria and Gram-negative bacteria, but also on fungi, protozoa, especially bacteria with drug resistance. With the continual emergence of new diseases, bacterial resistance has become a growing threat to human health. Due to their broad-spectrum of antimicrobial activities, the AMPs were considered to be excellent candidates for potential novel antibiotic agents. Some AMPs also exhibit tumouricidal and virucidal properties, and have potential of prevention and cure of parasite infectious disease. A few of these peptides show activity against pathogens causing sexually transmitted infection, including HIV. In agricultural production, they not only are used as animal feed additive or antistaling agent for food storage, but also in transgenic animals or plants to prevent diseases. The extensive studies on AMPs have helped us to understand the innate immunity well.Therefore, the studies in AMPs have important academic value and good application prospect.The houseflies (Musca domestica) that live in surroundings full of various microbes are closely correlative with human life. Houseflies have been implicated in the spread of over 100 pathogens that may cause diseases in humans and animals, including typhoid, cholera, bacillary dysentery, tuberculosis, anthrax ophthalmia and infantile diarrhea, as well as parasitic worms. But they can thrive without causing infection. Therefore it is presumed that the houseflies have unique immune defense mechanism and can product antimicrobial peptides with strong activity. Our laboratory has purified an antimicrobial peptide, with a molecular weight of 10 kDa. The peptide was heat stable, and had activities that were retained after 10 min incubation at 100 °C. It showed potential activities against Gram-positive and Gram-negative bacteria. After that, our lab also cloned a defensin gene (GenBank no. AY260152), named Md defensin {Mdde), and a cecropin gene (GenBank no. AF416602), named Md-Cecropin (Md-Cec), from housefly. Otherwise, attacin genes (GenBank no. AY460106 and GenBank no. DQ062744) were cloned by other authors form housefly.In this study, the expression patterns of the genes encoding cecropin, defensin and attacin were studied by semi-quantitative RT-PCR and in situ hybridization. We analysed the effects of challenge with the mixtures of Escherichia coli and Staphylococcus aureus on the antimicrobial peptides mRNA transcription in the larvae of M. domestica, and investigated the tissue distributions of the gene transcripts in larvae of M. domestica. The cDNA sequence encoding mature attacin peptide was cloned by RT-PCR, and then expressed in prokaryotic expression system.The results of the studies are as follows.1. RT-PCR was performed using Md-Cec specific primers MdcecF/MdcecR and total RNA from the unchallenged and challenged larvae at 5h, lOh, 24h and 48h after challenge respectively. The results showed that the Md-cec gene expression was detectable in challenged larvae from 5h to 24h after infection challenge, whereas not detectable in native larvae and challenged larvae at 48h. Quantitativeanalysis by Labwork v4.5 revealed that the quantity of Md-Cec transcript increased rapidly in 5h after challenge, then decreased slowly, maintained to lOh, distinctly reduced at 24h, no detectable again at 48h.2. In order to localize expression of Md-Cec mRNA in different tissues, we performed in situ hybridization with Md-Cec antisense DIG RNA probes and the tissue slices of larvae unchallenged and challenged with the mixtures of E. coli and 5. aureus. The results showed that there was strong transcription in the fat body, and that transcript was also detected in the epithelia of the body wall and epidermis of gut. However, transcript was not observed in muscles, or trachea in the challenged larvae. In unchallenged flies, no transcript was detected in any tissue.3. To analyze the effects of challenge with the mixtures of E. coli and S1. aureus on the Mdde transcripts in the larvae of M. domestica, RT-PCR was performed using Mdde specific primers Mdde F/Mdde R and total RNA from the unchallenged and challenged larvae respectively at 5h, lOh, 24h and 48h after challenge. The results of RT-PCR showed that the Mdde transcripts were found in either the challenged larvae with the mixtures of E. coli and S. aureus from 5h to 48h or unchallenged insects, and that the transcript level of the gene rapidly increased at 5h after challenge, largely increased at 48h.4. To study the tissue distributions of the Mdde transcripts in larvae of housefly, the in situ hybridization with Mdde antisense DIG RNA probes were carried out. The results of the in situ hybridization showed that the gene of Mdde was transcribed mainly in the epithelia of the body wall and the fat body, and no transcription signal was detected in tracheae, gut and muscles. The transcripts of Mdde in the epithelia of the body wall were found both in challenged larvae and unchallenged larvae, whereas the transcripts of Mdde in fat body were found only in challenged larvae.5. RT-PCR was performed using housefly attacin specific primers Attacin F/Attacin R and total RNA from the whole body of the unchallenged larvae and the tissues of challenged larvae at lOh after challenge by E.coli. The results showed that the attacin gene expression was not detectable in native larvae, and was detectable in fat body of challenged larvae, whereas not detectable in body wall andgut of challenged larvae.6. A cDNA encoding mature attacin peptide of housefly (Musca domestica) was isolated from total RNA of challenged larvae by E. coli, using RT-PCR, and named Mdatt. The sequence alignment showed that the nucleotide sequence of Mdatt is 97% identical to M. domestica attacin (GenBank no. DQ062744) and 96% to another M. domestica attacin(Gen&ank no. AY460106), while the deduced amino acid sequence of Mdatt is 99% and 98% identical respectively to GenBank DQ062744 and GenBank AY460106. The Mdatt was cloned into the pGEX-4T-l vector and expressed in E.coli BL21. The result of SDS-PAGE showed that the expressed production resided in the host cells in the form of inclusion bodies.Base on the analyses of the above results, the conclusions we come up with are as follows.1. The transcription of the Md-cec is inducible by microbe infections. The rapidly transcription occur in 5h after challenge by bacteria. The quantities of the transcript maintain to lOh, distinctly reduce at 24h, and come back to native level at 48h.2. The distribution of the Md-Cec transcript has tissue specificity. The transcript mainly produces in fat body, body wall and midgut, not in muscles and trachea.3. The transcription of Mdde in larvae of M domestica is constitutive. But the transcript quantity is inducible to increase by microorganism. The quantity gradually increases after challenge by bacteria, rapidly increases in 5h, largely increases at 48h.4. The Mdde transcript also has tissue specificity, mainly in body wall and fat body. The transcription is low level constitutive expression in body wall, whereas inducible in fat body. Differing from Md-cec, the transcript of Mdde is not found in gut. We consider that the transcription pattern of the Mdde in larvae of M. domestica is the results of the insect adapting to the natural environment.5. The transcription of the attacin is inducible by microbe infections in larvae of M. domestica. The transcript mainly produces in fat body, not in body wall and gut.6. The cDNA sequence encoding mature attacin peptide is conserved, and the amino acid sequence more conserved. The mature attacin peptide can be expressed in prokaryotic expression system.This study will enhance the understanding of the immune system in housefly;enrich the knowledge of the innate immunity in insects. The recombinant expressed antimicrobial peptides can be used in continued studies, and large-scale production may be used in genetic engineered medicine and animal feed additives.