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Chronic activation of the epithelial immune system of the fruit fly's salivary glands has a negative effect on organismal growth and induces a peculiar set of target genes.

Abdelsadik A, Roeder T - BMC Genomics (2010)

Bottom Line: Gene ontology analyses of regulated genes revealed a significant increase in genes associated with ribosomal and proteasomal function.Among the regulated genes, those that code for signaling associated protease activity are significantly modulated.Although they produce antimicrobial peptides, their overall response is highly tissue-specific.

View Article: PubMed Central - HTML - PubMed

Affiliation: Christian-Albrechts-University of Kiel, Zoophysiology, Kiel, Germany.

ABSTRACT

Background: Epithelial and especially mucosal immunity represents the first line of defence against the plethora of potential pathogens trying to invade via the gastrointestinal tract. The salivary glands of the fruit fly are an indispensable part of the gastrointestinal tract, but their contribution to the mucosal immunity has almost completely been neglected. Our major goal was to elucidate if the fly's salivary glands are able to mount an immune response and what the major characteristics of this immune response are.

Results: Ectopic activation of the IMD-pathway within the salivary gland cells is able to induce an immune response, indicating that the salivary glands are indeed immune competent. This reaction is characterized by the concurrent expression of numerous antimicrobial peptide genes. In addition, ectopic activation of the salivary gland's immune response induces morphological changes such as dwarfism throughout all developmental stages and a significantly decreased length of the salivary glands themselves. DNA-microarray analyses of the reaction revealed a complex pattern of up- and downregulated genes. Gene ontology analyses of regulated genes revealed a significant increase in genes associated with ribosomal and proteasomal function. On the other hand, genes coding for peptide receptors and some potassium channels are downregulated. In addition, the comparison of the transcriptional events induced following IMD-activation in the trachea and the salivary glands shows also only a small overlap, indicating that the general IMD-activated core transcriptome is rather small and that the tissue specific component of this response is dominating. Among the regulated genes, those that code for signaling associated protease activity are significantly modulated.

Conclusions: The salivary glands are immune-competent and they contribute to the overall intestinal immune system. Although they produce antimicrobial peptides, their overall response is highly tissue-specific. Our analysis indicates that chronic activation of the salivary gland's immune system is costly, as it induces severe reduction in growth throughout development. The IMD-regulated increase in expression levels of the fly's presenilin representatives opens the opportunity to use the salivary glands for studying the physiological and pathophysiological role of these genes in a simple but functional environment.

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Infection and ectopic activation of the IMD-pathway induces expression of antimicrobial peptides in the salivary glands. Oral infection of early 3rd instar with the insect pathogen Erwinia carotovora was used to monitor transcriptional changes in 3 selected antimicrobial peptide genes, drosomycin (drs), cecropin (cec) and diptericin (dipt)(white bars). Ectopic overexpression of the prgp-lc gene in the larval salivary glands was achieved using the Gal4/UAS system (sgs3::gal4 X UAS::pgrp-lc). Salivary glands of early 3rd instar larvae were isolated from these crossing and a parental line (responder line) used for control. Quantitative real-time PCR was performed with oligonucleotides comprising approximately 150 bp of the corresponding antimicrobial peptide genes; drosomycin (drs), cecropin (cec), diptericin (dipt), metchnikowin (metch), drosomycin (dros) and defensin (def) (grey bars). Results are the mean of at least 3 experiments performed in triplicate. Controls are set to 1. Statistically significant differences (compared with controls are marked by an asterik (p < 0.05).
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Figure 2: Infection and ectopic activation of the IMD-pathway induces expression of antimicrobial peptides in the salivary glands. Oral infection of early 3rd instar with the insect pathogen Erwinia carotovora was used to monitor transcriptional changes in 3 selected antimicrobial peptide genes, drosomycin (drs), cecropin (cec) and diptericin (dipt)(white bars). Ectopic overexpression of the prgp-lc gene in the larval salivary glands was achieved using the Gal4/UAS system (sgs3::gal4 X UAS::pgrp-lc). Salivary glands of early 3rd instar larvae were isolated from these crossing and a parental line (responder line) used for control. Quantitative real-time PCR was performed with oligonucleotides comprising approximately 150 bp of the corresponding antimicrobial peptide genes; drosomycin (drs), cecropin (cec), diptericin (dipt), metchnikowin (metch), drosomycin (dros) and defensin (def) (grey bars). Results are the mean of at least 3 experiments performed in triplicate. Controls are set to 1. Statistically significant differences (compared with controls are marked by an asterik (p < 0.05).

Mentions: To evaluate if the salivary glands of larval Drosophila are able to react to an immune challenge and to characterize this reaction type, we performed two types of experiments. First, conventional oral infection experiments were used to evaluate if this epithelial tissue is able to launch a classical immune response, characterized by increased expression of antimicrobial peptide genes. Using promotor indicator lines, where gfp expression is under transcriptional control of antimicrobial peptide gene promoters, no response could be observed using infection with Erwinia carotovora (data not shown). Using these oral infection experiments with Erwinia carotovora and qRT-PCR, we evaluated a set of three antimicrobial peptide genes; namely, drosomycin, cecropin and diptericin. Whereas expression of drosomycin is reduced, both, cecropin and diptericin expression is increased approximately 5 fold (Fig. 1). To show that ectopic pgrp-lc expression is able to induce an autonomous immune response in the larval salivary glands, we isolated salivary glands of control animals (parental lines) and those of the sgs3-Gal4 X UAS-pgrp-lc crosses. Isolation was performed manually from early 3rd larval instars and the salivary glands were thoroughly freed from attached fat body material. This material was used for quantitative real-time PCR experiments following standard procedures. We tested the expression levels of the canonical set of antimicrobial peptide genes, including metchnikowin (metch), defensin (def), drosocin (dros), drosomycin (drs), cecropin (cec) and diptericin (dipt). Expression of all of them, except that of drosomycin, is upregulated between 10 and more than hundred fold (Fig. 2). Greatest changes are seen for diptericin and defensin with approx. 80 and 160 fold increases, respectively. Expression of drosomycin on the other hand, is instead downregulated significantly.


Chronic activation of the epithelial immune system of the fruit fly's salivary glands has a negative effect on organismal growth and induces a peculiar set of target genes.

Abdelsadik A, Roeder T - BMC Genomics (2010)

Infection and ectopic activation of the IMD-pathway induces expression of antimicrobial peptides in the salivary glands. Oral infection of early 3rd instar with the insect pathogen Erwinia carotovora was used to monitor transcriptional changes in 3 selected antimicrobial peptide genes, drosomycin (drs), cecropin (cec) and diptericin (dipt)(white bars). Ectopic overexpression of the prgp-lc gene in the larval salivary glands was achieved using the Gal4/UAS system (sgs3::gal4 X UAS::pgrp-lc). Salivary glands of early 3rd instar larvae were isolated from these crossing and a parental line (responder line) used for control. Quantitative real-time PCR was performed with oligonucleotides comprising approximately 150 bp of the corresponding antimicrobial peptide genes; drosomycin (drs), cecropin (cec), diptericin (dipt), metchnikowin (metch), drosomycin (dros) and defensin (def) (grey bars). Results are the mean of at least 3 experiments performed in triplicate. Controls are set to 1. Statistically significant differences (compared with controls are marked by an asterik (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874812&req=5

Figure 2: Infection and ectopic activation of the IMD-pathway induces expression of antimicrobial peptides in the salivary glands. Oral infection of early 3rd instar with the insect pathogen Erwinia carotovora was used to monitor transcriptional changes in 3 selected antimicrobial peptide genes, drosomycin (drs), cecropin (cec) and diptericin (dipt)(white bars). Ectopic overexpression of the prgp-lc gene in the larval salivary glands was achieved using the Gal4/UAS system (sgs3::gal4 X UAS::pgrp-lc). Salivary glands of early 3rd instar larvae were isolated from these crossing and a parental line (responder line) used for control. Quantitative real-time PCR was performed with oligonucleotides comprising approximately 150 bp of the corresponding antimicrobial peptide genes; drosomycin (drs), cecropin (cec), diptericin (dipt), metchnikowin (metch), drosomycin (dros) and defensin (def) (grey bars). Results are the mean of at least 3 experiments performed in triplicate. Controls are set to 1. Statistically significant differences (compared with controls are marked by an asterik (p < 0.05).
Mentions: To evaluate if the salivary glands of larval Drosophila are able to react to an immune challenge and to characterize this reaction type, we performed two types of experiments. First, conventional oral infection experiments were used to evaluate if this epithelial tissue is able to launch a classical immune response, characterized by increased expression of antimicrobial peptide genes. Using promotor indicator lines, where gfp expression is under transcriptional control of antimicrobial peptide gene promoters, no response could be observed using infection with Erwinia carotovora (data not shown). Using these oral infection experiments with Erwinia carotovora and qRT-PCR, we evaluated a set of three antimicrobial peptide genes; namely, drosomycin, cecropin and diptericin. Whereas expression of drosomycin is reduced, both, cecropin and diptericin expression is increased approximately 5 fold (Fig. 1). To show that ectopic pgrp-lc expression is able to induce an autonomous immune response in the larval salivary glands, we isolated salivary glands of control animals (parental lines) and those of the sgs3-Gal4 X UAS-pgrp-lc crosses. Isolation was performed manually from early 3rd larval instars and the salivary glands were thoroughly freed from attached fat body material. This material was used for quantitative real-time PCR experiments following standard procedures. We tested the expression levels of the canonical set of antimicrobial peptide genes, including metchnikowin (metch), defensin (def), drosocin (dros), drosomycin (drs), cecropin (cec) and diptericin (dipt). Expression of all of them, except that of drosomycin, is upregulated between 10 and more than hundred fold (Fig. 2). Greatest changes are seen for diptericin and defensin with approx. 80 and 160 fold increases, respectively. Expression of drosomycin on the other hand, is instead downregulated significantly.

Bottom Line: Gene ontology analyses of regulated genes revealed a significant increase in genes associated with ribosomal and proteasomal function.Among the regulated genes, those that code for signaling associated protease activity are significantly modulated.Although they produce antimicrobial peptides, their overall response is highly tissue-specific.

View Article: PubMed Central - HTML - PubMed

Affiliation: Christian-Albrechts-University of Kiel, Zoophysiology, Kiel, Germany.

ABSTRACT

Background: Epithelial and especially mucosal immunity represents the first line of defence against the plethora of potential pathogens trying to invade via the gastrointestinal tract. The salivary glands of the fruit fly are an indispensable part of the gastrointestinal tract, but their contribution to the mucosal immunity has almost completely been neglected. Our major goal was to elucidate if the fly's salivary glands are able to mount an immune response and what the major characteristics of this immune response are.

Results: Ectopic activation of the IMD-pathway within the salivary gland cells is able to induce an immune response, indicating that the salivary glands are indeed immune competent. This reaction is characterized by the concurrent expression of numerous antimicrobial peptide genes. In addition, ectopic activation of the salivary gland's immune response induces morphological changes such as dwarfism throughout all developmental stages and a significantly decreased length of the salivary glands themselves. DNA-microarray analyses of the reaction revealed a complex pattern of up- and downregulated genes. Gene ontology analyses of regulated genes revealed a significant increase in genes associated with ribosomal and proteasomal function. On the other hand, genes coding for peptide receptors and some potassium channels are downregulated. In addition, the comparison of the transcriptional events induced following IMD-activation in the trachea and the salivary glands shows also only a small overlap, indicating that the general IMD-activated core transcriptome is rather small and that the tissue specific component of this response is dominating. Among the regulated genes, those that code for signaling associated protease activity are significantly modulated.

Conclusions: The salivary glands are immune-competent and they contribute to the overall intestinal immune system. Although they produce antimicrobial peptides, their overall response is highly tissue-specific. Our analysis indicates that chronic activation of the salivary gland's immune system is costly, as it induces severe reduction in growth throughout development. The IMD-regulated increase in expression levels of the fly's presenilin representatives opens the opportunity to use the salivary glands for studying the physiological and pathophysiological role of these genes in a simple but functional environment.

Show MeSH
Related in: MedlinePlus