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Fungal Peptide Destruxin A Plays a Specific Role in Suppressing the Innate Immune Response in Drosophila melanogaster

2007; Elsevier BV; Volume: 282; Issue: 12 Linguagem: Inglês

10.1074/jbc.m605927200

1083-351X

Curtis G. Hayes, Raymond J. St. Leger, Louisa P. Wu,

Invertebrate Immune Response Mechanisms

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects. Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects. Insects are the most diverse and prolific land animals, and a variety of pathogens have specialized to infect them. Unlike bacteria or viruses that usually need to be ingested, certain fungal species can directly breach the insect cuticle to cause disease. Fungi are the most commonly observed insect pathogens in nature, causing the largest percentage of deaths because of infection. As a result, methods of controlling insect populations using live fungal insecticides have attracted medical and agronomical interest (1Blanford S. Chan B.H. Jenkins N. Sim D. Turner R.J. Read A.F. Thomas M.B. Science. 2005; 308: 1638-1641Crossref PubMed Scopus (257) Google Scholar, 2Wright M.S. Raina A.K. Lax A.R. J. Econ. Entomol. 2005; 98: 1451-1458Crossref PubMed Scopus (47) Google Scholar). The ascomycete Metarhizium anisopliae is already in commercial use to control termites, grasshoppers, and thrips (2Wright M.S. Raina A.K. Lax A.R. J. Econ. Entomol. 2005; 98: 1451-1458Crossref PubMed Scopus (47) Google Scholar, 3Hunter D.M. Milner R.J. Spurgin P.A. Bull. Entomol. Res. 2001; 91: 93-99PubMed Google Scholar, 4Maniania N.K. Ekesi S. Lohr B. Mwangi F. Mycopathologia. 2002; 155: 229-235Crossref PubMed Scopus (46) Google Scholar). In some fungi, success in infecting a wide variety of insects can, at least in part, be attributed to secretion of virulence factors during pathogenesis. Destruxins were initially identified as toxic compounds secreted by Metarhizium and were later characterized as important virulence factors accelerating the deaths of infected insects (5Kodaira Y. Agric. Biol. Chem. 1961; 25: 261-262Crossref Scopus (2) Google Scholar, 6Kershaw M.J. Moorhouse E.R. Bateman R. Reynolds S.E. Charnley A.K. J. Invertebr. Pathol. 1999; 74: 213-223Crossref PubMed Scopus (180) Google Scholar, 7Dumas C. Robert P. Pais M. Vey A. Quiot J.M. Comp. Biochem. Physiol. Pharmacol. Toxicol. Endocrinol. 1994; 108: 195-203Crossref PubMed Scopus (52) Google Scholar, 8Fargues J. Robert P.H. Vey A. Entomophaga. 1985; 30: 353-364Crossref Scopus (26) Google Scholar).Chemically, Destruxins are cyclic hexadepsipeptides composed of an α-hydroxy acid and five amino acid residues. Five natural analogues (labeled A–E) have been isolated (5Kodaira Y. Agric. Biol. Chem. 1961; 25: 261-262Crossref Scopus (2) Google Scholar, 9Suzuki A. Kuyama A. Kodaira Y. Tamura S. Agric. Biol. Chem. 1970; 35: 1641-1643Google Scholar, 10Païs M. Das B.C. Ferron P. Phytochemistry. 1981; 20: 715-723Crossref Scopus (138) Google Scholar). These forms differ in the R-group of the hydroxyl acid residue and appear to have overlapping but different biological effects. Primarily, however, injection, ingestion, or topical application of a Destruxin on insects causes tetanic paralysis (11Samuels R.I. Reynolds S.E. Charnley A.K. Comp. Biochem. Physiol. 1988; 90C: 403-412Google Scholar). Destruxin-induced membrane depolarization due to the opening of Ca2+ channels has been implicated as a cause of paralysis and death (11Samuels R.I. Reynolds S.E. Charnley A.K. Comp. Biochem. Physiol. 1988; 90C: 403-412Google Scholar). Destruxin also causes signaling changes through the phosphorylative activation of certain proteins in lepidopteran and human cell lines. In addition, Destruxins cause morphological and cytoskeletal changes in insect plasmatocytes in vitro, and this adversely affects insect cellular immune responses, such as encapsulation and phagocytosis (12Vey A. Matha V. Dumas C. J. Invertebr. Pathol. 2002; 80: 177-187Crossref PubMed Scopus (85) Google Scholar, 13Vilcinskas A. Matha V. Goetz P. J. Insect Physiol. 1997; 43: 475-783Crossref Scopus (86) Google Scholar, 14Vilcinskas A. Matha V. Goetz P. J. Insect Physiol. 1997; 43: 1149-1159Crossref PubMed Scopus (80) Google Scholar). These could be indirect results of a calcium influx (15Dumas C. Matha V. Quiot J.M. Vey A. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 1996; 114: 213-219Crossref PubMed Scopus (50) Google Scholar). Destruxins also show biological activities against non-insects. They are particularly toxic to mammalian leukemia cells and spleen lymphocytes and have demonstrated anti-proliferative activity on mouse neoplasms in vitro (16Pedras M.S. Irina Zaharia L. Ward D.E. Phytochemistry. 2002; 59: 579-596Crossref PubMed Scopus (179) Google Scholar). Destruxins A, B, and E have also been shown to have antiviral properties in insect and human cell lines (17Vey A. Quiot J.M. Vago C. C. R. Acad. Sci. Ser. 1985; 3: 229-234Google Scholar, 18Huxham I.M. Lackie A.M. McCorkindale N.J. J. Insect Physiol. 1989; 35: 97-105Crossref Scopus (99) Google Scholar, 19Quiot J.M. Vey A. Vago C. Pais M. C. R. Acad. Sci. Ser. 1980; D291: 763-766Google Scholar). For example, Destruxin B has demonstrated a suppressive effect on hepatitis B surface antigen expression (20Chen H.C. Chou C.K. Sun C.M. Yeh S.F. Antiviral Res. 1997; 34: 137-144Crossref PubMed Scopus (43) Google Scholar, 21Yeh S.F. Pan W. Ong G.T. Chiou A.J. Chuang C.C. Chiou S.H. Wu S.H. Biochem. Biophys. Res. Commun. 1996; 229: 65-72Crossref PubMed Scopus (32) Google Scholar).Aside from their ability to open calcium channels, the mechanisms by which Destruxins achieve their varied biological activities have not been studied in vivo. Therefore, we used Drosophila melanogaster as an insect model to characterize the range of functions affected by Destruxins. Among insects, Drosophila has the best characterized immune response, and because of similarities in key signaling pathways, has been an invaluable model for understanding innate immunity in humans. Flies have three active innate immune mechanisms for dealing with an invading microorganism. Proteolytic cascades triggered by microbial determinants lead to the formation of melanotic clots at the site of infection (22Soderhall K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1082) Google Scholar). The toxic melanin along with encapsulating lamellocytes that circulate in the Drosophila hemolymph can neutralize many foreign microorganisms (23Braun A. Hoffmann J.A. Meister M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14337-14342Crossref PubMed Scopus (142) Google Scholar, 24Rizki R.M. Rizki T.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6154-6158Crossref PubMed Scopus (131) Google Scholar). Other hemocytes actively phagocytose invading pathogens (25Lanot R. Zachary D. Holder F. Meister M. Dev. Biol. 2001; 230: 243-257Crossref PubMed Scopus (465) Google Scholar, 26Elrod-Erickson M. Mishra S. Schneider D. Curr. Biol. 2000; 10: 781-784Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). In addition, pathogenic determinants activate Drosophila pattern recognition receptors, which initiate signal transduction cascades that trigger a humoral immune response. This response is marked by the production of antimicrobial peptides (AMPs) 2The abbreviations used are: AMP, antimicrobial peptide; PGRP, peptidoglycan recognition proteins; DmIKKβ, D. melanogaster IκB kinase β-subunit; NF-κB, nuclear factorκB; IκB, inhibitor ofκB; GO, gene ontology; PBS, phosphate-buffered saline; UAS, upstream activating sequence.2The abbreviations used are: AMP, antimicrobial peptide; PGRP, peptidoglycan recognition proteins; DmIKKβ, D. melanogaster IκB kinase β-subunit; NF-κB, nuclear factorκB; IκB, inhibitor ofκB; GO, gene ontology; PBS, phosphate-buffered saline; UAS, upstream activating sequence. that have activity against the invading pathogen. Two key pathways, named Toll and IMD, have been identified that mediate AMP expression (27Hoffmann J.A. Nature. 2003; 426: 33-38Crossref PubMed Scopus (1112) Google Scholar, 28De Gregorio E. Spellman P.T. Tzou P. Rubin G.M. Lemaitre B. EMBO J. 2002; 21: 2568-2579Crossref PubMed Scopus (628) Google Scholar, 29Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (2954) Google Scholar). The former involves the activation of the Toll receptor due to detection of primarily fungal or Gram-positive bacterial determinants by upstream pattern recognition receptors (30Gobert V. Gottar M. Matskevich A.A. Rutschmann S. Royet J. Belvin M. Hoffmann J.A. Ferrandon D. Science. 2003; 302: 2126-2130Crossref PubMed Scopus (296) Google Scholar, 31Levashina E.A. Langley E. Green C. Gubb D. Ashburner M. Hoffmann J.A. Reichhart J.M. Science. 1999; 285: 1917-1919Crossref PubMed Scopus (368) Google Scholar, 32Ligoxygakis P. Pelte N. Hoffmann J.A. Reichhart J.M. Science. 2002; 297: 114-116Crossref PubMed Scopus (277) Google Scholar). The activation of the Toll pathway leads to the phosphorylation of several adaptor proteins that culminate in the phosphorylation of Cactus, an IκB homologue in Drosophila (33Belvin M.P. Jin Y. Anderson K.V. Genes Dev. 1995; 9: 783-793Crossref PubMed Scopus (147) Google Scholar, 34Bergmann A. Stein D. Geisler R. Hagenmaier S. Schmid B. Fernandez N. Schnell B. Nusslein-Volhard C. Mech. Dev. 1996; 60: 109-123Crossref PubMed Scopus (92) Google Scholar, 35Reach M. Galindo R.L. Towb P. Allen J.L. Karin M. Wasserman S.A. Dev. Biol. 1996; 180: 353-364Crossref PubMed Scopus (100) Google Scholar). This leads to degradation of Cactus, freeing the NF-κB proteins Dorsal and Dif to translocate to the nucleus. There they activate transcription of a variety of genes important for the immune response, including the antimicrobial peptide Drosomycin (36Lemaitre B. Meister M. Govind S. Georgel P. Steward R. Reichhart J.M. Hoffmann J.A. EMBO J. 1995; 14: 536-545Crossref PubMed Scopus (196) Google Scholar, 37Meng X. Khanuja B.S. Ip Y.T. Genes Dev. 1999; 13: 792-797Crossref PubMed Scopus (209) Google Scholar). In the IMD pathway, activation of the receptor peptidoglycan recognition protein LC (PGRP-LC) by Gram-negative bacterial peptidoglycan (38Choe K.M. Werner T. Stoven S. Hultmark D. Anderson K.V. Science. 2002; 296: 359-362Crossref PubMed Scopus (477) Google Scholar, 39Kaneko T. Goldman W.E. Mellroth P. Steiner H. Fukase K. Kusumoto S. Harley W. Fox A. Golenbock D. Silverman N. Immunity. 2004; 20: 637-649Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar) leads to the phosphorylation of the adaptor Imd (40Georgel P. Naitza S. Kappler C. Ferrandon D. Zachary D. Swimmer C. Kopczynski C. Duyk G. Reichhart J.M. Hoffmann J.A. Dev. Cell. 2001; 1: 503-514Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) and subunits of Drosophila IKK (DmIKKβ and Kenny/IKKγ) (41Rutschmann S. Jung A.C. Zhou R. Silverman N. Hoffmann J.A. Ferrandon D. Nat. Immunol. 2000; 1: 342-347Crossref PubMed Scopus (221) Google Scholar, 42Silverman N. Zhou R. Stoven S. Pandey N. Hultmark D. Maniatis T. Genes Dev. 2000; 14: 2461-2471Crossref PubMed Scopus (245) Google Scholar) and finally the cleavage of the NF-κB-like protein Relish. Relish is responsible for the transcription of many proteins important for the immune response, including the antimicrobial peptide Diptericin (43Lemaitre B. Kromer-Metzger E. Michaut L. Nicolas E. Meister M. Georgel P. Reichhart J.M. Hoffmann J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9465-9469Crossref PubMed Scopus (462) Google Scholar, 44Meister M. Lemaitre B. Hoffmann J.A. BioEssays. 1997; 19: 1019-1026Crossref PubMed Scopus (147) Google Scholar). Drosomycin and Diptericin are often used as target genes to assay for the activation of the Toll and IMD pathways, respectively. Flies mutant in both the IMD and Toll pathways are unable to produce any of the characterized AMPs and are highly susceptible to infection from normally innocuous bacteria or fungi (28De Gregorio E. Spellman P.T. Tzou P. Rubin G.M. Lemaitre B. EMBO J. 2002; 21: 2568-2579Crossref PubMed Scopus (628) Google Scholar, 29Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (2954) Google Scholar).Here we report evidence that Destruxin A suppresses the Drosophila humoral immune response. We used cDNA microarrays and quantitative PCR to examine the effect of Destruxin A on adult Drosophila gene expression. The data revealed a significant proportion of AMP genes were down-regulated, suggesting that Destruxin may be suppressing components of the Drosophila immune system. The data further showed Destruxin had the ability to lower the expression of AMPs even when an immune response had been activated by Gram-negative bacterial infection. Destruxin also increased susceptibility of the fly to bacterial infection. The susceptibility could be rescued by ectopic expression of components of the IMD pathway. This result suggests that Destruxin mediates the specific down-regulation of AMPs through targeting a Drosophila innate immune signaling pathway and is the first evidence of such a phenomenon in vivo. In the evolutionary arms race between insect and fungus, Destruxins may thus be playing a novel role in facilitating fungal survival through specific suppression of host immune response components.EXPERIMENTAL PROCEDURESSpotted Microarray Construction and Analysis—From previous Affymetrix chip-based microarray experiments and a survey of the literature, we selected 464 genes important for Drosophila immune responses. We used Primer3 software to design primers to amplify unique regions of the selected genes, generating fragments between 200–600 bp in length (specific primer sequences can be obtained upon request). Fragments were amplified from whole genomic DNA of wild-type, Oregon R strains of D. melanogaster in a 96-well format. The reaction mixture to produce each amplicon contained 50 ng of Drosophila genomic DNA, 1 μm forward primer, 1 μm reverse primer, 1× Titanium Taq (Invitrogen), and 0.5 mm dNTP. The following PCR protocol was used. An initial 95 °C denaturation step for 5 min followed by 20 cycles of 30 s of denaturation at 95 °C, 30 s of annealing at 60 °C, and 45 s of extension at 75 °C. PCR products were run on agarose gels to confirm amplification success and specificity.Printing, hybridization, and scanning of slides were performed with an Affymetrix 417 Arrayer and 418 Scanner at the University of Maryland Biotechnology Institute Microarray Core facility located at the Center for Biosystems Research. PCR products were spotted in triplicate on poly-l-lysine-coated glass slides with a mean spot diameter of 100 μm and a spot spacing of 375 μm. Following printing and cross-linking, slides were washed with 1% SDS to remove background, treated with blocking solution (0.2 m succinic anhydride, 0.05 m sodium borate, prepared in 1-methyl-2-pyrrolidinone), and washed with 95 °C water and 95% ethanol. After drying, slides were kept in the dark at room temperature.For microarray experiments, RNA was extracted from a pooled sample of 20 flies with STAT-60 buffer, according to the manufacturer's protocols (Isotex Diagnostics). They were further purified using the Qiagen RNAeasy purification kit and directly labeled using the Cyscribe first strand labeling kit (Amersham Biosciences), according to the manufacturer's protocols. The raw scanned image files were analyzed using Spot-finder (TIGR), and data normalization, quality assurance and control, filtering, and clustering were performed using MIDAS (TIGR) and MS-Excel (45Saeed A.I. Sharov V. White J. Li J. Liang W. Bhagabati N. Braisted J. Klapa M. Currier T. Thiagarajan M. Sturn A. Snuffin M. Rezantsev A. Popov D. Ryltsov A. Kostukovich E. Borisovsky I. Liu Z. Vinsavich A. Trush V. Quackenbush J. BioTechniques. 2003; 34: 374-378Crossref PubMed Scopus (3964) Google Scholar). Standard deviation normalization and Lowess transformation was performed on the data using MIDAS software. Experiments were done in triplicate, and genes that had at least two readable spots were selected. A criteria of one standard deviation above or below the mean induction of all genes was used to select for up- or down-regulated genes. On normalized data, this represents the top (and bottom) 16% of all genes on the array. The genes were then classified according to available gene ontology classifications, and the major groups are presented in Fig. 1.Fly Stocks—OregonR flies were used as wild type. For ectopic expression of Toll and IMD pathway components, we used the transgenic c564-Gal4 line of flies that express Gal4 in various tissues throughout the fly, particularly in the lymph gland, fat body, salivary glands, imaginal discs, gut, and brain (46Harrison D.A. Binari R. Nahreini T.S. Gilman M. Perrimon N. EMBO J. 1995; 14: 2857-2865Crossref PubMed Scopus (377) Google Scholar). The c564-Gal4 flies were crossed into transgenic flies expressing upstream activating sequence (UAS)-DmIKKβ (provided by K. V. Anderson); UAS-PGRP-SA (provided by J. Royet); UAS-Imd, UAS-Diptericin, and UAS-Drosomycin (provided by B. Lemaitre) to ectopically express these components in the fly.Bacterial Infection, Survival, and Proliferation Assay—Escherichia coli DH5α strains were grown up in LB medium overnight, resuspended in an equal volume of filter-sterilized phosphate-buffered saline (PBS). Approximately 0.5 μl of the bacteria was then injected into the abdomens of female adult flies using a pneumatic picopump PV820 (World Precision Instruments) apparatus. A solution of 86 μm Destruxin A (Sigma-Aldrich) in 1× PBS was used for Destruxin injection. For gene expression studies, RNA was extracted 4 h after injection. The experiment was repeated a minimum of three times.Each survival experiment was performed with at least 20 flies and repeated three times. The total number of flies in each treatment was assessed for survival periodically over a five-day period. The Kaplan-Meier statistical model was used to compare fly survival, and p values <0.05 were deemed significant.For the bacterial proliferation assay, ampicillin-resistant E. coli were used. Twenty-four hours post-injection, the flies were anesthetized, surface sterilized by dipping in 95% ethanol, and homogenized in 1 ml of LB medium containing 1% Triton X-100 and 100 μg/ml ampicillin. The homogenized medium was incubated at 37 °C for 1 h, and 50 μl was plated on LB-Amp plates representing colony-forming units in 1/20 of a fly. Colonies were counted following an overnight incubation of the LB-Amp plates at 37 °C. The experiment was independently repeated 10 times for each treatment, and the error bars show standard deviation.Phagocytosis Assay—The assay was performed as described previously (26Elrod-Erickson M. Mishra S. Schneider D. Curr. Biol. 2000; 10: 781-784Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Adult flies are injected in the abdomen with fluorescein-labeled E. coli particles (Molecular Probes). After 30 min, trypan blue is injected to quench extracellular fluorescence. If the hemocytes are able to take up the fluorescent particles, the fluorescence can be visualized through the cuticle on the dorsal side of the abdomen. In cells deficient for phagocytosis, the fluorescence of the particles remains outside the phagocyte and is quenched by the trypan dye. This results in reduced visualization of particles inside flies deficient in phagocytosis.Quantitative PCR—RNA was isolated using STAT-60 buffer according to the manufacturer's protocol (Isotex Diagnostics). The RNA was digested with RNase-free DNase and subjected to reverse transcription using Superscript II (Invitrogen). The resulting cDNA was quantified using real time-PCR using LUX probes (Invitrogen) on an ABI 5700 real time-PCR system following the manufacturer's protocols. Gene expression was normalized using RP49 as an endogenous control. The data presented in this paper has been further normalized to set uninjected wild-type levels as the calibrator. The specific primers used can be obtained upon request. The experiments were repeated a minimum of three times and in some cases over five times.RESULTSDestruxin Injection Causes a Reduction in Expression of Some Antimicrobial Peptide Genes—To test the effect of non-lethal doses of Destruxin on Drosophila gene expression, we compared wild-type flies injected with 86 μm Destruxin A to flies injected with PBS using cDNA microarrays. The dose was determined experimentally as the highest dose that could be injected into the fly without causing significant difference in mortality compared with PBS-injected flies within five days (data not shown). The custom-made microarrays enabled the study of 464 Drosophila genes selected from an extensive literature survey of data collected by other groups through microarray experiments on genes predicted to be important for the immune response (28De Gregorio E. Spellman P.T. Tzou P. Rubin G.M. Lemaitre B. EMBO J. 2002; 21: 2568-2579Crossref PubMed Scopus (628) Google Scholar, 47Irving P. Troxler L. Heuer T.S. Belvin M. Kopczynski C. Reichhart J.M. Hoffmann J.A. Hetru C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15119-15124Crossref PubMed Scopus (329) Google Scholar). Genes that were significantly up- or down-regulated upon Destruxin injection were classified based on known gene ontology (GO) information (Table 1). MIAME (minimal information about a microarray experiment)-compliant raw data can be accessed from the NCBI GEO website (accession number GSE5767). Categories of genes for general metabolism were not affected by Destruxin. By contrast, 52% of all antibacterial humoral response genes (GO:0006961) on the array were down-regulated in Destruxin-injected flies (Table 1). This represents nearly 23% of all down-regulated genes on the microarray (Fig. 1B) and represents the largest category in the chart. In these microarray experiments, any observed change in gene expression is presumably because of the effect of Destruxin (signal) or noise in the microarray system compared with gene expression in the control. If it is all noise, using a one-standard-deviation criterion would result in ∼15% of genes down-regulated in any given gene ontology category on normalized microarray data assumed to follow a normal distribution of gene expression. This means that, of the 17 antibacterial humoral response genes represented on the microarray, we would expect to see 2.5 genes down-regulated by chance alone. However, we observed nine. A χ-square analysis with one degree of freedom shows that this observed value is significantly different from the expected 2.5, with a p value of 0.0343. In addition, none of the genes in this category were found to be induced upon Destruxin injection (Fig. 1A and Table 1). Thus, there is a statistically significant down-regulation of antibacterial humoral response genes.TABLE 1Genes affected upon Destruxin A injection The genes have been classified according to available gene ontology (GO) classification and have been separated into up-regulated and down-regulated categories. The total number of genes in each category is given next to the gene ontology number in the dark boxes, and the relative percentage of genes up- or down-regulated in each category is also provided. Open table in a new tab Similarly, 34% of genes having peptidoglycan receptor activity (GO:0004867) were down-regulated, representing 15% of all down-regulated genes (Fig. 1B). Only 5% of genes in this category were induced (Table 1). Proteolysis and peptidolysis genes (GO:0006508) represented the largest percentage of all up-regulated genes (Fig. 1A), but only 10% of all genes in this category were up-regulated (Table 1). Because 11% of these genes were down-regulated, there was no significant shift in either direction for this category (Table 1). Of note, most genes (nearly 85% of the genes on the array) were not affected by Destruxin injection, suggesting that the down-regulated antibacterial humoral genes represent a specific phenomenon and are not the result of general ill health brought about by Destruxin injection. Thus, compared with other categories, we observed the most significant and specific down-regulation of the antibacterial peptide response.To confirm that Destruxin caused down-regulation of antimicrobial peptide genes, quantitative real time-PCR was used to examine Diptericin, Cecropin, Attacin, and Metchnikowin expression (Fig. 2). In all cases, the injected flies had a significantly lower AMP production than PBS-injected flies within 4 h of injection (using a one-tailed Student's t test cutoff of p < 0.05), as predicted by the microarrays. Quantitative real time-PCR confirmed the suppressive effects of Destruxin on these AMPs in the absence of infection. We used the Gram-negative bacteria E. coli to determine whether Destruxins can also reduce AMP expression when the immune response has been activated. Co-injection of Destruxin A with the bacteria significantly reduced expression of Diptericin, Attacin, and Drosomycin in these immune-stimulated flies at 4 h as compared with injection with E. coli (Fig. 2). To address the question of whether Destruxin was inhibiting or merely delaying the expression of these AMPs, we also examined their expression at 8 and 24 h. For Drosomycin, Destruxin appears to suppress expression at the earlier time point but not at the later time points. Drosomycin expression typically peaks at 24 h after infection, and it is possible that the injected Destruxin is no longer effective at this later time point. Inhibition of Diptericin and Attacin expression by Destruxin was easier to interpret because of the relative transience in their expression. Inhibition by Destruxin also appeared to be specific to these three AMPs, as levels of some others, such as Cecropin, Drosocin, and Metchnikowin, induced by bacterial injection were not significantly affected by Destruxin.FIGURE 2Quantitative real time-PCR looking at the effect of Destruxin on specific antimicrobial peptides over time. Co-injecting Destruxin with E. coli causes a lowering of Drosomycin, Diptericin, and Attacin gene expression compared with E. coli-injected flies within 4 h of injection. Adult flies more than five days old were injected with PBS (solid gray), E. coli (solid black), 86 μm Destruxin (dashed gray), or E. coli + Destruxin (dashed black). RNA was isolated from pooled samples of 20 flies 4, 8, and 24 h after injection, and quantitative real time-PCR was done to examine gene expression. The data were normalized using Drosophila RP49 as an endogenous control, and the y-axis represents relative expression compared with uninjected transcript levels set as 1. The experiment was repeated at least three times and in some cases over five times. The error bars represent S.D. The error bars are only shown for the E. coli- and E. coli + Destruxin-injected flies. Statistically significant differences were assessed at all time points using an unpaired one-tailed Student's t test. Asterisks are used to denote significant differences (p < 0.05) between PBS and E. coli (gray) and between E. coli and E. coli + Destruxin injected flies (black).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Destruxin-injected Flies Are More Susceptible to Bacterial Infection—We performed survival assays to determine whether the decrease in antimicrobial peptide expression produced by Destruxins leaves flies more vulnerable to