Analysis of thioester-containing proteins during the innate immune response of Drosophila melanogaster

Abstract

Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1-Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila.

Fig. 1

Phylogenetic analysis of Tep genes. a Phylogenetic analysis of available protostomian Tep sequences. b Phylogenetic analysis of Tep sequences issued from the 12 sequenced Drosophila species. The references for all sequences used to generate the phylogenetic trees are provided in online suppl. table S1 (see also online suppl. fig. S1 for a phylogenetic tree of the species used for the interpretation of the Tep phylogenetic trees). Sequences indicated by an asterisk encode a nonfunctional thioester motif.

Fig. 2

Phylogenesis of exon 5 of Tep2. a Phylogenetic analysis of the exons 5 of Drosophila Tep2s. b Positions of the exons 5 on the genome of Drosophila species. The letters A, B and C refer to those given in FlyBase for melanogaster species; we have inverted D and E to follow the disposition on the chromosome.

Fig. 3

Modulation of the expression of Tep genes in adults after septic injury. a, b Drosophila Teps are located on the left arm of the second chromosome. a Tep2 (CG7052) and Tep3 (CG7068) map to cytogenetic position 28C1. Tep2 and Tep3 are 1.5 kb apart. The modified XP transposable element XP11521 is inserted 22 bp upstream of the transcription-starting site of Tep2, whereas the XP03976 element is inserted 121 bp downstream of the transcription- starting site of Tep3. We have generated a double mutant for Tep2 and Tep3, Tep2, 3, by FLP-mediated recombination between the FRT elements carried on the XP elements. b Tep4 (CG10363) maps to cytogenetic position 37F1. Transposable elements inserted in this region are shown. We have used EY04656 as a mutant for Tep4 as this modified P element is inserted in the initiation codon of the Tep4 gene. In the following experiments, we used an RNAi transgenic line to knock down Tep1 (CG18096). c-f Steadystate transcript levels of D. melanogaster Tep genes were measured by quantitative RT-PCR before and after infection with a mix of E. coli and M. luteus. These experiments are representative of at least two independent experiments. 0 h = Non-infected flies; 3 h/6 h/48 h = flies 3/6/48 h after infection; WT = wild type. Flies were frozen for experiments 3, 6, and 48 h after infection. Gene expression was normalized against rp49 gene expression and the results are expressed as percentage of maximal expression: 6 (Tep1; c) and 3 h after infection (Tep2 and Tep4; d, e). Expression of a Tep6 transgene under the control of a heat shock promoter using the UAS-GAL4 system (hsp>Tep6) after heat shock (f). ∗ p<0.05. c The induction of Tep1 is decreased by the expression of an RNAi transgene targeting specifically this gene.

Fig. 4

Expression of Tep genes in larvae. Whole-mount ISH on late third instar larvae. a-d Tep2. e-j Tep4. k-n Tep6. Left-hand panels = Tep gene expression; right-hand panels = controls. The probe used for ISH is indicated on the top right corner of each panel, whereas the genotype is indicated on the bottom right corner. The following tissues are shown: hemocytes (a, b, e, f, k, l); fat body (c, d, g, h); lymph glands (arrow), central nervous system, and imaginal discs (arrowhead; i, j, m, n). For Tep6, we used the sense probe on wild-type (WT) larvae as control, as Tep6 mutants (Mcr) are lethal.

Fig. 5

Expression of Tep genes in adults. ISH on paraffin-embedded longitudinal and transverse sections. a, b Tep2. c-h Tep4. i-n Tep6. Left-hand panels = Tep gene expression; right-hand panels = controls. The probe used for ISH is indicated on the top right corner of each panel, whereas the genotype is indicated on the bottom right corner. The following tissues are shown: crop (arrows) and hypoderm (arrowheads; a-f, i, j); head (g, h); proventriculus (cardium; k, l); mesophragma (arrowheads; e, f, m, n). For Tep6, we used the sense probe on wild-type (WT) flies as control, as Tep6 mutants (Mcr) are lethal.

Fig. 6

Tep mutants survive as wild-type (WT) flies to different types of infection. Survival experiments after distinct challenges in the septic injury model are presented and are representative of at least two independent experiments. The appropriate controls for the different microbes have been used: Gram-positive bacteria, fungi: mutants of the Toll pathway [Dif, spätzle (spz), PGRP-SA] and IMD pathway: kenny (key). None of the Tep mutants shows a reproducible susceptibility or resistance to infection phenotype, either in homozygous or hemizygous conditions. We used the logrank test to determine the significance between wild-type and mutant survival curves. ∗∗∗ p < 0.001 was the only constantly measured p value in several experiments.

Fig. 7

Tep mutants display normal phagocytosis activity. Adult Drosophila carcasses from WT (a, c) and Tep 2,3,4 mutant(b). pHrodo™ E. coli BioParticles® produce a red fluorescence when exposed to an acidic environment such as that present in the phagosome. It labeled sessile hemocytes (arrowheads; a, b) and pericardial cells (asterisk; a-c). Injection of latex beads, prior to pHrodo E. coli BioParticles injection (WT + Lxb), blocked phagocytosis. Note that the pericardial cells were unspecifically labeled.