Drosophila anti-nematode and antibacterial immune regulators revealed by RNA-Seq

Abstract

Background: Drosophila melanogaster activates a variety of immune responses against microbial infections. However, information on the Drosophila immune response to entomopathogenic nematode infections is currently limited. The nematode Heterorhabditis bacteriophora is an insect parasite that forms a mutualistic relationship with the gram-negative bacteria Photorhabdus luminescens. Following infection, the nematodes release the bacteria that quickly multiply within the insect and produce several toxins that eventually kill the host. Although we currently know that the insect immune system interacts with Photorhabdus, information on interaction with the nematode vector is scarce.

Results: Here we have used next generation RNA-sequencing to analyze the transcriptional profile of wild-type adult flies infected by axenic Heterorhabditis nematodes (lacking Photorhabdus bacteria), symbiotic Heterorhabditis nematodes (carrying Photorhabdus bacteria), and Photorhabdus bacteria alone. We have obtained approximately 54 million reads from the different infection treatments. Bioinformatic analysis shows that infection with Photorhabdus alters the transcription of a large number of Drosophila genes involved in translational repression as well in response to stress. However, Heterorhabditis infection alters the transcription of several genes that participate in lipidhomeostasis and metabolism, stress responses, DNA/protein synthesis and neuronal functions. We have also identified genes in the fly with potential roles in nematode recognition, anti-nematode activity and nociception.

Conclusions: These findings provide fundamental information on the molecular events that take place in Drosophila upon infection with the two pathogens, either separately or together. Such large-scale transcriptomic analyses set the stage for future functional studies aimed at identifying the exact role of key factors in the Drosophila immune response against nematode-bacteria complexes.

Fig. 1

Infection of adult flies with Heterorhabditis nematodes or their Photorhabdus bacteria elicits distinct transcriptomic profiles. a Transcriptome summary (number of reads and percentage mapped to the D. melanogaster genome) from flies infected by Heterorhabditis axenic or symbiotic nematodes, or Photorhabdus bacteria at 12 and 30 h post-infection. b Differential gene transcription (upregulated/downregulated genes) in flies at 12 h and 30 h post-infection with Heterorhabditis axenic or symbiotic nematodes, or Photorhabdus bacteria alone. c CUFFLINKS analysis of differentially expressed transcripts between the 12 and 30 h time-points in flies infected by Heterorhabditis axenic or symbiotic nematodes, or Photorhabdus bacteria alone. d Venn diagrams showing the number of Drosophila genes that are differentially expressed (upregulated or downregulated) at 12 h only or at 30 h only or at both time-points after infection with Heterorhabditis axenic or symbiotic nematodes, or their Photorhabdus bacteria alone. Expression patterns are indicated (UP/UP: gene upregulation at both 12 and 30 h, DOWN/UP: gene downregulation at 12 h and upregulation at 30 h, DOWN/DOWN: gene downregulation at both time-points, UP/DOWN: gene upregulation at 12 h and downregulation at 30 h)

Fig. 2

Infection of adult flies with Heterorhabditis nematodes or their Photorhabdus bacteria induces diverse physiological responses. Representative KEGG pathway categories in flies infected by a Heterorhabditis axenic nematodes, b Heterorhabditis symbiotic nematodes, or c Photorhabdus bacteria at 30 h post-infection. The number of genes represents those that were only found associated with a particular pathway. Representative PANTHER pathway categories in flies infected by d Heterorhabditis axenic nematodes, e Heterorhabditis symbiotic nematodes, or f Photorhabdus bacteria at 30 h post-infection

Fig. 3

Infection of adult flies with Heterorhabditis or Photorhabdus trigger the expression of diverse proteins. a Representative protein-based Gene Ontology (GO) groups for genes differentially induced by Heterorhabditis axenic or symbiotic nematodes, or Photorhabdus bacteria alone at 12 and 30 h post-infection with the pathogens. Each bar represents a subset of the most representative non-redundant upregulated and downregulated genes. Numbers of upregulated and downregulated genes upon infection with Heterorhabditis axenic (b), symbiotic (c) nematodes, or Photorhabdus bacteria (d, e) at 12 h and 30 h post-infection with the pathogens. Each bar includes genes that fall into the same molecular function category. GO analysis was performed using the global list of differentially expressed genes for each infection type and time-point

Fig. 4

Heterorhabditis and Photorhabdus induce the expression of diverse subsets of genes in Drosophila adults. The 25 most strongly induced genes upon infection with a, b Heterorhabditis axenic nematodes, c, d Heterorhabditis symbiotic nematodes and e, f Photorhabdus bacteria at 12 h and 30 h post-infection. X-axis represents the relative Log-Fold Change (LFC) for each gene after normalization against uninfected controls. All genes have a fold-change higher than 2 (LFC = 0.58 corresponds to 2-fold-change difference)

Fig. 5

Differential gene expression analysis using DESeq and GLM analysis to compare the different infection types. a Filtered list of genes that are common between all three infection types (p < 0.05); b Filtered list of genes that are common between the Axenic Heterorhabditis vs. Symbiotic Heterorhabditis and Axenic Heterorhabditis vs. Photorhabdus comparisons; c Filtered list of genes that are common between the Symbiotic Heterorhabditis vs. Photorhabdus and Axenic Heterorhabditis vs. Photorhabdus comparisons. The figure contains Drosophila genes with significantly altered expression upon infection of adult flies with the nematodes and their associated bacteria (separately or together), and their corresponding adjusted p-values for the two models used: one to determine the common genes between Heterorhabditis (Axenic/Symbiotic) infections and Photorhabdus infections, and a second to determine the common genes between Axenic Heterorhabditis infection and Photorhabdus infection after adjusting for the two time-points. Selected genes included those that appeared in all three comparisons. Red: LFC ≥ 2-fold downregulation; Yellow: LFC ≤ 2-fold downregulation and Green: LFC ≥ 2-fold upregulation

Fig. 6

Expression of immune-related genes in Drosophila flies infected by Heterorhabditis nematodes or their Photorhabdus bacteria. a Heat map showing immune-related genes that are differentially expressed by Heterorhabditis axenic nematodes, symbiotic nematodes and Photorhabdus bacteria at 12 h and 30 h post-infection. Genes selected from the Gene Ontology (GO) analysis correspond to the immune response category and have a positive expression level as an indication of their upregulation upon infection with the pathogens. Selected genes were assigned to the following immune pathways or immune-related groups: TNF, JAK/STAT, TOLL, JNK, IMD and VEGF pathways; immune induced molecules, PGRPs with unknown function, hematopoiesis, scavenger receptors, lysozymes and others. GO immune response categories identified in flies infected by b Heterorhabditis axenic nematodes, c Heterorhabditis symbiotic nematodes, or d Photorhabdus bacteria at 30 h post-infection. The Y-axis corresponds to the number of genes for each GO category and their relative level of expression (upregulation or down-regulation). All genes have a fold-change higher than 2 (Log Fold-Change = 0.58 corresponds to 2-fold-change difference)