Effects of doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

Abstract

Background: Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods: Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion: Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions: Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.

Figure 1

Transmission electron micrographs of Brugia malayi female worms. Panel A and C are sections of untreated female worm. Panel A shows numerous Wolbachia (arrows) in the hypodermis. Panel C shows, intact embryos in the uterus. Panel B and D are sections from doxycycline treated worms. The hypodermis is free of Wolbachia (Panel B) and only degenerate embryos were present in the uterus of treated worms (Panel D). U, uterus; C, cuticle; UW, uterine wall; MF, microfilaria; mtr, mitochondria. Scale bar = 1 μm.

Figure 2

Distribution of KEGG pathways mapped to up-regulated and down-regulated Brugia malayi genes after doxycycline treatment. Many genes involved in transport, signaling, tanslation, metabolism and cell communication pathways were affected. Significant canonical pathways (P ≤ 0.05) and number of genes mapped in each pathway and their percentages are shown in additional file 12 for regulated genes.

Figure 3

Correlation between expression values for Wolbachia and Brugia malayi genes obtained by microarray (y-axis) and qRT-PCR (x-axis). Normalized fold change values of microarray experiment and 2^{-ΔΔ Ct} values of qRT-PCR obtained for the same genes were Log transformed and analysed by the nonparametric Spearman rank correlation test (R = 0.4352; P = 0.007).