Proteomic analysis of the global response of an arthropod Wolbachia symbiont to doxycycline-induced stress
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ABSTRACT: The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. Additionally, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. For proteomic analysis, Wolbachia-enriched material was purified further using additional filtration and Percoll gradients. Proteins were solubilised by sonication in a MS safe detergent, reduced with dithiothreitol and alkylated with iodoacetamide. Proteomic grade trypsin was added at a protein:trypsin ratio of 50:1, and samples were incubated at 37 C overnight prior to removal of detergent by trifluoroacetic acid (TFA) precipitation. Peptide mixtures were analysed by nano LC-MSMS using a LTQ-Orbitrap Velos (ThermoFisher Scientific). In one control sample, peptides were fractionated by strong anion exchange (SAX) using commercially available SAX spin filters (Thermo Scientific). Data was imported into Progenesis LC-MS (Nonlinear Dynamics) and runs were time-aligned using default settings. Peaks were picked by the software. Peptide intensities were normalised against an auto-selected reference run and differences in protein expression and associated ANOVA P-values between seven control and nine doxycycline-treated biological replicates were calculated by Progenesis LC-MS. Spectral data were transformed to MGF files with Progenesis LC-MS and exported for peptide identification using the Mascot search engine (Matrix Science). Search parameters were set to a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.5 Da, while one missed tryptic cleavage was permitted. Carbamidomethylation (cysteine) was set as a fixed modification and oxidation (methionine) set as a variable modification. Mascot search results were further processed using the machine learning algorithm Percolator. The false discovery rate was less than 1%, and individual ion scores greater than 13 were considered to indicate identity or extensive homology (P less than 0.05). Data were imported into Progenesis LC-MS as XML files. For a protein to be considered differentially expressed, a FC of greater than 1.5 and a P-value of less than 0.01 was required, supported by greater than 2 unique peptides.
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Wolbachia Sp. Wmelpop
TISSUE(S): Permanent Cell Line Cell, Cell Culture
SUBMITTER: 
Stuart Armstrong
LAB HEAD: Stuart Armstrong
PROVIDER: PXD000345 | Pride | 2013-10-21
REPOSITORIES: Pride
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