Project : PXD000345

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Summary

Title

Proteomic analysis of the global response of an arthropod Wolbachia symbiont to doxycycline-induced stress

Description

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.

Sample Processing Protocol

See details in reference PMID : 24152719

Data Processing Protocol

See details in reference PMID : 24152719

Contact

Stuart Armstrong, Infection Biology

Submission Date

17/07/2013

Publication

    Darby AC, Christina Gill A, Armstrong SD, Hartley CS, Xia D, Wastling JM, Makepeace BL; Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress., ISME J, 2013 Oct 24, PubMed(s) : 24152719

Assay

Accession Title Protein Count Peptide Count Unique Peptide Count Spectrum Count Identified Spectrum Count
30115 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 1 782 3394 1808 9456 3120
30116 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 2 2798 14580 7854 25686 11657
30117 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 3 2029 10973 5701 21481 9319
30118 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 4 260 923 502 5354 845
30119 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 5 433 1657 928 6820 1489
30120 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 8 2317 8508 5793 16102 6505
30121 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 9 1956 5537 3864 9794 4075
30122 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 10 1780 6531 4213 11190 4853
30123 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 11 2471 8969 6025 16080 6763
30124 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 12 2403 9177 6147 15992 6961
30125 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 13 2955 10942 7285 18819 8295
30126 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 14 2193 7713 5257 15092 5892
30127 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 15 2253 7679 5170 14552 5686
30128 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 1 2396 7983 5381 15346 5868
30129 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 2 2963 11079 7212 19863 8374
30130 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 3 1615 5027 3494 10719 3837
30131 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 4 3098 11549 7403 20620 8616
30132 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 5 2189 7383 5050 13663 5612
30133 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 6 2809 10548 6787 18281 7859
30134 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 7 2843 10604 6860 18296 8053
30135 Proteomic analysis of the global response of an obligate intracellular symbiont (Wolbachia) to doxycycline-induced stress - Experiment 5 2310 8255 5548 13445 6372

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