Dataset Information

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress.

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. In addition, 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. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales.

SUBMITTER: Darby AC

PROVIDER: S-EPMC3960535 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress.

Darby Alistair C AC Gill A Christina AC Armstrong Stuart D SD Hartley Catherine S CS Xia Dong D Wastling Jonathan M JM Makepeace Benjamin L BL

The ISME journal 20131024 4

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. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics lea ...[more]

PMID: 24152719

Similar Datasets

Project 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.

Project description:Maize (Zea mays L.) is one of the major staple crops providing human food, animal feed, and raw material support for biofuel production. For its growth and development, maize requires essential macronutrients. In particular, nitrogen (N) plays an important role in determining the final yield and quality of a maize crop. However, the excessive application of N fertilizer is causing serious pollution of land area and water bodies. Therefore, cultivating high-yield and low-N-tolerant maize varieties is crucial for minimizing the nitrate pollution of land and water bodies. Here, based on the analysis of the maize leaf transcriptome and proteome at the grain filling stage, we identified 3957 differentially expressed genes (DEGs) and 329 differentially abundant proteins (DAPs) from the two maize hybrids contrasting in N stress tolerance (low-N-tolerant XY335 and low-N-sensitive HN138) and screened four sets of low-N-responsive genes and proteins through Venn diagram analysis. We identified 761 DEGs (253 up- and 508 down-regulated) specific to XY335, whereas 259 DEGs (198 up- and 61 down-regulated) were specific to HN138, and 59 DEGs (41 up- and 18 down-regulated) were shared between the two cultivars under low-N-stress conditions. Meanwhile, among the low-N-responsive DAPs, thirty were unique to XY335, thirty were specific to HN138, and three DAPs were shared between the two cultivars under low-N treatment. Key among those genes/proteins were leucine-rich repeat protein, DEAD-box ATP-dependent RNA helicase family proteins, copper transport protein, and photosynthesis-related proteins. These genes/proteins were involved in the MAPK signaling pathway, regulating membrane lipid peroxidation, and photosynthesis. Our results may suggest that XY335 better tolerates low-N stress than HN138, possibly through robust low-N-stress sensing and signaling, amplified protein phosphorylation and stress response, and increased photosynthesis efficiency, as well as the down-regulation of 'lavish' or redundant proteins to minimize N demand. Additionally, we screened glutathione transferase 42 (ZmGST42) and performed physiological and biochemical characterizations of the wild-type (B73) and gst42 mutant at the seedling stage. Resultantly, the wild-type exhibited stronger tolerance to low N than the mutant line. Our findings provide a better understanding of the molecular mechanisms underlying low-N tolerance during the maize grain filling stage and reveal key candidate genes for low-N-tolerance breeding in maize.

Dataset Information

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress.

Publications

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets