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.

Wolbachia small noncoding RNAs and their role in cross-kingdom communications

“…Two peaks of antisense expression were observed in a region of the Wolbachia genome corresponding to two hypothetical proteins and annotated as phage major capsid protein E. One of these two structures matched the location, sequence, and length of preWsnRNA-59. The results from Darby et al (15) are therefore consistent with our results. Secondary structure prediction revealed a stem and hairpin loop structure for both WsnRNAs (Fig.…”

“…S3). Interestingly, transcripts of preWsnRNA-59 were sequenced in a small RNA library of Aedes albopictus infected with Wolbachia strain wMelPop-CLA (15). Two peaks of antisense expression were observed in a region of the Wolbachia genome corresponding to two hypothetical proteins and annotated as phage major capsid protein E. One of these two structures matched the location, sequence, and length of preWsnRNA-59.…”

Enzymes Involved in AMPylation and deAMPylation

“…26 Since the original identification of Fic-1 in E. coli , 26,52,53 several Fic-1 homologues have recently been identified and characterized in other bacterial species. 31,36,37,43,54,55 The FicT toxins are typically coexpressed with their antitoxin-containing inhibitory α -helix as part of a toxin–antitoxin system in the cells. 31,36 When expressed without the corresponding antitoxin anti-Fic-1 (AntF), cellular growth is arrested 31,37 (Figure 16).…”

Wolbachia endosymbionts and human disease control

“…Finally, filarial Wolbachia are not just "passive passengers"; their increase in filarial infective stages (L3) and transcriptional profiles indicate a regulatory component of their presence, and likely interactions with the host. Whatever hypothesis one currently envisions, there is clearly a host-symbiont set of co-evolved interactions and caution is urged when trying to ascribe all Wolbachia-nematode interactions to only one particular mechanism [11,37].…”

“…Another method to investigate Wolbachia gene expression has been through the use of Wolbachia containing insect cell lines [11]. Enrichment of the insect Wolbachia from the insect cells prior to RNA extraction and sequencing provides a higher percentage of Wolbachia reads with less contaminating host material.…”