Ehrlichia ruminantium (ER), the causative agent of heartwater on ruminants, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma. Previous studies have shown that early stages of development may be critical for Ehrlichia pathogenicity. To gain insights into the biology of intracellular ER, we determined the genome-wide transcriptional profile of ER replicating inside bovine aortic endothelial cells using DNA microarrays. At intermediate and late stages of infection (reticulate and elementary bodies, respectively), a total of 54 genes were differentially expressed. Among them, we measured by q-RTPCR the overexpression of 11 of 14 genes. A number of genes involved in metabolism, nutrient exchange, and defense mechanisms, including those involved in resistance to oxidative stress, were significantly induced in ER reticulate bodies. This is consistent with the oxidative stress condition and nutrient starvation that seem to occur in Ehrlichia-containing vacuoles. During the lysis stage of development, when ER is infectious, we showed the overexpression of a transcription factor, dksA, which is also known to induce virulence in other pathogens such as Salmonella typhimurium. Our results suggest a possible role of these genes in promoting ER development and pathogenicity.

BackgroundWhole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood.ResultsWe successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively.ConclusionsWe conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.

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