Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium

Abstract

Background: Whole 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.

Results: We 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.

Conclusions: We 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.

Figure 1

NKpnI PCR amplicons of cDNAs of Gardel strain passage 39 after SCOTS. 0×, 1×, 2× and 3×: cDNA amplicons of NKpnI PCR after 0, 1, 2 or 3 rounds of capture. C+: positive control cDNA with NKpnI tag. C-: Negative control (Water). MW: Molecular weight 100 pb DNA ladder. hpi: hours post infection

Figure 2

Southern blot using DIG labeling cDNAs of Gardel strain at 96 hpi. 0×, 1×, 2× and 3×: Southern blots showing cDNAs of Gardel strain passage 39 before and after one, two and three rounds of SCOTS. rRNA: Southern blot using DIG-rDNA 23S+5S and 16S.

Figure 3

Quantitative RT-PCR targeting ER rRNA 16S on cDNAs of Gardel strain after SCOTS at different time point post infection: 24, 72, 96 and 120 hours post infection.

Figure 4

Quantitative RT-PCR of map1, recA and rpoD genes on cDNA samples of Gardel strain before and after 3 rounds of capture. hpi: hours post infection.

Figure 5

Detection of probes using Gardel strain cDNAs generated after Ribominus (RM) and SCOTS treatment. A: ER Microarray Hybridization with gDNA of Gardel strain passage 40, with cRNAs from Gardel strain passage 47 using RiboMinus purification (RM), with cDNAs from Gardel strain passage 39 before SCOTS (0×) and after 3 rounds of capture (3×). B: Percentage of ER detected probes at 24 and 96 hpi for cRNAs from Gardel strain passage 47 using RiboMinus purification (RM) with cDNAs from Gardel strain passage 39 before SCOTS (0×) and after 3 rounds of capture (3×).