Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium.
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.
Emboulé
Loïc
L
UMR 15 CIRAD-INRA, Contrôle des maladies animales exotiques et émergentes, Site de Duclos, Prise d'Eau 97170, Petit Bourg, Guadeloupe. loic.emboule@cirad.fr
Daigle
France
F
Meyer
Damien F
DF
Mari
Bernard
B
Pinarello
Valérie
V
Sheikboudou
Christian
C
Magnone
Virginie
V
Frutos
Roger
R
Viari
Alain
A
Barbry
Pascal
P
Martinez
Dominique
D
Lefrançois
Thierry
T
Vachiéry
Nathalie
N
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016995:Ehrlichia ruminantium / Q000737:chemistry* / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D006041:Goats; D017422:Sequence Analysis, DNA / Q000379:methods*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D018076:DNA, Complementary