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GALLETTI, M. F. B. M. Effects of the temperature and blood feeding on the gene expression profile of Rickettsia rickettsii during infection of its tick vector Amblyomma aureolatum. 2013. 148 p. Ph. D. thesis (Doctorate in Biology of HostPathogen Relation) – University of São Paulo, Institute of Biomedical Sciences, São Paulo, 2013. Rickettsia rickettsii is the causative agent of Rocky Mountain Spotted Fever, which is the most lethal spotted fever rickettsiosis that affects humans. The main tick species that transmits R. rickettsii in the metropolitan area of São Paulo’s city is Amblyomma aureolatum. When an infected and starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. The main aim of the present work was to determine the effects of these two environmental stimuli on the R. rickettsii transcriptional profile during A. aureolatum infection. We initially established an effective system for rickettsia propagation to generate a substantial quantity of genetic material for microarray standardization. For that, for the first time, we established an in vitro infection of the virulent Brazilian R. rickettsii strain in the BME26 tick embryonic cell line from Rhipicephalus (Boophilus) microplus. Using customized oligonucleotide microarrays, we analyzed the effects of a 10°C temperature elevation and a blood meal on the transcriptional profile of R. rickettsii infecting whole organs of Amblyomma aureolatum female ticks. This is the first bacterial transcriptome study of the Rickettsia genus when infecting a natural tick vector. Although both stimuli significantly increased the bacterial load, blood feeding had a greater effect, also modulating five-fold more genes than the temperature upshift. Among the genes induced by blood-feeding, some encode virulence factors, such as Type IV Secretion System (T4SS) components, suggesting that this important bacterial transport system is used to secrete effectors during the acquisition of the blood meal by the tick. Using an in silico conserved domain analysis of hypothetical proteins, we identified additional T4SS components of R. rickettsii that were never previously described. Blood-feeding also up-regulated the expression of antioxidant enzymes, which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. Finally, we studied the transcriptional profile of selected genes of R. rickettsii on the salivary glands and midguts of male and female ticks by microfluidic RT-qPCR. Results showed that temperature upshift and blood feeding modulate specific sets of genes in each tissue, allowing for the establishment of a tissue-specific transcriptional signature. The modulated genes identified in this study require further functional analysis and may have potential as future targets for vaccine development.

Efeitos da temperatura e da alimentação sanguínea sobre o perfil de expressão gênica de Rickettsia rickettsii durante a infecção do carrapato-vetor Amblyomma aureolatum.