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Functional characterization of protein disulfide isomerases in Amblyomma sculptum tick cell colonization by Rickettsia rickettsii

Grant number: 21/11118-9
Support type:Scholarships in Brazil - Master
Effective date (Start): April 01, 2022
Effective date (End): September 30, 2023
Field of knowledge:Biological Sciences - Parasitology - Entomology and Malacology of Parasites and Vectors
Principal researcher:Andréa Cristina Fogaça
Grantee:Vitoria Vaz Bibiano
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Rickettsia rickettsii is the etiologic agent of Rocky Mountain spotted fever. After the transmission through the bite of an infected tick, the bacterium infects the endothelial cells of the vertebrate host, where it proliferates, causing vasculitis, with a potentially fatal evolution. In Brazil, the main vector of R. rickettsii is Amblyomma sculptum [a species of the Amblyomma cajennense species complex]. Specifically, in the metropolitan region of São Paulo, Amblyomma aureolatum is the vector. R. rickettsii presents both transstadial and transovarial transmissions, so it can be perpetuated for consecutive generations in natural tick populations. Thus, besides being vectors, ticks are also reservoirs of R. rickettsii. Pathogens ingested with the blood meal first reach the tick midgut, where they must resist the effects of the effector molecules on the immune system. If they succeed in evading the attack of antimicrobial factors in the midgut, pathogens need to migrate to the hemolymph and reach the salivary glands, so that they are transmitted, via saliva, to a healthy vertebrate host in a subsequent blood meal. In this context, a better understanding of the interactions between R. rickettsii and its tick vectors is important, and may lead to the identification of potential targets for the development of strategies to block transmission. Despite belonging to the same genus, A. aureolatum and A. sculptum exhibit marked differences in relation to the susceptibility to infection by R. rickettsii, with A. aureolatum being more susceptible than A. sculptum. Our research group previously determined the effects of infection on the gene expression profile of A. aureolatum salivary glands. Among the CDSs upregulated by infection, we highlight a protein disulfide isomerase (PDI; Ambaur-64469). The knockdown of the PDI of A. aureolatum by RNAi made ticks more susceptible to infection. PDIs are redox proteins of the thioredoxin superfamily, which are necessary for the correct formation of disulfide bonds and folding of proteins in the endoplasmic reticulum (ER). In A. sculptum, a PDI similar to the CDS Ambaur-64469 was detected (AcajSigP-77638). In addition, RNA-seq analyzes showed five other PDI CDSs in A. sculptum, among which Acaj-77698 and AcajSigP-77333 also possess the conserved active sites of PDIs. The development of tick cell lines has contributed significantly to the advancement of scientific research on the interactions between ticks and tick-borne pathogens, as they allow that experiments be conducted more quickly and under controlled conditions before experiments involving the use of animals. Thus, the general objective of this proposal is to determine the functional role of PDIs on the colonization of an embryonic cell line of A. sculptum (IBU/ASE-16) by R. rickettsii. To that end, the effects of infection of the gene expression of PDIs AcajSigP-77638, Acaj-77698 and AcajSigP-77333. In addition, PDIs will be silenced by RNAi and the percentage of gene silencing will be assessed by RT- qPCR and the total number of R. rickettsii will be determined by qPCR.

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