Functional characterization of the Toll pathway and potential effectors in the colonization of Amblyomma sculptum tick cells by Rickettsia rickettsii

• Grant number: 21/03369-1
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): August 01, 2021
• Effective date (End): April 30, 2023
• Field of knowledge: Biological Sciences - Parasitology - Entomology and Malacology of Parasites and Vectors
• Principal Investigator: Andréa Cristina Fogaça
• Grantee: Beatriz Iglesias Alonso
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Rickettsia rickettsii is etiologic agent of Rocky Mountain spotted fever. After being transmitted by the bite of an infected tick, the bacterium infects the endothelial cells of the vertebrate host, where it proliferates, causing vasculitis, which evolution is potentially fatal. In Brazil, the main vector of R. rickettsii is Amblyomma sculptum [a species of the Amblyomma cajennense complex]. Specifically in the metropolitan region of São Paulo, the transmission is given by Amblyomma aureolatum. R. rickettsii presents transstadial and transovarial transmissions, so that it can be perpetuated for consecutive generations in the natural populations of ticks. Thus, besides vectors, ticks are also reservoirs of R. rickettsii. Pathogens ingested within the blood meal first reach the tick midgut, where they must resist the effects of effector molecules of the tick immune system. If pathogens are successful in evading the attack of these antimicrobial factors, they need to migrate to the hemolymph and reach the salivary glands, so that they can be transmitted, via saliva, to a healthy vertebrate host in a subsequent blood meal. In this context, understanding the interactions between R. rickettsii and its vector ticks is important, and may lead to the identification of targets for the development of strategies to block transmission. Despite belonging to the same genus, A. sculptum and A. aureolatum have marked differences in susceptibility to infection by R. rickettsii, where A. aureolatum is much more susceptible than A. sculptum. Our research group previously determined the effects of infection on the gene expression profile of the midgut and salivary glands of Amblyomma spp. ticks. Among the CDSs of immune system factors modulated by the infection, we highlight GILTs (gamma-interferon-inducible lysosomal thiol reductase) and microplusins. Interestingly, two GILT CDSs were upregulated in the midgut of A. sculptum and one was downregulated in the midgut of A. aureolatum, which might be one of the factors responsible for the differences in susceptibility to infection. Gene silencing of the microplusin CDS Ambaur-69859 by RNA interference (RNAi) resulted in an increase in the prevalence of infected ticks and in the infection level, indicating that this antimicrobial peptide is important for the protection of A. aureolatum against infection. A microplusin CDS similar to A. aureolatum Ambaur-69859 was also upregulated by infection in the salivary glands and in the midgut of A. sculptum (Acaj-57400). In addition, the expression of another microplusin CDS (Acaj-64186) was higher in the midgut of A. sculptum ticks fed on infected hosts, but which were not infected (refractory), in relation to those that were infected (susceptible). In arthropods, the effectors of the immune system can be constitutively expressed or their expression can be induced after the recognition of molecular patterns associated with pathogens by the Toll, IMD and JAK/STAT immune signaling pathways. Few effectors associated with each signaling pathway were identified in ticks. Thus, this proposal aims to determine the effects of gene silencing of components of the Toll pathway, GILTs and microplusins on the proliferation of R. rickettsii in an embryonic cell line of A. sculptum (IBU/ASE-16). For this, the gene expression of Dorsal, Cactus, MyD88, GILTs (Acaj-71750 and Acaj-74441) and microplusins (Acaj-57400 and Acaj-64186) of A. sculptum will be silenced by RNAi. The total number of R. rickettsii and the percentage of gene silencing will be determined by qPCR. In addition, the gene expression of CDSs of GILTs and microplusins in the groups with Dorsal, Cactus and MyD88 silenced to assess whether they are effectors of the Toll pathway.