Determination of the effects of pro- or antioxidant agents on Amblyomma sculptum apoptosis and evaluation of the apoptosis inhibitor protein as a vaccine target

Abstract

Ticks are obligate hematophagous ectoparasites that transmit various pathogens to both humans and other animals. Brazilian spotted fever (BSF) is a serious disease that affects humans. It is caused by the bacterium Rickettsia rickettsii and mainly transmitted by the tick Amblyomma sculptum in Brazil. In recent years, the importance of A. sculptum has increased because, in addition to being a vector of R. rickettsii, this species has been progressively more found infesting cattle, causing damage to livestock. Despite the harmful effects of ticks and the pathogens transmitted by them on both human and animal health and on the economy, their control is mainly based on the use of acaricides. However, acaricides can contaminate the environment and products derived from animals that are intended for human consumption. In addition, acaricides can select resistant populations of ticks. In this context, the elucidation of the molecular mechanisms of the interaction of R. rickettsii with its tick vectors is important and may lead to the identification of potential targets for the development of new strategies to control ticks and FMB. Apoptosis is a programmed cell death process that plays an important role in the control of pathogens, since the death of the infected cell prevents the spread of infection to adjacent cells. Our research group has previously demonstrated that R. rickettsii inhibits tick cell apoptosis and that this inhibition is important for bacterial proliferation. Coding sequences (CDS) of regulators that exert an inhibitory action on apoptosis are induced in MG from infected A. sculptum. Among them, the CDS Acaj-73060 encodes a member of the Inhibitor of Apoptosis Proteins (IAPs) family, which inhibit the activation of executor caspases. The knockdown of this CDS mediated by RNA interference (RNAi), followed by blood feeding, caused tick mortality rates close to or equal to 100%. Furthermore, the expression of this CDS, as well as other negative regulators of apoptosis, is repressed in the MG of fed ticks, reinforcing the hypothesis that this death process is activated by the acquisition of a blood meal. It is possible that the heme released by digestion of the blood meal, among other factors, induces the production of reactive oxygen species (ROS) in the midgut of A. sculptum, which, in turn, can activate apoptosis. Thus, determining the effects of administration of pro- or antioxidant agents on the mortality of A. sculptum ticks that received dsIAP Acaj-73060 may provide information about the role of ROS in the activation of apoptosis. Furthermore, the high mortality rates of ticks that received dsIAP Acaj-73060 and were subsequently fed point this protein out as a potential target for the development of a vaccine against A. sculptum. Based on literature data and results previously obtained by our research group, the general objective of this project is to determine the effects of the knockdown of IAP Acaj-73060 associated with pro- or antioxidant agents on the activation of apoptosis in A. sculptum and to evaluate the potential of IAP Acaj-73060 as a target for the development of a vaccine. To this end, we propose as specific objectives: 1. to evaluate the effects of IAP Acaj-73060 silencing associated with the administration of pro- or antioxidant agents on viability, caspase-3 activity and phosphatidylserine exposure in IBU/ASE-16 cells and on the mortality of A. sculptum ticks; 2. to obtain recombinant IAP Acaj-73060 and anti-IAP Acaj-73060 antiserum and 3. to analyze the effects of antiserum administration on tick fitness. (AU)