Expression and biochemical characterisation of components of an acaricide metabolising cytochrome P450 from the cattle tick, Rhipicephalus microplus, to improve the efficacy of control methods.

Abstract

The Southern cattle tick, Rhipicephalus microplus Canestrini (1888), is a highly burdensome pantropical ectoparasite of domestic cattle. This threat to economic and food security is exacerbated by the increasing prevalence of acaricide (pesticide) resistance in the cattle tick. One mechanism by which R. microplus is evolving resistance is upregulation of detoxifying enzymes, such as Cytochrome P450. A recently isolated P450, CYP3006G8, was previously identified as a metaboliser of the pyrethroid class of acaricide. Biochemically characterising components of this enzyme complex is an important means of understanding how this complex can be targeted and its effects mitigated. The approach to this research took the aim of biochemically characterising CYP3006G8 and its redox partners, P450 oxidoreductase (POR) and cytochrome b5, and divided it into three major objectives: (1) comparative enzyme kinetic studies of the FAD binding domain (FADBD) of POR from R. microplus, from the tick’s host species Bos taurus, from the malarial mosquito Anopheles gambiae, and from the mosquito’s host Homo sapiens. (2) Optimisation of the co-expression of active CYP3006G8 and POR in an E. coli JM109 cell model. (3) Quantification of POR and cytochrome b5 expression in 11 strains of R. microplus with known resistance profiles. These investigations found the kinetic profiles of the arthropod FADBD, assessed using NADPH and inhibitory NADPH analogues, to be sufficiently distinct from the mammalian FADBD as to tentatively indicate tick POR and mosquito POR as promising targets for inhibition by chemical control methods. CYP3006G8 and its POR were co-expressed in the E. coli using 1 mM IPTG and incubation at 20 °C for 24 hours, with a yield of 0.18 μM active P450, and POR activity of 114.29 μM. This represented the first successful expression of CYP3006G8 and its POR in a prokaryotic cell model.

Date of Award	25 Jul 2024
Original language	English
Awarding Institution	Northumbria University
Supervisor	Meng Zhang (Supervisor) & Robert Finn (Supervisor)