Functional analysis of Mu-class glutathione S-transferase HdGSTm1 from Haemaphysalis doenitzi with a focus on the roles in the tick blood sucking.

Abstract

During blood feeding, ticks face significant oxidative stress due to hemoglobin breakdown. In this context, GSTs are proposed to act as critical antioxidants, possibly by detoxifying lipid peroxides. The transcriptome of H. doenitzi was screened for GST genes, followed by the recombinant expression and functional analysis of these genes. Specifically, HdGSTm1 was cloned by PCR, and the recombinant protein rHdGSTm1 was expressed and purified. The properties of rHdGSTm1 were analyzed, gene transcription was assessed, and RNA interference was employed to evaluate its functional impact. As results, the open reading frame of HdGSTm1 is 693 base pairs (bp) in length and number of coding amino acids is 230 and it belongs to Mu-class GSTs. And the activity of GST for GSH was measured (V_max =1.39 ± 0.07 µmol/(min·mg), K_m=1.254 ± 0.12 µM). It has the highest enzyme activity at pH 7.0, 30 ℃. Antioxidant assay showed that rHdGSTm1 had concentration-dependent antioxidant capacity. The relative expression of HdGSTm1 was higher in adult ticks and the highest in Malpighian tubules. RNAi results demonstrated that HdGSTm1 had a significant effect on bloodsucking (t-test, t₍₃₄₎ = 4.296, p = 0.0001). In summary, this study isolated and characterized a Mu-class GST gene from H. doenitzi. The recombinant protein exhibits free radical scavenging activity. Evaluation of its antioxidant capacity facilitates analysis of the active oxidative stress response mechanism during blood feeding in adult Malpighian tubules, and provides a basis for elucidating the molecular mechanism. Notably, it impacts blood feeding behavior. HdGSTm1, even at low expression levels, can significantly reduce the blood-feeding duration in H. doenitzi.