Molecular characterization and tissue localization of glutathione S-transferase from adult Ancylostoma ceylanicum

Abstract

Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.

Molecular characteristics of glutathione transferase gene family in a neglect medical Spirometra tapeworm

“…Hence, many parasitic helminth GSTs are considered to be important targets for the treatment or immune intervention of parasitic infections ( 11 ). Current studies have confirmed that glutathione transferase displayed effective protective immunity in experimental animals for antihelminth ( 12 , 13 ). According to their amino acid conservation and phylogenetic inferences, the GST superfamily can be subdivided into four independent and unrelated families: cytosolic GSTs (cGSTs), microsomal GSTs (MAPEG), mitochondrial GSTs (kappa GSTs or GSTK) and bacterial GSTs ( 14 ).…”

Soil-Transmitted Helminth Vaccines: Are We Getting Closer?

“…Further, the same vaccine nanoparticle, when incorporated into natural and unnatural hydrophobic amino acids, also significantly reduced both worm and egg burden in orally vaccinated mice following N. brasiliensis challenge without the need for adjuvant ( 93 ). Pre-clinical studies also suggested that GSTs from N. americanus, A. caninum , or A. ceylanicum to be promising vaccine candidates ( 230 ). For example, Na-GSTs from the dog hookworm A. caninum (Ac-GST-1) elicited a significant reduction in adult hookworm burdens following challenge infection compared to control animals ( 225 , 231 ).…”

Twenty-five-year research progress in hookworm excretory/secretory products

“…Goud et al [92] expressed Na-GST-1 in the yeast Pichia pastoris and purified it by three chromatography phases, producing a 23.7-kDa protein with a 51% yield and a 98% purity. Furthermore, a 24 kDa Ac-GST-1 was cloned from adult A. caninum [93], and its homologous enzyme has been isolated from A. ceylanicum [94]. Ac-gst-1 mRNA was detectable in all developmental stages, but its protein was only detectable in adult worm extracts and ES products [93].…”