Molecular characterization of the carboxypeptidase B1 of Anopheles stephensi and its evaluation as a target for transmission-blocking vaccines

Abstract

Malaria is one of the most important infectious diseases in the world, and it has many economic and social impacts on populations, especially in poor countries. Transmission-blocking vaccines (TBVs) are valuable tools for malaria eradication. A study on Anopheles gambiae revealed that polyclonal antibodies to carboxypeptidase B1 of A. gambiae can block sexual parasite development in the mosquito midgut. Hence, it was introduced as a TBV target in regions where A. gambiae is the main malaria vector. However, in Iran and neighboring countries as far as China, the main malaria vector is Anopheles stephensi. Also, the genome of this organism has not been sequenced yet. Therefore, in this study, carboxypeptidase B1 of A. stephensi was characterized by genomic and proteomic approaches. Furthermore, its expression pattern after ingestion of Plasmodium falciparum gametocytes and the effect of anti-CPBAs1 antibodies on sexual parasite development were evaluated. Our results revealed that the cpbAs1 expression level was increased after ingestion of the mature gametocytes of P. falciparum and that anti-CPBAs1 directed antibodies could significantly reduce the mosquito infection rate in the test group compared with the control group. Therefore, according to our findings and with respect to the high similarity of carboxypeptidase enzymes between the two main malaria vectors in Africa (A. gambiae) and Asia (A. stephensi) and the presence of other sympatric vectors, CPBAs1 could be introduced as a TBV candidate in regions where A. stephensi is the main malaria vector, and this will broaden the scope for the potential wider application of CPBAs1 antigen homologs/orthologs.

Fig 1

Full-length sequence of cpbAs1 cDNA and its predicted amino acid sequence. Nucleotide and amino acid numbers are shown on the left. The solid arrow shows the signal-peptide cleavage site, and the open arrow shows the zymogen activation site. The amino acids that are predicted to include the active site and are involved in zinc binding are shown in boxes, and residues that are involved in substrate binding and cleavage are shown in bold. The aspartic acid residue that is predicted to determine the substrate specificity is underlined. The lowercase letters at the begining and the end of the sequences are related to 3′- and 5′-untranslated regions, respectively.

Fig 2

Alignment of CPBAs1 with 10 CPB enzymes of other organisms. The residues that are involved in zinc and substrate binding are shown in boxes. The GenBank nucleotide sequence accession numbers of the aligned sequences are as follows: carboxypeptidase B of Anopheles stephensi, ADD31639.1; carboxypeptidase B precursor of Anopheles gambiae, AAS99341.1; carboxypeptidase B preproprotein of Homo sapiens, NP_001862; carboxypeptidase B2 of Homo sapiens, AAT97987.1; carboxypeptidase B precursor of Rattus norvegicus, NP_036665.1; carboxypeptidase B precursor of Sus scrofa (porcine), P09955.5; carboxypeptidase B of Aedes aegypti, AAT36734.1; carboxypeptidase B of Aedes aegypti, AAT36733.1; carboxypeptidase B of Aedes polynesiensis, AAT36736.1; zinc carboxypeptidase of Glossina morsitans morsitans, ADD19124.1; Drosophila sechellia, XP_002035702.1; carboxypeptidase B of Culex quinquefasciatus, XP_001856154.1; carboxypeptidase of Eupolyphaga sinensis, AEI58636.1.

Fig 3

Three-dimensional structures of CPBAs1 (A) and CPBAg1 (B). The model visualization was performed by the SWISS-MODEL web server (http://swissmodel.expasy.org/).

Fig 4

SDS-PAGE and Western blotting of CPBAs1. (A) SDS-PAGE analysis of recombinant CPBAs1 that was expressed in E. coli Origami after purification with Ni-NTA agarose. (B) Western blotting of midgut-extracted protein cocktail (lane 1) and purified recombinant CPBAs1 (lane 2). To perform Western blotting, rabbit anti-CPBAs1 polyclonal antibody and horseradish peroxidase-conjugated anti-rabbit antibody were used as first and second antibodies, respectively.

Fig 5

The effect of GEMSA (2-guanidinoethylmercaptosuccinic acid) and 1,10-phenanthroline on CPBAs1 activity. The activity of the recombinant CPBAs1 was assayed after incubation with the zinc-carboxypeptidase inhibitor 1,10-phenanthroline and GEMSA (the active site-directed inhibitor), using Hyp-Arg as the substrate. The activity of 100% corresponds to 15,125 and 14,840 units/mg for 1,10-phenanthroline and GEMSA experiments, respectively.

Fig 6

Expression pattern analysis of cpbAs1 gene in A. stephensi midgut after ingestion of mature gametocytes. The expression pattern of the cpbAs1 gene was analyzed in different time frames after blood feeding on day 5 postemergence. The S7 ribosomal protein gene was used as the housekeeping gene for normalizing the data. Asterisks indicate statistically significant differences compared to the noninfected blood-fed group, based on the P value from the Wilcoxon test (<0.05). Bars indicate standard deviations from the three independent experiments.

Fig 7

Evaluating the effect of anti-CPBAs1 antibodies on sexual P. falciparum development. The frequency of infected mosquitoes (A) and intensity of infection (B) were evaluated on day 7 postinfection after midgut dissection. The proportion of the infected mosquitoes was determined by mean value from each series of three independent infections. Asterisks indicate statistically significant differences at the 95% confidence level, based on the P value from the chi-square test (A) and ANOVA (B). Numbers above the columns indicate the sample size.