Identification, characterization and analysis of expression of gene encoding carboxypeptidase A in Anopheles culicifacies A (Diptera: culicidae)
Graphical abstract
Introduction
Malaria considered as one of the deadliest disease of mankind was responsible for more than 207 million cases of malaria and nearly 627000 deaths worldwide during year 2012 (World Health Organization, 2013). Despite the worldwide efforts to counter the disease, the mortality due to malaria has increased due to widespread resistance by both vector and parasite towards insecticides and drugs, respectively. Genetic transformation of disease vectors seems to be promising approach at present. Various tools those are required for efficient transgene expression has been developed during last several years. List of anti-parasitic molecules has also been enlarged. Efficient expression of such genes in the vector requires time and tissue specific gene regulatory elements. Promoters specific for gut, salivary gland, fat body & hemolymph have been shown to drive transgene expression in tissue specific manner. Mosquito midgut is the first site where parasite interacts and later develops into further stages. Before successful transformation into further stages the parasite has to cross tough physical and chemical barriers offered by gut lumen. (Abraham et al., 2005). Thus, the hostile environment of gut lumen mounts strong selection pressure on parasite development and provides ideal site where expression of anti-parasitic molecule should complement mosquito innate defence more effectively than any other tissue. Blood meal induced gut transcribed regulatory elements are ideal candidates to drive the expression of anti-parasitic genes. Different mosquito gut specific gene regulatory elements available today have been successfully used in transgene expression. Transgenic mosquitoes using promoter of carboxypeptidase gene (Franz et al., 2006, Ito et al., 2002, Kim et al., 2004, Meredith et al., 2011; Moreira et al., 2000, Moreira et al., 2002), peritrophic matrix protein (AgAper1) (Abraham et al., 2005) and recently trypsin (Antryp 1) & G12 (Nolan et al., 2011) have been constructed in last decade.
Carboxypeptidases are zinc-metalloproteases that cleave one amino acid at a time from c-terminus of the protein. These have been categorised as A/B (Digestive) and N/E (Regulatory) carboxypeptidases. Digestive carboxypeptidases have been divided into A and B classes depending upon their preference for aliphatic and basic residues (Vendrell and Aviles, 1999).
The first carboxypeptidase cloned & characterized from mosquito was from Anopheles gambiae and named AgCP. The expression of Carboxypeptidase A is regulated by blood feeding and its presence is detectable within half hour of the blood feeding (Edwards et al., 1997). Later Lavazec et al. (2005) characterized the expression of 23 CP-like genes in the same species. The carboxypeptidase gene family has been extensively studied in Ae. aegypti and it is family of 18 genes. Eleven out of these 18 belongs to carboxypeptidase A (Isoe et al., 2009). Interestingly, the expression pattern of CPA in both species is quite dissimilar (Edwards et al., 2000). The differences between expressions pattern of An. gambiae carboxypeptidase A (AgCP) and Ae. aegypti carboxypeptidase A (AeCPA) raises the possibility that there are different upstream elements that drive their expression. In similar way, there are possibilities that some differences in upstream elements may exist between species to species in same genus.
An. culicifacies is principal vector of malaria in India and neighbouring countries and exists as a species complex of five sibling species named A, B, C, D and E and is responsible for 60–65% of the annual malaria cases. Sibling species A, C, D and E are vectors while B is non vector (Subbarao and Sharma, 1997, Kar et al., 1999). Here we describe the cloning, comparative sequence analysis and expression of carboxypeptidase A (AcCP) in An. culicifacies A.
Section snippets
Mosquitoes
Anopheles culicifacies A, mosquitoes were reared in an insectary maintained at a temperature of 28 ± 2 °C and 75 ± 5% relative humidity and fitted with simulated dusk and dawn machine with a photoperiod of 14 h day and 10 h night as described by Chugh et al. (2011).
Adults mosquitoes were fed upon 5% glucose soaked pads. After 5 days of eclosion female mosquitoes were offered rabbit blood for ovarian development. Mosquitoes were starved by denying access to sugar and water for 8 h before blood feeding.
Identification of carboxypeptidase A encoding Anopheles culicifacies gene
Based on the conserved regions of the An. gambiae CPA, we attempted to isolate the carboxypeptidase A gene from An. culicifacies. Initially able to isolate 300 base pairs of coding region, by primer walking we cloned 3 kb of the gene. The gene harbours an open reading frame of 1290 bp which codes for a 429 amino acid protein with a predicted molecular weight of 48.5 kDa. BLAST searches revealed that the predicted gene shares high similarity with carboxypeptidases from other organisms. It is more
Discussion
The carboxypeptidases are the first enzymes which are induced in mosquito gut in response to blood meal and initiate preliminary digestive process. In the present study, the coding sequence of carboxypeptidase A of An. culicifacies A (AcCP) shares 82% and 69% identity to An. gambiae and Ae. Aegypti carboxypeptidase A coding sequences, respectively. However, the upstream sequence of AcCP does not share so much similarity. The position of TATA box from start codon was found to be −60 in An.
Conclusion
The expression pattern of AcCP shows that the gene is expressed rapidly in response of blood meal. The expected role of early induction of AcCP has been ascribed to the initiation of protein digestion process and triggering of endopeptidase activity. Sufficient tools for genetic transformation of mosquitoes including Anopheles exist these days. The study of carboxypeptidase A in An. culicifacies provides another tool for construction of transgenic mosquitoes whose upstream regulatory elements
Acknowledgements
The author Ashwani Kumar highly acknowledges the INSPIRE-SRF awarded by DST (Grant No. DST/INSPIRE Fellowship/2009/[xxvii]), Govt. of India.
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