Malaria control is heavily reliant on the use of insecticides for interventions such as Indoor Residual Spraying (IRS) and Long Lasting Insecticide Nets (LLINs). Widespread resistance to insecticides in the malaria vector Anopheles gambiae threatens these interventions. To ensure the continued effectiveness of malaria control strategies, novel insecticides and insecticide synergists must be formulated, and in order to do so, new insecticide targets must be investigated. Digestion of huge volumes of vertebrate blood in the midgut of An. gambiae mosquitoes results in the production of large concentrations of free haem: a potentially cytotoxic molecule. Amongst the adaptations haematophagous arthropods have for limiting haem mediated toxicity is haem degradation by the haem oxygenase system. Haem oxygenase, with its obligate redox partner, cytochrome P450 reductase, catalyses the hydrolysis of haem into biliverdin, producing carbon monoxide and ferrous iron. To investigate the biochemical properties of An. gambiae haem oxygenase (AgHO), the enzyme was cloned and expressed in E. coli. The purified AgHO was observed to catalyse the degradation of haem to biliverdin, releasing CO and Fe2+, in the presence of insect and human CPR reducing systems. AgHO bound haem stoichiometrically, with a KD closer to that of H. sapiens and C. diphtheriae than that of D. melanogaster. Application of the HO inhibitors Sn-protoporphyrin and Zn-protoporphyin to mosquitoes as a bloodmeal supplement revealed a dose-dependent reduction in egg laying capability of An. gambiae. It is unclear whether this is due to a detrimental effect on mosquito health because of haem mediated cytotoxicity, or if this reduction in reproductive fitness is due to a key role for AgHO in the reproductive system. A range of truncated CPRs from insects (An. gambiae, A.mellifera, C. quinquefasciatus, D. melanogaster, R. prolixus) and H. sapiens were also cloned and expressed. The six enzymes were biochemically characterised side-by-side using cytochrome c reduction assays in order to determine whether there are any differences in activity between those CPRs in blood-sucking insects and others. There were no clear trends in terms of enzyme kinetics which associated with a phenotype of haematophagy. The insect enzymes studied had higher Vmax values than H. sapiens with respect to both NADPH (1.6-7.8 fold higher) and cytochrome c (1.5-5.8 fold higher). Also compared were the enzymes' sensitivity to nucleotide analogue inhibitors and DPIC. HsCPR more sensitive to the nucleotide analogues than insect CPRs (by 2-30 fold). DPIC however was more inhibitory to insect CPRs by up to 20 times, giving it potential as an insecticide synergist. Again, there were no clear trends aligning inhibition profiles with a blood-feeding habit. In conclusion, AgHO has been found to be a true haem oxygenase, with biochemical characteristics in keeping with well characterised HOs such as H. sapiens HO-1 and C. diptheriae HmuO. AgHO inhibition appears to play a key role in oviposition and mosquito fecundity, and therefore may be a potential target for insecticides and insecticide synergists. Taken together, these results suggest that knowledge of these enzyme targets may aid the future development of novel insecticides.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:706835 |
| Date | January 2016 |
| Creators | Spencer, Christopher Stephen |
| Contributors | Paine, M. ; Lycett, G. ; Lian, L.-Y |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/3001871/ |
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