Identifying and Validating Pantothenate Kinase Isoforms and Identifying the Insertion Site of PanK Transgenes in Anopheles Stephensi

Abstract

Malaria, a mosquito-borne illness, is one of the leading causes of death in Africa and a major epidemic in nearly every continent. This disease is transmitted by female Anopheles mosquitoes, primarily A. gambiae in Africa and A. stephensi in the Indian subcontinent. Since 2012, A. stephensi has become widespread across horn of Africa, posing a great risk as an “urban mosquito” residing in city centers and having near-constant human interaction. This spread is accompanied by a rise in insecticide resistant mosquitoes and drug resistant parasites, demonstrating an urgent need for novel malaria control strategies. Coenzyme A (CoA), an essential coenzyme in multiple biological pathways, provides an alternative method for malaria control within the mosquito. Malaria parasites require CoA for growth and development but can only uptake the primary substrate for CoA synthesis, pantothenate, from the mosquito or vertebrate hosts. To limit malaria parasite growth, pantothenate stores can be shifted to bio-unavailable CoA for the parasite inside of the mosquito host. To do so, the rate-limiting enzyme pantothenate kinase (PanK) can be activated or upregulated to rapidly increase flux through the CoA synthesis pathway. The primary use of pantothenate in the mosquito is for the biosynthesis of CoA, so reduced pantothenate levels shouldn’t impact mosquito fitness. In mammals and other arthropods, the PanK gene has multiple isoforms with unique structures and functions. To successfully manipulate PanK in A. stephensi, a further understanding of the gene is required. In silico analysis of the A. stephensi PanK gene predicted three unique isoforms that hadn’t been experimentally validated. Using 5’ rapid amplification of cDNA ends (5’ RACE), two isoforms were validated, and one eliminated as a biologically relevant isoform. Isoform-specific qPCR of different tissues and time points following a blood meal found that the short form isoform peaks at 48 hours post-blood meal with a ten fold increase from sugar fed with a relative mRNA level of five, with a smaller peak at 6 hours post-blood meal, about a three fold increase from sugar fed. It also showed that the long form isoform peaks at 48 hours in the thorax with a three fold increase from sugar fed, which had a relative mRNA level of 2. Long form expression in the ovaries had a relative expression level of 5 before blood feeding but drops to 1 immediately following a blood meal, then increases gradually to a peak of 5 at 48 hours post-blood meal. Subcellular fractionation of mosquito tissues into cytosol and mitochondrial fractions found that the long form is primarily cytosolic while the short form is primarily mitochondrial. Following these studies, a PanK transgene using the long form isoform was randomly inserted into the A. stephensi genome, generating 7 transgenic lines. Inverse PCR of these lines found that 2 midgut lines - M5A and M5VA - and 2 fat body lines - VGM2 and VGM4, - have favorable insertions away from gene coding regions and are utilizable for future studies on malaria parasite development and mosquito fitness.