Characterization of Anopheles gambiae (African Malaria Mosquito) Ferritin and the Effect of Iron on Intracellular Localization in Mosquito Cells.

Ferritin is a 24-subunit molecule, made up of heavy chain (HC) and light chain (LC) subunits, which stores and controls the release of dietary iron in mammals, plants, and insects. In mosquitoes, dietary iron taken in a bloodmeal is stored inside ferritin. Our previous work has demonstrated the transport of dietary iron to the ovaries via ferritin during oogenesis. We evaluated the localization of ferritin subunits inside CCL-125 [Aedes aegypti Linnaeus (Diptera: Culicidae), yellow fever mosquito] and 4a3b [Anopheles gambiae Giles (Diptera: Culicidae), African malaria mosquito] cells under various iron treatment conditions to further elucidate the regulation of iron metabolism in these important disease vectors and to observe the dynamics of the intracellular ferritin subunits following iron administration. Deconvolution microscopy captured 3D fluorescent images of iron-treated mosquito cells to visualize the ferritin HC and LC homologue subunits (HCH and LCH, respectively) in multiple focal planes. Fluorescent probes were used to illuminate cell organelles (i.e., Golgi apparatus, lysosomes, and nuclei) while secondary probes for specific ferritin subunits demonstrated abundance and co-localization within organelles. These images will help to develop a model for the biochemical regulation of ferritin under conditions of iron exposure, and to advance novel hypotheses for the crucial role of iron in mosquito vectors.

The effect of bacterial challenge on ferritin regulation in the yellow fever mosquito, Aedes aegypti.

Secreted ferritin is the major iron storage and transport protein in insects. Here, we characterize the message and protein expression profiles of yellow fever mosquito (Aedes aegypti) ferritin heavy chain homologue (HCH) and light chain homologue (LCH) subunits in response to iron and bacterial challenge. In vivo experiments demonstrated tissue-specific regulation of HCH and LCH expression over time post-blood meal (PBM). Transcriptional regulation of HCH and LCH was treatment specific, with differences in regulation for naïve versus mosquitoes challenged with heat-killed bacteria (HKB). Translational regulation by iron regulatory protein (IRP) binding activity for the iron-responsive element (IRE) was tissue-specific and time-dependent PBM. However, mosquitoes challenged with HKB showed little change in IRP/IRE binding activity compared to naïve animals. The changes in ferritin regulation and expression in vivo were confirmed with in vitro studies. We challenged mosquitoes with HKB followed by a blood meal to determine the effects on ferritin expression, and demonstrate a synergistic, time-dependent regulation of expression for HCH and LCH.

Differential regulation of transferrin 1 and 2 in Aedes aegypti.

Available evidence has shown that transferrins are involved in iron metabolism, immunity and development in eukaryotic organisms including insects. Here we characterize the gene and message expression profile of Aedes aegypti transferrin 2 (AaTf2) in response to iron, bacterial challenge and life stage. We show that AaTf2 shares a low similarity with A. aegypti transferrin 1 (AaTf1), but higher similarity with mammalian transferrins and avian ovotransferrin. Iron-binding pocket analysis indicates that AaTf2 has residue substitutions of Y188F, T120S, and R124S in the N lobe, and Y517N, H585N, T452S, and R456T in the C lobe, which could alter or reduce iron-binding activity. In vivo studies of message expression reveal that AaTf2 message is expressed at higher levels in larva and pupa, as well as adult female ovaries 72h post blood meal (PBM) and support that AaTf2 could play a role in larval and pupal development and in late physiological events of the gonotrophic cycle. Bacterial challenge significantly increases AaTf1 expression in ovaries at 0 and 24h PBM, but decreases AaTf2 expression in ovaries at 72h PBM, suggesting that AaTf1 and AaTf2 play different roles in immunity of female adults during a gonotrophic cycle.

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells.

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by (59)Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.

The unique regulation of Aedes aegypti larval cell ferritin by iron.

Mosquitoes must blood feed in order to complete their life cycle. The blood meal provides a high level of iron that is required for egg development. We are interested in developing control strategies that interfere with this process. We report the temporal effects of iron exposure on iron metabolism of Aedes aegypti larval cells. These cells take up iron in linear relationship to exposure time and distribute the iron primarily to the membranes. Iron uptake increases cytoplasmic, membrane and secreted ferritin. Membrane ferritin is abundant in cells treated with iron, increases in cells in the absence of iron exposure and is associated with the secretory pathway. Our data suggest that in contrast to mammals, mosquitoes control intracellular iron levels by producing membrane ferritin in anticipation of an iron load such as that provided by a blood meal and support the hypothesis that secreted ferritin is a primary iron storage protein for these animals.

Regulation of the ribonucleotide reductase small subunit (R2) in the yellow fever mosquito, Aedes aegypti.

Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides, a rate limiting step in DNA synthesis. Class I RNR is a tetramer that consists of two subunits, R1 and R2; enzymatic activity requires association of R1 with R2. The R2 subunit is of special interest because it dictates the interaction with R1 that is required for enzymatic activity expression, and it is expressed only during the S phase of the cell cycle. We previously sequenced an R2 cDNA clone from the yellow fever mosquito, Aedes aegypti. We found the message was upregulated by blood feeding. We now report the sequence of an R2 genomic clone. The gene consists of 4 introns and 5 exons. Both major and minor transcriptional start sites have been identified, and their use differs in sugar-fed versus blood-fed females. The gene contains putative cis-regulatory sites for E2F, Caudal (Cdx) and Dearolf (Dfd). The mosquito R2 gene contains iron-specific regulatory elements immediately upstream of the minimal promoter region. Binding of a factor to the distal putative Cdx site in the -400 region is altered by iron treatment of cells. Further, following blood feeding, R2 message is significantly induced in mosquito ovaries (tissues that are involved in oogenesis--a process requiring DNA synthesis).

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage.

Inhalation of airborne pollution particles that contain iron can result in a variety of detrimental changes to lung cells and tissues. The lung iron burden can be substantially increased by exposure to cigarette smoke, and cigarette smoke contains iron particulates, as well as several environmental toxins, that could influence intracellular iron status. We are interested in the effects of environmental contaminants on intracellular iron metabolism. We initiated our studies using lung A549 type II epithelial cells as a model, and we evaluated the effects of iron dose and smoke treatment on several parameters of intracellular iron metabolism. We show that iron at a physiological dose stimulates ferritin synthesis without altering the transferrin receptor (TfR) mRNA levels of these cells. This is mediated primarily by a reduction of iron regulatory protein 2. Higher doses of iron reduce iron regulatory protein-1 binding activity and are accompanied by a reduction in TfR mRNA. Thus, for A549 cells, different mechanisms influencing IRP-IRE interaction allow ferritin translation in the presence of TfR mRNA to provide for iron needs and yet prevent excessive iron uptake. More importantly, we report that smoke treatment diminishes ferritin levels and increases TfR mRNA of A549 cells. Ferritin serves as a cytoprotective agent against oxidative stress. These data suggest that exposure of lung cells to low levels of smoke as are present in environmental pollutants could result in reduced cytoprotection by ferritin at a time when iron uptake is sustained, thus enhancing the possibility of lung damage by iron-mediated oxidative stress.