involved in two projects. I will start by describing the ferritin
project and close with the ribonucleotide reductase project.
long-term objective is to understand
human disease transmission by blood-feeding insect vectors. I am
specifically interested in the role that iron plays during pathogenic
invasion. Data in vertebrates suggest that pathogenic infectivity may
be linked to the availability of iron in the host. In vertebrates, some
iron-binding proteins are upregulated under pathological conditions.
This upregulation is hypothesized to cause chelation of iron and thus
reduce iron availability to the pathogen. The actual machinery that
activates this response remains unknown. My aim is to elucidate the
mechanisms by which pathogenic infectivity is affected by the host iron
Global Map for Aedes aegypti, CDC, 1999
The host organism for my research is the hematophagous (blood feeding) mosquito, a vector of numerous diseases. Worldwide, millions of people die yearly from diseases transmitted by hematophagous insects. In the U.S., for the past few years, the West Nile virus carried by the mosquito Culex pipiens has caused epidemic outbreaks nationwide and cost the U. S. hundreds of thousands of dollars; West Nile virus is moving westward and southward. With all the public health concerns, not much is known about the basic biology of hematophagous insects. My work has a foundation for understanding how these disease vectors regulate their iron metabolism.
Molecular Model of
The iron-dependent enzyme, ribonucleotide reductase (RNR), catalyses the de novo synthesis of deoxyribonucleotides. The subunits of Class I RNR are denoted R1 and R2. Class I RNR activity depends on the cell cycle and is regulated at the transcriptional level. RNR enzymatic activity completely depends on the association of R1 with R2, and the interaction between R1 and R2 is dictated by the C-terminal sequence of R2. R2 is considered a good drug target because, in principle, if the C-terminus of the pathogen is sufficiently different from that of the host, specific inhibition of pathogenic growth could be achieved by interference with the C-terminus. This theory was successfully tested in herpes viruses, where synthetic peptides designed against the viral R2 C-terminus caused a complete inhibition of the viral RNR, but showed no cross-inhibition of the human host RNR. Seven amino acid residues at the C-terminus of R2, FTLDADF, contain the minimum length necessary for full inhibitory activity of RNR.
It occurred to me that RNR would be a good target for new strategies to inhibit the ability of pathogens to propagate in mosquitoes. As a first step toward this effort, my laboratory together with Dr. Winzerling’s laboratory have cloned and sequenced the cDNAs encoding the R1 and R2 subunit in the yellow fever mosquito Aedes aegypti. The mosquito enzyme appears to be a Class I RNR as deduced amino acid sequences of the R1 and R2 subunits share significant identities with the amino acid sequences of other Class I R1 and R2 proteins. CLUSTAL W analysis reveals stretches of amino acid sequence in the A. aegypti R2 C-terminus that are notably different from the malarial parasite Plasmodium falciparum. These regions thus could serve as potential sites for new control strategies. In the mammalian work, killing the pathogen through specific peptide targeting of its RNR has been achieved.
My laboratory together with Dr. Winzerling’s laboratory have shown that messages for both R1 and R2 are in very low amounts in both larvae and adult males. These messages are expressed at low levels in sugar-fed females, but are induced in blood-fed females, implicating that transcriptional regulation is important in blood feeding. Using semi-quantitative PCR, we found that expression of R1 and R2 messages are highest in the ovaries of both blood-fed and sugar-fed females. However, induction of R1 message by blood feeding is highest in the gut, whereas, induction of R2 message by blood feeding is highest in the ovaries. In mammalian cells, R1 message is constitutively present, while R2 expression is S phase specific, suggesting that R2 is induced with DNA synthesis. In mosquitoes, R1 message is also constitutively present in most tissues, and R2 message is induced in tissues that are involved in oogenesis (such as the ovaries) and vitellogenesis (such as the fat body)Ñtissues where the level of DNA synthesis is induced with blood feeding.
My laboratory has identified the genomic clone for R2 and characterized the R2 promoter. We will prepare an ovary-specific cDNA expression library (a yeast one-hybrid library) from sugar-fed female mosquitoes; this library will be used for the identification of trans-regulatory factors involved in the basal control of R2 genes. Our approach is to use the R2 promoter as a bait for basal transcriptional factors in the yeast one-hybrid system, then use the basal factors to trap the blood-meal inducible factors in a yeast two-hybrid system; the yeast two-hybrid system will be made from ovary-specific mRNA of blood-fed female mosquitoes. We will follow similar studies for R1. Once both R1 and R2 genes are well characterized, we will use specific peptides to block the pathogenic invasion (such as that of the malaria parasite) without interfering with the insect host gene expression or protein synthesis.