David J. Lampe - US grants

The long-term goals of this project are to understand the structure and function of the Himar1 transposase. A series of in vitro and in vivo E. coli assays and screens will be used to determine the amino acids responsible for mediating the several activities of Himar1 transposase that constitute transposition. In vitro mutagenesis will be used to create a series of C-terminal truncations of transposase each of which will be screened by gel shift assays to determine how much of the protein can be deleted before DNA binding activity is eliminated. Random and specific point mutations in the DNA binding domain will then be introduced and the mutations assayed for their effects in vivo and in vitro. The boundaries of the catalytic domain, the part of the protein responsible for the DNA breakage and joining reactions, will be mutated randomly and at conserved positions based on an alignment of many mariner-family transposons and the mutants screened for the ability to either excise the transposon or insert it into a target. The motif responsible for mediating target site TA dinucleotide identification will be localized with an E. coli screen which will allow the identification of randomly mutated Himar1 transposase constructs that complete insertion into improper target sequences. Finally, the DNA sequence of the inverted terminal repeat (ITR) will be mutated to determine the minimum sequences required for transposition. High-resolution chemical footprinting will be carried out to determine which base pairs are specifically contacted by transposase. Finally, a random mutagenesis screen will be performed to determine the effects of each position in the ITR on transposition frequency. By understanding the structure and function of this transposable element it is hoped that its utility in a variety of species will be dramatically improved.

[unreadable] DESCRIPTION (provided by applicant): Malaria is a major human disease that has shown no evidence of declining despite years of drug therapy to treat infected humans and efforts to control its mosquito vectors. Alternative strategies to deal with this problem are clearly needed. Work has progressed in the area of antimalarial effector genes that can prevent the establishment of Plasmodium parasites in their mosquito hosts, but there is currently no way to deliver these genes and their products to mosquitoes in the wild. One method to deliver such genes is by transforming bacteria that normally live in mosquito midguts, like the gamma proteobacterium Enterobacter agglomerans, into gene product delivery vehicles. This proposal seeks to tackle one aspect of modifying E. agglomerans into an effective antimalarial reagent, namely the development of efficient protein secretion systems for this bacterium and the testing of these systems to determine whether E. agglomerans that secrete antimalarial proteins can block the transmission of malaria from mosquitoes to their hosts in a rodent malaria model system. Both Type I and Type II secretion systems will be developed and tested. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The fight against the intolerable burden of malaria is restricted to the use of insecticides that kill the mosquito vector and drugs that kill the parasite in humans. Considering that recent estimates place the number humans infected with malaria at over 500 million (nearly 1 in 12 humans), the introduction of new means to counter the disease is urgently needed. We propose to develop a novel strategy to prevent the spread of malaria parasites by anopheline mosquitoes, based on genetic modification of bacteria that inhabit the gut of these insects. We will evaluate in detail the ability of two candidate bacterial species (Pantoea agglomerans and Asaia sp.) to colonize larval and adult mosquitoes and their ability to be transmitted vertically from one generation to the next. We will develop robust methods to secrete antimalarial effector proteins from each bacterial species to ensure that the effector proteins will reach the intended targets on the parasite or on the midgut epithelium. Different bacterial strains that secrete effector proteins will be evaluated for their efficacy to interfere with parasite development in the mosquito. Using the data obtained from engineered bacterial strains producing single effectors, we will create an optimal combination of strains that combine multiple effectors for maximum efficacy. Given that this funding mechanism is restricted in its time frame, no field trials are proposed although discussions with the relevant regulatory agency (US-EPA) are already underway. This research is expected to lead to the development of a novel weapon that can be used in combination with traditional control strategies (drugs, insecticides, vaccines) to combat malaria. PUBLIC HEALTH RELEVANCE: Malaria is one of the deadliest infectious diseases and kills an estimated 2 million persons every year. The mosquito is the obligatory vector for transmission. This project will devise new ways to interfere with the mosquito capacity to transmit the parasite, by genetically modifying bacteria that live in the mosquito midgut.

DESCRIPTION (provided by applicant): Malaria is a disease caused by protozoan parasites that are transmitted to humans by mosquitoes in the genus Anopheles. There are nearly 500 million new cases of malaria every year, and from 1-2 million people die from the disease while others are severely debilitated. About half the human population is at risk of contracting malaria, and its range may spread as global warming accelerates. The broad, long term objectives of this proposal are to create new methods of combating malaria to complement the current methods of control, namely insecticides to kill mosquito vectors and drugs to kill parasites in infected people. This project seeks to develop the means to create strains of bacteria that can interfere with the ability of mosquitoes to transmit malaria thus reducing its overall health burden and aiding in the goal of eradicating this disease. The specific aims of this research project are as follows: Aim 1: Creation of strains of Asaia SF2.1 that secrete anti-Plasmodium effector proteins using native secretion signals. Asaia SF2.1 is intimately associated with Anopheles mosquitoes in the field, colonizing the midgut, salivary glands, and gonads of these insects. Thus it has optimal microbial ecology on which to build a paratransgenesis system against malaria. We will develop native secretion systems using genomic data and a genetic screen for the secretion of anti-malarial effector proteins from Asaia SF2.1 to ensure efficient secretion of these proteins in mosquito midguts. Aim 2: Isolation of strong conditional promoters from Asaia SF2.1. Antimalarial effector proteins must be expressed when the parasite is present in the mosquito midgut and in sufficient quantities to be effective. We will isolate strong conditional promoters from Asaia SF2.1 that are active when bacteria are present during a blood meal using a genetic screen, genomic homology searches, and RNAseq. Aim 3: Creation of genetically stable strains of transgenic Asaia SF2.1. For eventual field use, paratransgenic strains of bacteria must be genetically stable. They cannot be based on laboratory plasmids that are maintained with drug selection. We will develop methods to create strains that contain genes inserted in the chromosome or borne on plasmids that need no drug selection.