Paratransgenesis for malaria using Asaia sp. bacteria

描述（由申请人提供）：疟疾是一种由原生动物寄生虫引起的疾病，通过按蚊属蚊子传播给人类。每年有近5亿新的疟疾病例，1-2百万人死于这种疾病，而其他人则严重虚弱。大约一半的人口有感染疟疾的危险， 而且随着全球变暖的加速，其范围可能会扩大。这项建议的广泛、长期目标是创造防治疟疾的新方法，以补充目前的控制方法，即杀死蚊子媒介的杀虫剂和杀死受感染者体内寄生虫的药物。该项目旨在开发制造细菌菌株的方法，这些菌株可以干扰蚊子传播疟疾的能力，从而减少其总体健康负担，并有助于实现根除这一疾病的目标。本研究项目的具体目标如下：目标1：创建Asaia SF2.1菌株，其使用天然分泌信号分泌抗疟原虫效应蛋白。Asaia SF2.1与野外的按蚊密切相关，定殖于这些昆虫的中肠、唾液腺和性腺。因此，它具有最佳的微生物生态学，在此基础上建立抗疟疾的副转基因系统。我们将使用基因组数据和Asaia SF2.1抗疟疾效应蛋白分泌的遗传筛选来开发天然分泌系统，以确保这些蛋白在细胞中的有效分泌。 蚊子中肠目的2：从Asaia SF2.1中分离强条件启动子。当寄生虫存在于蚊子中肠中时，抗疟效应蛋白必须以足够的量表达才能有效。我们将从Asaia SF2.1中分离出强条件性启动子，这些启动子在血餐期间存在细菌时具有活性，使用遗传筛选，基因组同源性搜索和RNAseq。目的3：建立遗传稳定的转基因Asaia SF2.1菌株。对于最终的现场使用，细菌的副转基因菌株必须是遗传稳定的。它们不能基于通过药物选择维持的实验室质粒。我们将开发方法来创造含有插入染色体或携带在质粒上的基因的菌株，而不需要药物选择。

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.