Using the fruit fly to study malaria

 Malaria is caused by parasites of the genus Plasmodium. These protozoans have a complex lifecycle in which they must grow in both a vertebrate and a mosquito to complete their lifecycle. Both parts of the lifecycle are required for the transmission of the disease in the wild. Four Plasmodium species are known to cause disease in humans and the most virulent, Plasmodium falciparum is the cause of over a million deaths a year and up to half a billion cases. The cost of this disease in terms of human lives and sickness is enormous. Comparing this disease to others in the news, malaria kills more people each year than breast cancer, prostate cancer, melanoma and leukemia combined. Only in the last five years has AIDS matched malaria in terms of the number of people it kills.

There is currently no useful vaccine for the disease. The parasites have become resistant to many useful drugs and continue to develop resistance against new drugs. Chemically treated bed nets are useful in reducing the intensity of malaria cases but even these are too expensive to be used everywhere they are needed.

We have begun studying the basic interactions the malaria parasite has with its insect host with the hope of learning what the parasite requires to grow and how the insect can kill the parasite. We hope that this information will lead to new ways of fighting this scourge

Our plan is to use the fruit fly, Drosophila melanogaster in our studies. We have developed a method of injecting Plasmodium into the fly and monitoring the development of the parasites. Remarkably, the parasites complete much of their lifecycle in the fly, which diverged from the natural host of the parasite, the anopheles mosquito, hundreds of millions of years ago. The advantage of using the fly is simple - a large variety of genetic tools are available to study its biology. We are currently performing genetic screens that will identify mutant flies that are either better or worse at supporting the growth of Plasmodium. In this way we will identify insect factors that the parasites require to grow and develop and we will identify immune mechanisms that the fly can use to fight the Plasmodium infection. We will use this information to bootstrap back to the mosquito and determine whether these genes are also important in the native host.

The injectin video demonstrates how we inject flies with various microorganisms. In this example, blue dye is injected into the open circulation system of the fly through a glass needle. The advantage of this technique over the use of a solid wire needle is that we can measure the dose of the microbe and deliver precise quantities of material.

Microbial Pathogenesis

Flies have a multifaceted immune response. Most infectious agents must breach an epithelial sheet to gain entry into the host. The epithelium itself raises an immune response and produces antimicrobial peptides. A clotting reaction occurs to close the wound and a reactive oxygen generating reaction produces melanin at the site of injury.

 In the circulation of the fly, microbes will encounter a variety of immune cells. If the microbes are small enough, they will be phagocytosed by a cell called the plasmatocyte. This cell can also produce antimicrobial peptides, though it is unclear whether these are secreted or directed toward the vesicles containing the phagocytosed material.

By far, the best characterized immune response in the fly concerns the organ called the fat body. The fat body is the liver-like organ in the fly that is responsible for synthesizing the majority of circulating proteins. During an immune response the fat body secretes large quantities of antimicrobial peptides into the circulation. This is under the control of two signal transduction pathways that both regulate the nuclear import or NFkB like molecules.

 Much of our current work is focused on the role of the phagocytes on the immune response of the fly. To do this we have developed methods of infecting flies with microbial pathogens that target phagocytic cells. The three microbes we use most are Listeria monocytogenes, Mycobacterium marinum and Salmonella typhimurium. In humans, the species of Listeria and Salmonella we use infect the gut, causing diarrhea. Mycobacterium marinum forms granulomas on the extremeties as it prefers to grow in cold blooded organisms such as fish. We use this bacterium as a model for Mycobacterium tuberculosis, the causative agent of TB.

 Listeria was chosen as it is able to break out of vesicles and live in the cytoplasm of a macrophage. We have found that Listeria is able grow in fly phagocytes in much the same way it grows in vertebrate macrophages.  In the picture Listeria cells (green) are shown moving on actin tails (red).  In contrast, M.marinum and S.typhimurium redirect vesicle transport within a cell and create a specialized membrane bound compartment in which they can grow. By studying how these different microbes alter the biology of Drosophila phagocytes and determining how the fly fights these effects, we hope to learn about microbial virulence and innate immune responses.

The advantage of performing this work in the fly as compared to a mouse of a fish is the ability to perform large scale forward genetic screens in both host and pathogen to determine factors regulating the virulence of the microbes and the responses of the hosts.