RNA Interference in Cryptococcus neoformans

As technology advances, so does our knowledge about the world around us, and perhaps more importantly, the world within us. One of the most important fields of research today is in the area of pathogens and human disease. This summer, I was able to experience the life of a scientist working to understand a lethal organism while working under Dr. Tamara Doering studying Cryptococcus neoformans.

Cryptococcus neoformans is a fungus similar to yeast. It can be found naturally all around us, especially in pigeon excrement. Though a healthy person can breathe in spores from this organism daily and not get sick, people who are immune-deficient aren't so lucky. Cryptococcus neoformans is an opportunistic killer that preys on people with A.I.D.S., cancers of the immune system, and those who have had their immune systems suppressed by drugs because of an organ transplant. This organism begins by infecting the lungs and goes on to cause fatal meningitis if not stopped. Current drugs against this fungus have several disadvantages. These drugs have serious side effects and don't eliminate infection in very ill patients. In addition, some strains of Cryptococcus neoformans have become drug resistant.

It is for these reasons that the Doering lab is studying how Cryptococcus lives and how we can manipulate it. My project dealt with preventing gene expression at the RNA level. The approach we are using, first developed in worms, is known as RNA interference (RNAi). The premise behind RNAi is that when double stranded RNA corresponding to the coding sequence of a particular gene is inserted into a cell producing that gene product, the gene product is no longer produced. It is a relatively simple and little understood method of turning off genes without manipulating the cell's DNA. Though RNAi has been tried in plants, C. elegans, Drosophila, and T. brucei, it has not been tried in Cryptococcus neoformans or any other fungus. This method would be especially valuable when dealing with this organism because technical problems make it very difficult to manipulate genes at the DNA level.

Before trying RNA interference in Cryptococcus neoformans, there were several decisions to be made. The first was which genes to target. We decided to knock out the ADE2 and CAP59 genes. The ADE2 gene is in the pathway which controls the metabolism of adenine, a metabolite essential for cell survival. Because this gene can control whether or not the cells live or die on certain media, it is easy to test. The CAP59 gene acts in the formation of a capsule around each cell. This capsule is unique among pathogenic fungi and essential for virulence, making it an important part of the cell to understand and control. Once we decided which genes to target, we had to figure out how to get double stranded RNA for those genes into the cells, since this never occurs naturally. We decided to build a plasmid containing a sequence of DNA and then an inverted piece of the same DNA separated by an unrelated DNA sequence. The plasmid would also contain elements to turn the expression of this sequence on and off. In 'on' conditions, when the cell transcribes the sequence as RNA, the corresponding basepairs on the RNA sequences will bond to each other forming double stranded RNA. The question remaining was how to get the special plasmid into the cells. We decided to use a method known as electroporation, which sends a quick electric shock into the cells causing the pores to open in the cell membrane, allowing the entrance of our plasmid. Once the plasmid is in the cell, all we have to do is put the cells in conditions to turn on the double stranded RNA synthesis, and then observe cell growth to see if the method worked.

During my short time in the lab, I was able to isolate and amplify all of the DNA fragments involved, ligate and transform the fragments into E. coli cells, and test the method of electroporation on Cryptococcus with a control plasmid. All of those steps were successful; the next step will be to use electroporation to transform the cells. After the double stranded RNA is expressed in the cells, their growth and development will be observed on different media as well as under a microscope.

Though we are now close to learning whether or not RNA interference will work in Cryptococcus neoformans, there is still much more to learn, both about the method and the organism. Future experiments may be designed to discover how RNA interference works and under what conditions. Researchers may also try to find other ways to manipulate the function of genes in Cryptococcus neoformans or to further understand how the organism functions. In any case, it is clear that this experiment is only one step in the quest to understand and stop this lethal organism.