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Reese Lab Research
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The goal of this project is to find a new model for comparing the virulence (disease-causing ability) of different strains of cryptococcus. Systems are desired for testing strains that grow at room temperature but that cannot grow at mammal body temperature. Three room temperature models are available, but these have data interpretation problems. Developing a new system will involve feeding cryptococcus to organisms (tobacco worm, planaria, or butterflies). Virulent strains should negatively affect the organisms, whereas avirulent strains should not. Strains with varying virulence can then be evaluated.

The focus of this project is to learn more about the function of alpha-1,3-glucanase. We predict that this enzyme regulates and rearranges alpha-1,3-glucan in the cell wall. What if there is too much of the enzyme? Will it decrease the alpha-1,3-glucan material and therefore reduce the amount of capsule material that can bind to the cryptococcal cell wall? To examine the effects of too much of this enzyme, we will overexpress it in C. neoformans by putting the gene for the protein in a plasmid driven by a specific C. neoformans promoter that can be turned off and on for control. Referring to the figure, when copper (green sphere) is present, the Cuf1 protein (Cuf1p) binds it and cannot bind to the Cu sensing element (CuSE). The CRT4 promoter is transcribed at basal level only. When copper is chelated by bathocuproinedi-sulphonic acid (BCS), then Cuf1p binds to CuSE and the machinery is turned on to transcribe the CRT4 promoter. Overexpression of this enzyme in bacteria will provide a needed cell wall degrading enzyme that will be useful in cell wall composition assays.

1. Overexpression of alpha-1,3-glucan synthase in C. neoformans or other yeast
2. Characterization of alpha-1,3-glucan synthase in budding and hyphae formation
4. Comparison of alpha-1,3-glucan synthase between related fungal strains
5. Screening of cryptococcal libraries for strains differing in growth and survival under cell wall stressing conditions
6. Overexpression of alpha-1,3-glucanase in bacteria to use as a reagent
7. Use RNAi on alpha-1,3-glucan synthase or glucanase in other strains
8. Looking at other environmental fungi

Reese Lab Research _____________________________________________________________________
Project 1
Localizing the enzyme responsible for making the critical sugar for capsule attachment

The goal of this project is to find out where in the cell the alpha-1,3-glucan cell wall component is made. Is it made in the cell cytoplasm, in a membrane bound organelle, or in the cell wall? (In the adjacent cell slice, this inner cell material is pink). Alpha-1,3-glucan is needed for the capsule material (purple) to stick to the cell wall (teal). The enzyme that makes this sticky cell wall sugar is called alpha-1,3-glucan synthase (since it synthesizes the glucan). In order to find the enzyme (localize it) in the cell, we will introduce a marked copy of the gene into cells that do not have a copy of the gene. The marked gene will lead to a marked protein product that can be detected and localized with a fluorophore-labeled antibody (to the marker) and use of the fluorescent microscope.
		Colorized version of quick-freeze deep-etch electron micrograph of C. neoformans cell wall edge (Tamara Doering & John Heuser, Washington University)

		Project 2
		Using RNA interference to make the levels of a cell wall regulation enzyme go down

		The goal of this project is to look at regulation of alpha-1,3-glucan in the cell wall. Alpha-1,3-glucanase is thought to be the opposite of alpha-1,3-glucan synthase, and takes glucan apart. What if the cell cannot regulate the cell wall glucan in this way? To test this, we are using RNA interference (a rather new technique that works in some organisms) to reduce the amount of alpha-1,3-glucanase in the cell. Doubled-stranded RNA is put into the cell, the DICER complex dices it up, making small pieces which bind with the RNA interference silencing complex. This prevents translation of the targeted gene, leading to no functional protein. The mutant cells can be screened for how this change affects their growth. Enzymes like alpha-1,3-glucanase could be potential drugs
Image from: Tricia R. Cottrell and Tamara L. Doering. Silence of the strands: RNA interference in eukaryotic pathogens. Trends in Microbiology, 11, 37-43, 2003. This is an excellent review of RNAi in eukaryotic pathogens as of 2003. A .pdf format of this paper can be downloaded from the Tamara Doering lab papers.

Project 3
Clearing a critical cell wall component as a possible therapeutic treatment of cryptococcosis.

The goal of this project is to clear alpha-1,3-glucan from the cell wall by inhibiting its synthesis. This could be done by preventing the alpha-1,3-glucan synthase enzyme from making the sugar polymer (chain of sugars) by having it bind something that mimics the next glucose required for the polymer, but that will halt the polymer process. This would not change the glucan content of the parent generation of fungal cells, but daughter cells formed in the presence of the inhibitor should be absent of alpha-1,3-glucan. We can test this by tagging the parent cell walls fluorescent green (left). These cells would have capsule that can be detected by a capsule antibody that has a red-fluorescent tag outside of the green (right). If the daughter cells do not make alpha-1,3-glucan and can not bind capsule, they will not fluoresce at all. Inhibiting alpha-1,3-glucan synthase is a potential drug target, as no capsule could bind these cells and the human immune system could clear the infection.
		Image from: Amy J. Reese and Tamara L. Doering. Cell wall alpha-1,3-glucan is required to anchor the Cryptococcus neoformans capsule. Molecular Microbiology, 50, 1401-1409, 2003. A .pdf format of this paper can be downloaded from the Tamara Doering lab papers. Left side = Acapsular C. neoformans cell walls have been tagged directly with fluorescein. (Method described in Lynda Pierini and Tamara L. Doering. Spatial and temporal sequence of capsule construction in Cryptococcus neoformans. Molecular Microbiology, 141, 105-115, 2001. A .pdf format of this paper can be downloaded from the Tamara Doering lab papers.) Right side = Acapsular cells have been exposed to capsular material and then to an anti-capsule antibody tagged with Cy3 red dye (Reese & Doering, 2003).

		Project 4
		Looking for a room temperature virulence model for C. neoformans

		The goal of this project is to find a new model for comparing the virulence (disease-causing ability) of different strains of cryptococcus. Systems are desired for testing strains that grow at room temperature but that cannot grow at mammal body temperature. Three room temperature models are available, but these have data interpretation problems. Developing a new system will involve feeding cryptococcus to organisms (tobacco worm, planaria, or butterflies). Virulent strains should negatively affect the organisms, whereas avirulent strains should not. Strains with varying virulence can then be evaluated.
The current room temperature models available are for Acanthamoeba castellani, a protozoan amoeba (Steenbergen JN, et al., PNAS. 2001, 98(26):15245-50), Dictyostelium discoideum, a slime mold (Steenbergen JN, et al. Infect Immun. 2003, 71(9):4862-72), and Caenorhabditis elegans, small worms (Mylonakis E, et al., PNAS. 2002, 26;99(24):15675-80)

Project 5
*Identifying natural reservoirs of Cryptococcus neoformans* on the Cedar Crest College Campus**

The focus of this project is to gather information as to the natural campus locations of yeast/fungal organisms, particularly habitats of Cryptococcus neoformans. Although C. neoformans is found all over the environment, bird droppings and particular tree species have been linked to high concentrations of fungal cells. It is also clear that not all natural habitats have been determined. The steps of this project involve sample collection, and a series of sample platings. Microbial plates that encourage fungal growth over bacterial growth will used. Then, conditions that are specific for a C. neoformans characteristic will allow for determination of this fungus over other yeast species. Mapping of C. neoformans habitats on campus will be the final step of this project.
		Image from Indrani Bose, Doering lab. Left side: C. neoformans colonies on minimal media Right side: C. neoformans colonies on niger seed agar to enhance melanin pigment production in the fungal cell walls. To learn more about melanin and other virulence factors for C. neoformans, see Indrani Bose, Amy J. Reese, Jeramia J. Ory, Guilhem Janbon, and Tamara L. Doering. A yeast under cover: The capsule of Cryptococcus neoformans. Eukaryotic Cell, 2, 655-663, 2003. A .pdf format of this paper can be downloaded from the Tamara Doering lab papers.

		Project 6
		*Overexpression of alpha-1,3-glucanase in C. neoformans* to determine function**

		The focus of this project is to learn more about the function of alpha-1,3-glucanase. We predict that this enzyme regulates and rearranges alpha-1,3-glucan in the cell wall. What if there is too much of the enzyme? Will it decrease the alpha-1,3-glucan material and therefore reduce the amount of capsule material that can bind to the cryptococcal cell wall? To examine the effects of too much of this enzyme, we will overexpress it in C. neoformans by putting the gene for the protein in a plasmid driven by a specific C. neoformans promoter that can be turned off and on for control. Referring to the figure, when copper (green sphere) is present, the Cuf1 protein (Cuf1p) binds it and cannot bind to the Cu sensing element (CuSE). The CRT4 promoter is transcribed at basal level only. When copper is chelated by bathocuproinedi-sulphonic acid (BCS), then Cuf1p binds to CuSE and the machinery is turned on to transcribe the CRT4 promoter. Overexpression of this enzyme in bacteria will provide a needed cell wall degrading enzyme that will be useful in cell wall composition assays.
Promoter plasmids were created by Jeramia Ory and Cara Griffith. This figure is from Jeramia J. Ory, Cara L. Griffith, and Tamara L. Doering. Copper regulation of gene transcription in Cryptococcus neoformans. Yeast, 21, 919-926, 2004. A .pdf format of this paper can be downloaded from the Tamara Doering lab papers.

Future projects may include:

1. Overexpression of alpha-1,3-glucan synthase in C. neoformans or other yeast 2. Characterization of alpha-1,3-glucan synthase in budding and hyphae formation 4. Comparison of alpha-1,3-glucan synthase between related fungal strains 5. Screening of cryptococcal libraries for strains differing in growth and survival under cell wall stressing conditions 6. Overexpression of alpha-1,3-glucanase in bacteria to use as a reagent 7. Use RNAi on alpha-1,3-glucan synthase or glucanase in other strains 8. Looking at other environmental fungi
		C. neoformans stained with India ink (carbon particles). The capsule-inducing conditions have made the capsule very large, excluding ink around the cells to produce a characteristic halo. This method was originally used in clinics to identfy cryptococcus in spinal fluid.