Reese Lab Research
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Cell Wall Research
  
Understanding enzymes involved in synthesis and regulation of cell wall components affecting capsule attachment
 
 

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). We are interested in learning more about this enzyme in terms of its location in the cell and how it is regulated.

It is likely that another enzyme, alpha-1,3-glucanase, degrades alpha-1,3-glucan. This enzyme could be important for the cell if cell wall material needs to be repaired or a bud forms? We would like to understand the importance of these proteins.

These enzymes are not found in humans, and may make safe and reasonable drug targets. Our research will help investigate these possibilities.

  
Colorized version of quick-freeze deep-etch electron micrograph of C. neoformans cell wall edge (Tamara Doering & John Heuser, Washington University)

 

  

 

 
 
Bio122 Research
    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 this project involve sample collection, and then phenotypic analysis by colony and cell morphology. Genotypic analysis by polymerase chain reaction and fungal primers can also be performed. If C. neoformans samples can be found, further study can identify types of isolates.

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.

 

  

 

 
Capsule Binding Research
  
Preventing capsule from binding is a possible therapeutic treatment of cryptococcosis.    
     

The goal of this project is to prevent capsule from binding to the cell, making them avirulent (non-disease causing). This could be done by preventing the alpha-1,3-glucan synthase enzyme from making the sugar polymer (chain of sugars) or by blocking the capsule's ability to bind. Or by preventing capsule synthesis all together. We can test capsule binding 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 cells can not bind capsule, they will not fluoresce. Methods of inhibiting capsule synthesis or removing capsule is a potential drug aproach, as 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).
   

 

 
   
Fungal Ecology Research
    What types of fungal organisms live in extreme environments? And is C. neoformans there?
     
 
What types of fungi live on the strange food-stuffs of caves? What fungi grow on shower curtains? How to fungi share their habitats with bacteria or other fungi? We'd like to look into some of these fungal ecology diversity questions. For example Hazel Barton's laboratory at Northern Kentucky University has been studying the bacteria of cave environments and growth in starvation conditions (visit http://www.cavescience.com). She was even featured in the Amazing Caves IMAX movie! What fungal organisms live in these communities? Can Cryptococcus neoformans be found there? Our inquiring minds want to know.
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.
   

 

 
Microbial Forensics Research
    
Can microbial profiles be used to help link suspects as forensics evidence?    
     

When you bite into something, are microbes from your mouth left behind? If so, how long will they stay there? Can you tell who bit the object from this microbial profile? Can you tell who left one behind at the scene of the crime? We are looking into these questions to add microbial forensic evidence to help link suspects with crime scenes, etc.

When a body decomposes, there are methods used to determine the time of death, but each of these has issues. Another microbial forensic project in the lab is looking at the role of microbes in decomposition and how they might be used to estimate time of death.

  

If you look closely, can you see the lip print? This image is from Cathy Massafra and Heather Jubinsky's poster that was presented at the 2006 Pennsylvania Academy of Sciences meeting.