Research

The Elmendorf Lab
Department of Biology, Georgetown University

Investigations into the
Pathogenesis and Evolutionary Importance
of Giardia lamblia

Transmission Electron Micrographs of Giardia lamblia
Images taken by Michael McCaffery

Why Giardia?

Giardia lamblia is one of the most prevalent intestinal protozoan pathogens worldwide. In the U.S., infections are most common among campers and children in daycare centers, although contamination of municipal water supplies has also been noted. In much of the world, however, limited access to clean water results in virtually universal infection by the age of three. Giardia lamblia also infects a wide range of mammals, including humans, cattle, sheep, horses, pigs, dogs and cats, and is therefore of significant veterinary concern.

After ingestion of the infective cyst stage by the host, the parasite differentiates in the lumen of the small intestine into the trophozoite form, attaches to intestinal epithelial cells and replicates. Its presence often results in severe gastrointestinal symptoms, including diarrhea, vomiting and weight loss. The parasite then redifferentiates into the cyst and is excreted to perpetuate the fecal-oral route of transmission. Studies of its basic biology are essential to define the ability of Giardia to adapt to and manipulate its host environment and cause disease.

In addition to its medical importance, Giardia is also a representative of one of the earliest diverging eukaryotic lineages. Phylogenetic analyses have consistently defined it as a member of the archaezoan family Hexamitidae - one of the earliest known branches to diverge from the eukaryotic tree. Its parasitic lifestyle and extremely early divergence from other eukaryotes have permitted Giardia to evolve independent and diverse solutions to common eukaryotic cellular problems as is true for many other parasitic protists. Indeed the biological diversity displayed by parasitic protists is instrumental in their ability to survive within hosts and cause disease.

Giardia is also an exceptionally malleable system

• it can easily be cultured in the lab (indeed the trophozoite stage with which we work primarily is generally not infectious) and several animal models also exist for in vivo anlyses of host-pathogen interactions
• molecular tools are available, including cDNA and genomic libraries, an almost-completed genome project, and a serial analysis of gene expression project
• reverse genetic manipulations are routine, including both transient and stable transfection, antisense technology to reduce mRNA and protein levels
• biochemical and cellular biology studies benefit from the axenic growth of the parasite, ease of preparation for microscopy, and the ability to examine GFP-fusion proteins in living cells

Our Research

Our laboratory focuses on two main questions:

• How is the cytoskeleton involved in parasite attachment to the small intestine?
• How can we explain the unusual genome structure and patterns of expression found within the parasite?

Cytoskeletal Structure and Function in Pathogenesis
Giardia has a highly complex and sophisticated cytoskeleton. In addition to many less-flamboyant cytoskeletal elements, the parasite is characterized by eight flagella, an elaborate spiral array of microtubules and affiliated ribbons termed the ventral disk, and a microtubule cluster referred to as the median body. Giardia’s ability to attach to intestinal epithelial cells is a necessary prerequisite for parasite virulence. Work by this lab and others have firmly established the microfilament cytoskeleton as critical for attachment. We therefore seek to answer four questions:

1. How can the biophysics of attachment in Giardia be characterized?
2. By what biomechanical process do the dynamics of actin filaments control attachment?
3. What proteins are part of the actin cytoskeleton in Giardia?
4. How can the nature of the microfilament proteins in Giardia be used to design new chemotherapeutics?

To address these questions, we have established a broad research program that involves several important collaborations:

1. In collaboration with the Dynamics Imaging Laboratory of Dr. Jeff Urbach in the Physics Department at Georgetown University, we are investigating the force and dynamics of Giardia attachment. Our studies have started characterizing and quantifying attachment to glass substrates in a flow cell, but we are eager to move our work into studies involving more relevant substrates, including explants of intestinal epithelium. This work is done by Ryan McAllister, a post-doctoral fellow, in the Urbach lab and Jesse Cohen, a reserach assistant, in my laboratory. Early studies were performed by Aliza Apple '05, an undergraduate Physics major.
2. We are collaborating with the Georgetown Advanced Electronics Laboratory of Dr. Mak Parajape in the Physics Department at Georgetown University to develop and test a variety of artificial substrates in Giardia attachment assays. This work will help us distinguish between various attachment theories. Additional work examining the dynamics of actin filaments in Giardia is being performed by Samantha Bowen '07 and Trey Picou '08 in my laboratory.
3. In collaboration with three labs from the MBL (Dr. Mitch Sogin), Berkeley (Dr. Zac Cande), and Notre Dame (Dr. Holly Goodson), we have been engaged in significant bioinformatics analyses to characterize the Giardia cytoskeleton from a genomics perspective. Although many cytoskeletal proteins are recognizable as homologs to those characterized in other systems, Giardia also clearly does not possess numerous 'ubiquitous' eukaryotic cytoskeletal proteins and concomitantly has many novel cytoskeletal proteins.
   Thus, we are currently using a two-pronged approach to biochemically purify actin-associated proteins: column chromatography and sedimentation assays using polymerized heterologous F-actin to capture filament-binding proteins and column chromatogrpahy assays using recombinant Giardia G-actin produced in E. coli to capture monomeric binding proteins. This work is done by Haibei Luo (GS), Kristin Yates '06, and Jesse Cohen. A newer angle on the project involves the production of recombinant Giardia actin in a baculovirus system to obtain sufficient quantities of 'native' protein for both monomeric and filamentous binding studies. This work is done in my laboratory by Amanda Munson, a post-doctoral fellow, and Nick Stoler '08.
4. We are collaborating with Dr. Nagarajan Pattabiraman's laboratory to perform in structure based drug design. Our current target is actin itself, which in Giardia has sufficient structural differences from mammalian actin to affect binding of phallicidin drugs. This work has been initiated by Rasha Khoury '04, Nirica Borges '05, and Rajendram V. Rajnarayanan, a post-doctoral fellow with Dr. Pattabiraman.