The Landfear laboratory studies the biological functions of membrane transport proteins in parasitic protozoa including Leishmania, African trypanosomes, and malaria. Because these parasites live within either mammalian or insect hosts during their entire life cycles, they are dependent upon uptake of a plethora of essential nutrients from their hosts for survival. Uptake is mediated by integral membrane proteins designated transporters, carriers, or permeases. Our research focuses on transporters that play especially important roles in the life cycles of these parasites, particularly in the infectious stages. These studies employ a synthesis of molecular biology, biochemistry, genetics, cell biology, and pharmacology.
Transporters for the sugar glucose play important roles in the biochemistry of many cells, since glucose is a key nutrient for both energy and biosynthesis. The glucose transporters of Leishmania, trypanosomes, and malaria are essential for life in the mammalian host. We have identified 3 distinct glucose transporter isoforms in Leishmania and studied their different functions and subcellular locations. Deletion of the 3 glucose transporter genes is lethal to the infectious stage of the life cycle but not to the stage that lives within the insect vector, and we are determining why glucose uptake is essential for the infectious stage. We are also developing a high throughput screen to identify small molecule inhibitors of Leishmania, trypanosome, and malaria glucose transporters that could represent leads for development of anti-parasitic drugs.
Purines are also essential nutrients that parasites need to salvage from their hosts. We are studying purine transporters in Leishmania and trypanosomes and attempting to determine which of these transporters are essential for parasite viability. Current evidence suggests that one of the purine transporters may be specialized to function in the infectious stage of the Leishmania parasite that lives inside host macrophages and that one of the trypanosome transporters may localize to intracellular membranes of the parasite rather than to its surface membrane.
ContactProfessor Scott M. Landfear DEPARTMENT OF MOLECULAR MICROBIOLOGY AND IMMUNOLOGY OREGON HEALTH & SCIENCE UNIVERSITY PORTLAND, OREGON 97239, U.S.A.
Telephone: (503) 494-2426 FAX: (503) 494-6862 E-mail: email@example.com
What Do We Study?
Promastigotes (left) and amastigotes (right) of Leishmania parasites. Parasites have been fixed and Giemsa stained.
The Molecular Biology of Parasitic Protozoa Including Leishmania and Trypanosomes.
Parasitic protozoa are single cell eukaryotic organisms that cause a variety of devastating diseases. In particular, our laboratory focuses on Leishmania species, the causative agents of leishmaniasis, and Trypanosoma brucei, the causative agent of African sleeping sickness.
Leishmania parasites have two principal stages in their life cycles, the promastigotes that live in the gut of the sandfly insect vector, and the amastigotes that live within phagolysosomal vesicles of the vertebrate host macrophages (Fig. 1). The promastigotes are slender, spindle-shaped cells with a single flagellum that endows them with motility. This form of the parasite can be easily cultured in tissue culture medium. The amastigotes are smaller, oval-shaped cells that have only a residual flagellum and lack motility. They are specialized for survival and growth in the acidic, oxidative environment of the macrophage phagolysosome. Amastigotes can be grown in animals, in tissue culture macrophage-like cells, or axenically in tissue culture medium (without macrophages). Several species of Leishmania parasites are responsible for disease in humans. The severity of the disease ranges from the disfiguring cutaneous form, in which nodules form at the site of infection (the insect bite) leaving scars upon healing, to the lethal visceral leishmaniasis. It is estimated that at least 12 million people worldwide suffer from one form or another of leishmaniasis.
African trypanosomes (Trypanosoma brucei) also have two principal stages in their life cycle. The procyclic forms (PFs) live in the tsetse fly vector, while the bloodstream forms (BFs) live free in the bloodstream of the vertebrate host (Fig. 2). African trypanosomaisis is widely distributed throughout equatorial Africa and causes a fatal disease in the absence of effective treatment. Both life cycle forms of this parasite are flagellated and motile and can be grown in tissue culture medium.
Figure 2. A scanning electron micrograph of a bloodstream trypanosome next to a red blood cell. Trypanosomes live extracellularly in the bloodstream of the vertebrate host. Photograph courtesy of Dr. Andreas Seyfang.
Although drugs are available for treating both leishmaniasis and African sleeping sickness, these drugs are expensive, toxic, and suffer from increasing occurrence of drug-resistant strains in the field. Hence, there is a compelling need for the development of improved drugs or other therapies for treatment and control of these prevalent infections.
The Importance of Membrane Transport Proteins in the Biology and Pharmacology of Leishmania and Trypanosomes.
Both Leishmania and trypanosomes spend their entire life cycles within an insect or a vertebrate host. Consequently, they are dependent upon their host to provide a variety of nutrients that are essential for their survival. These nutrients are delivered to the parasite via transport proteins that are located in the surface membrane of the microbe. Each membrane transport protein typically transports one or a few related nutrients by shuttling these molecules across the otherwise impermeable barrier of the parasite membrane. Consequently, these transporters play a central role in the biology and biochemistry of these parasites.
In addition, some of these transporters (most notably the nucleoside transporters) also mediate the uptake of drugs or experimental drugs that could otherwise not be efficiently delivered to their site of drug action within the parasite. Consequently, transporters (also called permeases) can be of great importance for the pharmacology of the parasites.
The Study of Glucose Transporters and Nucleoside Transporters in these Parasites
Much of our current work focuses on glucose transporters in Leishmania species and nucleoside transporters in both Leishmania donovani and Trypanosoma brucei.
Glucose is an important nutrient for the promastigote form of Leishmania parasites. The promastigotes living in the sandfly gut are exposed to sugars such as sucrose, which is present in the plant sap and nectar that is the main diet of the insect. The sucrose is cleaved to glucose and fructose within the insect alimentary tract and provides an important energy source for the parasite. Once the parasite enters the phagolysosomes of the macrophage, there is less free glucose available, and the amastigotes utilize fatty acids and amino acids present within the lysosomal vesicles for energy production. Both glucose transport and glucose metabolism are developmentally regulated during the parasite life cycle, being much more active in the promastigotes than the amastigotes. Hence, Leishmania glucose transporters offer examples of strongly developmentally regulated genes.
Purine nucleosides and nucleobases are important nutrients for all known parasitic protozoa, including Leishmania and trypanosomes, because the parasites cannot synthesize purines de novo. As a result, these parasites are absolutely dependent upon salvage of purines (as either nucleosides or nucleobases) from their hosts. This reliance upon purine salvage is one of the major biochemical differences between the parasites and their mammalian hosts (which can synthesize purines de novo), and purine salvage pathways thus represent promising sites for therapeutic intervention. The first step in purine salvage is the uptake of purines across the parasite plasma membrane by purine transporters. Furthermore, several drugs or experimental drugs (both nucleoside analogs and other compounds) enter the parasites through nucleoside transporters. Consequently, parasite nucleoside transporters are of great importance for both the biochemistry and the pharmacology of these parasites.
An Interdisciplinary Approach to Studying Parasite TransportersThe study of membrane transport proteins and the genes that encode them offers the opportunity to investigate many aspects of parasite biology. In our studies, we employ an interdisciplinary approach using molecular biology, biochemistry, genetics, cell biology, and chemical biology to understand these transporters and their role in the biology of the parasites.
Thus we are interested in studying:
- the genes that encode the transporters, their multiplicity and arrangement within the genome, and their regulation during the parasite life cycle (molecular biology)
- the mechanism of action of the transporters themselves, the relationship of their structure to biochemical function, and their function as facilitative (passive) or active (concentrative) permeases (biochemistry)
- the molecular and cellular mechanisms for targeting different transporters to distinct membranes within the parasite (cell biology)
- the role of each of these transporters in the biology of the parasite, typically approached by targeted gene deletion to generate null mutants (genetics)
- small molecules that may serve as selective inhibitors of parasite glucose transporters and that can serve as leads for development of novel anti-parasitic drugs (chemical biology).
In a broader sense, we are interested in studying the role of transporters in the physiology of the parasites, i.e. how do these transporters allow the parasite to adapt to the very different environments of the insect vector and the vertebrate host or to live with changing nutritional conditions within the vector or host.
SPECIFIC RESEARCH PROJECTS IN THE LANDFEAR LAB
Figure 3. Topology of facilitative glucose transporter in the membrane.
1. Multiple Differentially Expressed Glucose Transporters in Leishmania mexicana
Glucose transporters are integral membrane proteins with 12 transmembrane segments connected by hydrophilic segments on both the extracellular and cytosolic sides of the membrane (Fig. 3). These proteins fold into a 3-dimensonal structure that contains a central pore that allows transport of glucose and related hexose sugars across the parasite membrane.
Three distinct glucose transporters
Figure 4. Three glucose transporters encoded as a gene cluster.
In the parasite Leishmania mexicana, glucose transporters are encoded by a cluster of genes encompassing 3 distinct glucose transporters, LmxGT1, LmxGT2 and LmxGT3 (Fig. 4, hereafter referred to as GT1, GT2, and GT3). Sequence differences between the open reading frames of these 3 genes lie largely in the amino-terminal and carboxy-terminal hydrophilic domains. The remainder of the coding regions are quite similar but do contain a few differences in sequence.
Heterologous expression of the 3 genes in Xenopus oocytes reveals that they all encode functional glucose transporters. The GT2 mRNA is expressed preferentially in promastigotes, whereas the GT1 and GT3 mRNAs are constitutively expressed in both life cycle stages. This observation suggests that each LmxGT isoform is likely to have unique properties.
One study in the laboratory has focused on a genetic analysis of the LmxGT glucose transporter gene cluster to define the physiological role of glucose transporters in this parasite. In this approach, the entire LmxGT1-LmxGT2-LmxGT3 gene cluster has been ‘knocked out’ by homologous gene replacement to create a glucose transporter null mutant. Indeed, this null mutant is unable to take up glucose, fructose, mannose, or galactose. This null mutant is viable in the promastigote (insect) stage of the life cycle, although it grows considerably more slowly than wild type parasites. However, unexpectedly the null mutant is not viable in the amastigote stage of the life cycle that infects mammalian macrophages, and we have subsequently shown that glucose is an essential nutrient for amastigotes. Consequently, glucose transporters are essential for the infectious stage of the life cycle, suggesting that they might represent potential targets for development of novel anti-parasite drugs.
Recent studies have focused on the biochemical consequences of glucose transporter gene deletion. We have determined that the null mutants synthesize considerably lower amounts of the principal storage carbohydrate and virulence factor beta-mannan, and this deficiency in maintaining an energy reserve may in part explain why the null mutants do not survive in the nutrient-poor environment of the macrophage phagolysosome. In addition, the null mutants are considerably more susceptible to oxidative stresses that they would encounter with the macrophage (possibly due to reduced flux of glucose through the pentose phosphate pathway that generates the principle intracellular reducing agent NADPH), suggesting another reason why these mutants do not survive within these mammalian host cells.
We have recently determined that there are several suppressor mutations that support the viability of the glucose transporter null mutants to survive. In particular, a linear subchromosomal amplification occurs in several independently isolated glucose transporter null mutants, suggesting that this amplification may be necessary for viability of the null mutant. We have identified a single open reading frame on this linear amplicon that supports survival of the glucose transporter null mutant. We have also observed that other suppressor mutants arise spontaneously from the glucose transporter null mutants following passage in culture. These mutants have amplified another transporter, designated LmxGT4 (which has very poor but measurable glucose transport activity), on a circular amplicon and have re-acquired the ability to transport hexoses and metabolize them as sources of metabolic energy.
- Burchmore, R.J.S. and Landfear, S.M. (1998) Differential regulation of multiple glucose transporter genes in Leishmania mexicana. J. Biol. Chem. 273:29118-29126.
- Burchmore, R.J.S. et al. (2003) Genetic characterization of glucose transporter function in Leishmania mexicana. Proc. Natl. Acad. Sci. U.S.A. 99:3901-3906.
- Rodriguez-Contreras, D. and Landfear, S.M. (2006) Metabolic changes in glucose transporter-deficient Leishmania mexicana and parasite virulence. J. Biol. Chem. 281:20-68-20076.
- Feng, X., Rodgriguez-Contreras, D., Buffalo, C., Bouwer, A., Kruvand, E., Beverley, S.M., and Landfear, S.M. (2009) Amplification of an alternate transporter gene suppresses the avirulent phenotype of glucose transporter null mutants in Leishmania mexicana. Mol. Microbiol. 71:369-381.
- Feng, X., Rodriguez-Conreras, D., Polley, T., Lye, L.-F., Scott, D., Burchmore, R.J.S., Beverley, S.M., and Landfear, S.M. (2013) Transient genetic suppression facilitates generation of hexose transporter null mutants in Leishmania mexicana. Mol. Microbiol, In press.
2. LmxGT1 is a unique flagellar glucose transporter
Localization of the 3 glucose transporters (referred to hereafter as GT1, GT2, and GT3 for simplicity) using Green Fluorescent Protein (GFP) chimeras and fluorescence microscopy revealed that GT1 is localized to the flagellar membrane (Fig. 5), whereas GT2 and GT3 traffic to the plasma membrane surrounding the cell body but are excluded from the flagellar membrane. Recent observations suggest that several flagellar (or ciliary) membrane proteins both in these protozoa and in other eukaryotes are involved in signal transduction pathways involved in sensing the environment. By analogy, one current speculation is that the GT1 glucose transporter might be involved in sensing glucose concentrations outside the cell and relaying this information to the interior of the cell. This hypothesis might provide a ‘reason’ why the parasite expresses a specific flagellar glucose transporter/sensor. Current experiments are directed toward testing this hypothesis.
Another important question is how LmxGT1 and other flagellar membrane proteins are selectively targeted to the flagellar membrane. We have demonstrated that the unique amino terminal hydrophilic domain (Fig. 3) of GT1 (the most divergent region of GT1 compared to GT2 or GT3) encompasses the flagellar targeting information. Indeed, mutagenesis of the GT1 transporter demonstrates that 3 sequential amino acids, N95, P96, and M97, are required for efficient flagellar targeting. A computational model of the amino terminal hydrophilic domain of GT1 (Fig. 6) predicts that these 3 critical amino acids are located at the tip of a loop (red residues on Fig. 6) that is at the surface of this domain. It is likely that these residues interact with another protein that is part of the flagellar targeting machinery. Crosslinking and mass spectrometry experiments have now identified a novel protein, designated KHARON1, that interacts with the flagellar targeting motif of GT1. Knocking out the Kharon1 gene prevents GT1 from targeting to the flagellum, confirming that KHARON1 is essential for flagellar targeting. KHARON1 appears to be one component of a large (>1 megadalton) multiprotein complex that mediates flagellar transport of GT1 and potentially other flagellar membrane proteins.
Figure 6. Rosetta computational model of the N-terminal domain of LmxGT1 that confers flagellar targeting. The 3 critical amino acids are shown in red.
- Piper, R.C. et al. (1995) Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain. J. Cell Biol. 128:499-508.
- Abdel Nasser, M.I. and Landfear, S.M. (2004) Sequences required for the flagellar targeting of an integral membrane protein. Mol. Biochem. Parasitol. 135:85-100.
- Tran, K., Rodriguez-Contreras, D., Shinde, U., and Landfear, S.M. (2012) Both sequence and context are important for flagellar targeting of a glucose transporter. J. Cell Sci. 125:3292-3298.
3. Cloning, Expression, and Functional Analysis of Nucleoside and Nucleobase Transporter Genes in Leishmania donovani and Leishmania major.
Figure 7. NT1 and NT2 are nucleoside transporters, and NT3 and NT4 are nucleobase transporters. The substrates taken up by each transporter are indicated below. NT4 is activated by acid pH (i.e. pH 5-6). Click to enlarge.
Work in the Landfear and Ullman laboratories resulted in the cloning from L. donovani of the first genes for purine nucleoside transporters NT1 and NT2. Subsequent work revealed that the genomes of both L. donovani and L. major encode two more transporters, NT3 and NT4, that mediate the uptake of purine nucleobases. Hence the parasites can satisfy their purine requirements by importing either purine nucleosides such as adenosine or guanosine or by taking up purine nucleobases such as adenine, guanine, or hypoxanthine (Fig. 7). All four of these permeases are members of the SLC29 family (also called the equilibrative nucleoside transporter family) and contain 11 transmembrane segments (Fig. 8).
Gene Knockout Studies
Figure 8. Topology of NTs in the membrane. The numbered open rectangles represent transmembrane domains, the gray rectangle represents the plasma membrane.
Gene knockout studies performed on all four NT genes are now determining which ones are critical for survival of both promastigotes and amastigotes. Of interest, NT4 is a very poor transporter at neutral pH but acquires the ability to transport purine nucleobases with high affinity at pH 5-6, i.e. it is activated by acid pH. Thus NT4 appears to be designed to function optimally in the amastigote stage of the parasite life cycle that lives within the acidified phagolysosomal vesicles of the host macrophage. Indeed the NT4 null mutant is quantitatively compromised in its ability to survive within primary murine macrophages and is strongly impaired in its ability to cause infection in a mouse model of leishmaniasis.
Modeling the Structure
Understanding the function of transporters requires knowledge of their 3-dimensional structure. However, crystallographic studies on membrane proteins are usually difficult. To overcome the lack of a current crystal structure for any member of the SLC29 family, we have collaborated with Dr. Ujwal Shinde in the Department of Biochemistry and Molecular Biology at OHSU to generate a computational model of NT1 (Fig. 9). The top image is a side view of the structure with cytosol above and extracellular surface at the bottom. The bottom image is rotated 90° to provide a view from the cytosolic face toward the extracellular space. The red spheres are space filling models of 3 aromatic residues that are predicted to interact to close the 'extracellular gate' of the transporter in this (outside closed, inside open) conformation.
Of particular interest, this model has successfully predicted functionally important residues (e.g., gating residues that close off the permeation pore in one orientation of the transporter) in NT1 that would not have been identified prior to the availability of a structural model. The 3-dimensional model provides a platform for further probing the structure and function of these transporters at the molecular level.
- Ortiz, D., Sanchez, M.A., Koch, H., Larsson, P., and Landfear, S.M. (2009) An acid-activated nucleobase transporter from Leishmania major. J. Biol. Chem. 284:16164-16169.
- Valdés, R., Arastu-Kapur, S., Landfear, S.M., and Shinde, U. (2009) An ab initio structural model of a nucleoside permease predicts functionally important residues. J. Biol. Chem. 284:19067-19076.
- Valdés, R, Shinde, U., and Landfear, S.M. (2013) Cysteine crosslinking defines the extracellular gate for the Leishmania donovani nucleoside transporter 1.1. (LdNT1.1). J. Biol. Chem., In press.
4. Searching for small molecules that selectively inhibit essential parasite glucose transporters.
The glucose transporters expressed by Leishmania mexicana, Trypanosoma brucei, and Plasmodium falciparum (malaria parasite) are critical for survival of the disease-causing stages of the parasite life cycles. Hence, compounds that selectively inhibit the parasite glucose transporters may provide leads that can be further developed into desperately needed anti-parasitic drugs. We are collaborating with the laboratory of Dr. Kip Guy (St. Jude Children’s Research Hospital, Memphis, TN) to identify such selective inhibitors. Our laboratory has developed an assay that is being employed in the high-throughput screening of an ~600,000 compound chemical library to search for such selective inhibitors.
- Feistel, T, Hodson, C.A., Peyton, D.H., and Landfear, S.M. An expression system to screen for inhibitors of parasite glucose transporters. Mol. Biochem. Parasitol. 162:71-76 (2008).