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What Do We Study? 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.

Figure 1. Promastigotes (left) and amastigotes (right) of Leishmania parasites. Parasites have been fixed and Giemsa stained. 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 little free glucose available, and the amastigotes utilize fatty 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 Transporters.

The 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, and cell biology to understand these transporters and their role in the biology of the parasites. Thus we are interested in studying: i) the genes that encode the transporters, their multiplicity and arrangement within the genome, and their regulation during the parasite life cycle (molecular biology); ii) 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); iii) the molecular and cellular mechanisms for targeting different transporters to distinct membranes within the parasite (cell biology); and iv) the role of each of these transporters in the biology of the parasite, typically approached by targeted gene deletion to generate null mutants (genetics). 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 WITHIN THE LABORATORY

1. Glucose Transporters in Leishmania enriettii: Distinct Transporter Isoforms are
Targeted to the Flagellar Membrane and the Pellicular Plasma (Cell Body) Membrane.

Glucose transporters, whether in lower eukaryotes such as Leishmania or in mammals, appear to have 12 hydrophobic transmembrane segments interspersed with hydrophilic loops that are located on either the cytoplasmic or the extracellular side of the surface membrane (Fig, 3). Our previous studies on Leishmania enriettii demonstrated that this parasite encodes two distinct glucose transporter isoforms, one of which (ISO1) is targeted to the flagellar membrane and the other of which (ISO2) is targeted to the pellicular plasma membrane that surrounds the body of the parasite (Fig. 4). Intriguingly, these two isoforms are identical throughout most of their sequence. However the hydrophilic amino-terminal domains that are located in the cytoplasm are completely different (Fig. 3).

Figure 3. Schematic representations of ISO1 (flagellar isoform) and ISO2 (pellicular plasma membrane isoform). The filled rectangles represent hydrophobic segments that are thought to form transmembrane α-helices, while the thinner lines represent hydrophilic loops on either side of the membrane. The region indicated in black is identical between ISO1 and ISO2. The only regions of sequence that differ between the two proteins are the hydrophilic amino-terminal domains, indicated in red for ISO1 and blue for ISO2. These domains are completely different from each other in sequence; the ISO1 domain is 132 amino acids, and the ISO2 domain is 46 amino acids. Follow this link to see larger topological figures of ISO1 and ISO2

Figure 4. Confocal images of L. enriettii promastigotes expressing epitope tagged ISO1 (top 3 images) or epitope tagged ISO2 (bottom 2 images). The ISO1 protein is located in the flagellar membrane, whereas the ISO2 protein is located in the pellicular plasma membrane surrounding the cell body. Follow this link to see a larger version of these micrographs.

How are these two very similar proteins targeted to distinct domains of the surface membrane? Since the only regions of difference are the amino-terminal hydrophilic domains, it is likely that at least one of these domains contains essential targeting information. We have demonstrated that the amino-terminal domain of ISO1 contains a dominant flagellar targeting sequence. Chimeric proteins containing this amino-terminal domain of ISO1 fused to the body of an otherwise pellicular plasma membrane protein will retarget this chimeric protein to the flagellar membrane. Extensive analysis of deletion mutants and chimeric constructs suggests that an two separate segments of the hydrophilic amino-terminal domain of ISO1 (between amino acids 84-100 and 110-118) are involved in flagellar targeting.

The flagellar targeting machinery is probably important for routing a variety of proteins into the flagellum. Thus, several other proteins have been identified in Trypanosoma brucei or Trypanosoma cruzi that are restricted to the flagellar membrane. Hence, the flagellar targeting pathway is likely to be of general importance in these parasites.

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 ISO1 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.

Relevant Publications:

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.

Snapp, E.L. and Landfear, S.M. (1997) Cytoskeletal association is important for differential targeting of glucose transporter isoforms in Leishmania. J. Cell Biol. 139:1775-1783.

Snapp. E.L. and Landfear, S.M. (1999) Characterization of a targeting motif for a flagellar membrane protein in Leishmania enriettii. J. Biol. Chem. 274:29543-29548.

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.

2. Multiple Differentially Expressed Glucose Transporters in Leishmania mexicana.

We have also investigated glucose transporter genes in the human pathogen Leishmania mexicana. In contrast to Leishmania enriettii, this species contains a cluster encompassing 3 distinct glucose transporter genes, LmGT1, LmGT2 and LmGT3. 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.

Figure 5. Genomic cluster of glucose transporter genes in Leishmania mexicana. Three distinct isoforms are encoded by a cluster of the LmGT1, LmGT2 and LmGT3 genes.

Heterologous expression of the 3 genes in Xenopus oocytes reveals that they all encode functional glucose transporters. In contrast to the situation in L. enriettii where both the ISO1 and ISO2 genes are developmentally regulated, the LmGT2 mRNA is expressed preferentially in promastigotes, whereas the LmGT1 and LmGT3 mRNAs are constitutively expressed in both life cycle stages. This observation suggests that each LmGT isoform is likely to have unique properties.

One study in the laboratory has focused on a genetic analysis of the LmGT glucose transporter gene cluster to define the physiological role of glucose transporters in this parasite. In this approach, the entire LmGT1-LmGT2-LmGT3 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 β-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 intracellular reducing agent NADPH), suggesting another reason why these mutants do not survive within these mammalian host cells.

Recently, we have 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 are currently attempting to identify which component of this linear amplicon 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 a minor glucose transporter, designated LmGT4, on a circular amplicon and have re-acquired the ability to transport hexoses and metabolize them as sources of metabolic energy.

Relevant Publications:

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.

3. Gene Cloning, Expression, and Functional Analysis of Nucleoside and Nulceobase Transporters from Leishmania Parasites.

In collaboration with the laboratory of Dr. Buddy Ullman in the Department of Biochemistry and Molecular Biology at OHSU, we are engaged in a detailed analysis of nucleoside transporter genes in both Leishmania donovani and Leishmania major. As explained above, purine nucleoside and nucleobase transporters are central to both the biochemistry and pharmacology of these parasites. While both laboratories are working on various aspects of nucleoside transporters in these and other parasitic protozoa, the work described here is being performed primarily in the Landfear lab, while related projects are being pursued primarily in the Ullman lab. Both laboratories engage in joint group meetings and frequent collaborative discussions regarding the nucleoside transporter projects.

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 6). All four of these permeases are members of the SLC29 family (also called the equilibrative nucleoside transporter family) and contain 11 transmembrane segments (Fig. 7).

Gene knockout studies performed on all four NT genes have revealed that any single gene can be deleted without compromising viability of promastigotes. However a dual knockout of the two nucleobase transporter genes, NT3 and NT4, could not be obtained, suggesting that these two nucleobase transporters together are probably essential for parasite viability. Of additional 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. Current studies are probing how NT4 is activated at acid pH and whether this nucleobase transporter is essential for infectivity of the parasite in a murine model of leishmaniasis.

Figure 6.The four distinct purine transporters in Leishmania species. 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).
	Figure 7. Schematic figure representing the predicted topology of the LdNT1.1 permease.Circles represent individual amino acids, with red circles indicating amino acids that are identical compared to the human equilibrative nucleoside transporter hENT1. The boxes indicate predicted transmembrane domains. Follow this link to see a larger LdNT1 Transporter topology figure.

Understanding the function of transporters requires knowledge of their three-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, the Landfear and Ullman laboratories have both engaged in computational studies to generate models for the three-dimensional structure of NT1 and NT2. These studies have been done in collaboration with Dr. Ujwal Shinde in the Department of Biochemistry and Molecular Biology at OHSU. Two independent models, one employing homology modeling or ‘threading’ and the other employing ab initio modeling, produced similar models for the topology of helix packing for NT family members (Fig. 8, ab initio model for NT1). These models predict that NT family members assume helix packing similar to that of Major Facilitator Superfamily members such as the bacterial lactose permease. Extensive mutagenesis studies of NT1 are consistent with and support the general accuracy of the computational models. Of particular interest, these models have 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. These three-dimensional models provide a platform for further probing the structure and function of these transporters at the molecular level.

 

Figure 8. Ab initio structural model of NT1 predicting the folding of transmembrane helices. The tan cylinders in A and B represent transmembrane helices and are numbered 1 through 11 in part B. Part B represents a 90° rotation of A around a horizontal axis. Residues where mutation resulted in strong phenotypes (loss of function, green; change in substrate specificity, red; failure to traffic to the cell surface, yellow) are indicated as space-filling side chains on various helices. The transport ‘pore’ can be seen in B as an open cavity at the center of the helices.

Relevant Publications

Valdés, R., Vasudevan, B, Conklin, D., and Landfear, S.M. (2004) Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway. Biochemistry 43:6793-6802.

Valdés, R., Liu, W., Ullman, B., and Landfear S.M. (2006) Comprehensive examination of charged intramembrane residues in a nucleoside transporter. J. Biol. Chem. 281:22647-22655.

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. An ab initio structural model of a nucleoside permease predicts functionally important residues. J. Biol. Chem., EPub ahead of print, jbc.M109.017947.

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