A major objective of the Landfear laboratory is to investigate membrane transport proteins that provide parasitic protozoa with important nutrients that they acquire from their hosts. The ability and requirement to salvage nutrients from both the invertebrate vectors and mammalian hosts is a key feature of the parasitic mode of existence.
Our laboratory has focused heavily upon transporters for:
- Glucose and related hexoses, which represent both central fuel sources and metabolic precursors for a variety of anabolic pathways.
- Purine nucleosides and nucleobases, key building blocks in many metabolic pathways, including RNA and DNA synthesis, and compounds that cannot be synthesized by any of the parasitic protozoa and must be salvaged from their hosts.
In addition, our laboratory is engaged in several drug development projects, both those that target critical parasite transporters and those that entail broader based phenotypic screens.
Glucose transporters in Leishmania mexicana
L. mexicana and other Leishmania species contain three clustered genes that encode three related but distinct glucose (hexose) transporters: GT1, GT2, and GT3 (see figure one).
Figure one shows the glucose transporter gene cluster in Leishmania mexicana that encodes three glucose transporter isoforms: GT1, GT2 and GT3. The image at the right shows the topology of glucose transporters in the membrane. Numbered white rectangles represent transmembrane segments.
Each of the GTs encoded by these genes is a high affinity transporter for glucose and a somewhat lower affinity transporter for the related hexoses fructose, mannose, and galactose. The high affinity for glucose allows the parasite to compete effectively for uptake of this nutrient from the host environment.
Studies in the laboratory have demonstrated that GT2 is the principal glucose transporter expressed in the flagellated insect stage of the parasite called the promastigote. GT2 mRNA is abundant in promastigotes but is downregulated in level ~20-fold in the intracellular amastigotes that live inside the acidified phagolysosomes of mammalian host macrophages (see figure two), and this regulation occurs at the level of mRNA stability.
GT1 is a hexose transporter with especially interesting properties. It is localized almost exclusively to the membrane of the flagellum of the promastigote (see figure three). In contrast, GT2 is located in the plasma membrane surrounding the cell body but is excluded from the flagellar membrane. Recent studies have established that GT1 functions to sense the level of extracellular glucose in promastigotes and that this sensory function is critical for the viability of the parasite in this life cycle stage. Null mutants, ∆gt1, in which the GT1 genes have been deleted are unable to sense the depletion in glucose that occurs when parasites grow to high density.
Figure two: The two principal life cycle stages of Leishmania parasites. Promastigotes, top, live within the sand fly vector in nature. Amastigotes, bottom, infect mammalian macrophages and reside within acidified phagolysosomal vesicles. Smaller dark spots represent the nuclei of amastigotes inside the larger macrophages.
Consequently, these null mutants fail to enter the stationary phase of growth and undergo catastrophic cell death. The selective location of GT1 to the flagellar membrane is consistent with a sensory function for this protein, as many ciliary or flagellar membrane proteins in other organisms appear to play roles as sensory receptors. In addition, the ability of transporters to function as sensors or signal transduction receptors has been observed in a variety of other systems and has led to the concept of ‘transceptors’, transporters or transporter-like membrane proteins that bind their ligands and send a sensory signal to the interior of the cell.
This function for GT1 is particularly important, because so little is known about how parasites such as Leishmania and trypanosomes sense their environment. These parasites universally lack G-protein coupled receptors or tyrosine-kinase linked receptors, major families of sensory receptors on most other eukaryotes. Hence, GT1 may offer one of the first insights into a membrane protein in trypanosomatid parasites that binds a known ligand (glucose) and has a known biological function (transition of the parasite from growth to stationary phase) in sensing the changing environment to which parasites are continually exposed. By contrast, GT3 is the principal glucose transporter expressed in amastigotes within mammalian host macrophages. Unexpectedly, this permease is expressed in the endoplasmic reticulum (ER) membrane of the amastigote, whereas expression of GT1 and GT2 essentially ceases in amastigotes.
This pronounced developmental regulation occurs at the level of protein stability, with GT1 and GT2 being degraded in amastigotes while GT3 is stable. The unique C-terminal region of GT3 is likely responsible for its stable expression in amastigotes and for targeting to the ER. The mechanisms for the organellar targeting and the differential regulation of GT3 are currently under investigation.
Gene knockout studies have also emphasized the critical nature of GTs in the parasite life cycle. As mentioned above, GT1 is crucial during the growth to stationary phase transition of promastigotes and is thus likely critical to the parasite during its colonization of the sand fly insect vector. In a knockout of the entire glucose transporter gene cluster, the ∆gt1-3 null mutants are strongly impaired as intracellular amastigotes, and they form lesions in BALB/c mice that are small and develop very slowly compared to lesions induced by wild type parasites.
Identification of the Kharon1 protein that mediates flagellar targeting of
GT1 and is also essential for viability of Leishmania amastigotes and African trypanosomes
To understand how membrane proteins are selectively targeted to the flagellar membrane, we initiated tagging and mass spectrometric studies to identify proteins that specifically interacted with the flagellar targeting signal of GT1. A protein designated KHARON1 (named after the figure from Greek mythology who transports souls across the barrier of the River Styx) was identified that was required for migration of the GT1 transporter into the flagellar membrane. Targeted deletion of the Kharon1 gene (∆kharon1 mutants) prevented flagellar targeting of GT1 but was not lethal to culture form promastigotes. Strikingly, the ∆kharon1 null mutant was not viable as intracellular amastigotes, and this mutant was completely avirulent in mice (figure four). Hence, targeting of membrane proteins into the small ‘residual’ flagellum of the amastigote is likely essential for the ability of these parasites to survive inside the host macrophage.
Similar experiments with the Kharon1 ortholog from the African trypanosome Trypanosoma brucei revealed that expression of the KHARON1 protein is essential for viability of both the bloodstream (mammalian) and procyclic (Tsetse fly vector) stages of trypanosomes and that KHARON1 thus plays a vital role in multiple trypanosomatid parasites.
Furthermore, while an ortholog of the Leishmania GT1 is not expressed in African trypanosomes, other flagellar membrane proteins, such as a putative Ca2+ channel, require KHARON1 for targeting to the flagellum. Ongoing studies focus on the role of KHARON1 in transport of multiple flagellar membrane proteins in both parasites and the reasons for its requirement for viability of the infectious forms of each parasite.
Leishmania purine nucleoside and nucleobase transporters
One of the most important metabolic differences between parasitic protozoa, such as Leishmania and trypanosomes, and their mammalian hosts is that the parasites are unable to synthesize the purine ring and must rely upon their hosts to provide these critical building blocks. Hence, there has been great interest in understanding how parasites salvage essential purines and whether blocking such salvage could represent a novel therapeutic strategy for control of infection.
The Landfear and Ullman (Department of Biochemistry and Molecular Biology) laboratories have collaborated closely on studies of Leishmania purine transporters. Our laboratories employed a genetic strategy to clone the genes for the first parasite purine transporters, the NT1 and NT2 transporters from L. donovani that mediate uptake of purine nucleosides such as adenosine (NT1), as well as guanosine and inosine (NT2). In addition, we identified the NT3 and NT4 genes that code for transporters that take up purine nucleobases such as adenine, guanine, hypoxanthine, and xanthine.
Many studies in the Landfear laboratory have focused on discerning the detailed structure and function of the NT1 adenosine transporter. Since no crystal structures are available for any NT, our laboratory collaborated with that of Ujwal Shinde (Biochemistry and Molecular Biology) to develop several computational 3-dimensional models of NT1, in both the outward closed/inward open and the outward open/inward closed conformations.
These studies have allowed us to understand how this and related transporters fold within the membrane and to identify functionally important amino acids and domains of the permease (e.g., the permeation channel and the outer and inner ‘gates’ that close off each end of this channel in the two alternating conformations of the permease) (see figure five). Functional studies have also demonstrated that NT3 and NT4 both transport the purine nucleobases adenosine, guanine, hypoxanthine, and xanthine, but that NT3 is optimized to function in the acidic environment of the macrophage phagolysome, whereas NT4 functions optimally at neutral pH. For this reason, L. major parasites in which the NT3 gene has been deleted, ∆nt3, are strongly impaired in ability to generate lesions in BALB/c mice. Similar gene knockout studies on NT1 and NT2 of L. major have demonstrated that a double ∆nt1/∆nt2 gene knockout that cannot transport purine nucleosides is especially impaired in lesion formation. These studies underscore the critical roles that purine nucleoside and nucleobase transporters play in the ability of Leishmania parasites to survive inside the phagolysosome of the host macrophages and to cause disease.
The malaria hexose transporter PfHT as a promising drug target
Malaria parasites such as Plasmodium falciparum represent one of the most burdensome global health problems, with an estimated ~300 million infections per year and as many as 1 million deaths per year. Since development of drug resistance has been a problem with malaria chemotherapy, there is an acute need for new drugs that target distinct processes from those targeted by current drugs. The malaria hexose transporter PfHT has been widely recognized as a promising novel target. This is because malaria parasites rely upon uptake of large amounts of glucose from the blood followed by inefficient glucose metabolism that occurs solely by glycolysis. This heavy reliance upon import of glucose from host blood through a single transporter, PfHT, implies that selective inhibition of this central permease could be a highly effective therapeutic strategy. Indeed both genetic and pharmacological studies from several laboratories, including that of Sanjeev Krishna at St. George’s Medical School in London that first identified the PfHT gene, have validated PfHT as a worthy drug target and have established the dependency of malaria parasites upon PfHT function, not only in their intraerythrocytic stages but also in liver and mosquito stages of the life cycle.
Our laboratory established a heterologous expression system in which the PfHT permease was expressed as the sole glucose transporter in the ∆gt1-3 null mutant of L. mexicana. In collaboration with the laboratory of Kip Guy at St. Jude Children’s Hospital in Memphis, this expression system was employed in high throughput screens of several libraries of anti-malarial compounds, those that had been identified in phenotypic screens as having the ability to kill P. falciparum intraerythrocytic parasites in vitro. Several of these anti-malarial compounds were identified that selectively inhibited the glucose uptake activity of PfHT, versus the human glucose transporter GLUT1, at nM concentrations. Hence, these compounds represent starting points for development of leads and ultimately drugs that inhibit growth of malaria parasites by starving them for glucose.
Targeting the Leishmania mitochondrial electron transport chain for development of novel drugs
Many organisms generate the majority of their metabolic energy (ATP) in the mitochondria via movement of electrons down the mitochondrial electron transport chain (ETC). Our collaborator Michael Riscoe at the Portland Veterans Administration Medical Center has developed a library of compounds called Endochin-like Quinolones (ELQs) some of which have proven highly effective against malaria parasites. ELQs are structural analogs of the compound coenzyme Q or ubiquinone that mediates transport of electrons between several protein components of the mitochondrial electron transport chain. Some of these ELQs proved to be highly specific for the malarial cytochrome bc1, a multisubunit component of the ETC, but did not inhibit the human protein, and they are highly efficacious at killing malaria parasites (where the ETC is required for synthesis of essential pyrimidines but not for generation of ATP). Hence, the Landfear, Ullman, and Riscoe laboratories initiated a collaborative project to determine whether any of these ELQs might selectively inhibit the ETC of Leishmania parasites, potentially starving the parasites for ATP and also generating toxic Reactive Oxygen Species that are released from the inhibited ETC. By screening over 100 ELQs, we have identified several that kill amastigotes inside host macrophages when applied at nM or low µM concentrations. These studies underscore the potential of targeting the Leishmania ETC for development of new desperately needed anti-leishmanial drugs and suggest that further modification of the top ELQ hits may lead to development of such novel therapeutics.
Phenotypic screens for compounds that kill Leishmania parasites
In the process of screening for compounds that selectively inhibit PfHT using a heterologous expression system in Leishmania parasites (see section on malaria hexose transporter above), we also identified ~2800 compounds from a 600,000 compound library that inhibited growth of Leishmania parasites. This screen effectively represented an additional ‘phenotypic screen’ against Leishmania in which this ‘focused library’ of anti-leishmanial compounds kill the parasites by unknown mechanisms of action. Further ongoing studies with this focused library have uncovered various compounds that kill amastigotes inside macrophages at nM concentrations (see figure six). We are completing the amastigote screen of these 2800 compounds to identify the cohort of compounds that kill intra-macrophage amastigotes with the highest potency and with the most desirable chemical properties for further drug development. This approach may identify a cohort of compounds that can serve as leads for development of new anti-leishmanial drugs.
Figure six: Dose-response curves for killing of intracellular amastigotes of L. mexicana by compound SJ551674-2 (open circles) compared to effect on host macrophages (filled circles). This compound selectively kills the intracellular parasites with an EC50 value of ~140nM, but it is not toxic to host macrophages.