Nutrient sensing in Leishmania: Flagellum and cytosol.

Parasites are by definition organisms that utilize resources from a host to support their existence, thus, promoting their ability to establish long-term infections and disease. Hence, sensing and acquiring nutrients for which the parasite and host compete is central to the parasitic mode of existence. Leishmania are flagellated kinetoplastid parasites that parasitize phagocytic cells, principally macrophages, of vertebrate hosts and the alimentary tract of sand fly vectors. Because nutritional supplies vary over time within both these hosts and are often restricted in availability, these parasites must sense a plethora of nutrients and respond accordingly. The flagellum has been recognized as an "antenna" that plays a core role in sensing environmental conditions, and various flagellar proteins have been implicated in sensing roles. In addition, these parasites exhibit non-flagellar intracellular mechanisms of nutrient sensing, several of which have been explored. Nonetheless, mechanistic details of these sensory pathways are still sparse and represent a challenging frontier for further experimental exploration.

A cytoskeletal protein complex is essential for division of intracellular amastigotes of Leishmania mexicana.

Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, which associate with KHARON. KHAP1 is located only in the subpellicular microtubules, whereas KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

Leishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models.

Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1-3 mutant which lacks the major glucose transporters LmxGT1-3. We identified a novel suppressor line (Δlmxgt1-3s2 ) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1-3s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non-essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources.

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes.

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability.

Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs.

Endochin-like quinolones (ELQs) are potent and specific inhibitors of cytochrome bc1 from Plasmodium falciparum and Toxoplasma gondii and show promise for novel antiparasitic drug development. To determine whether the mitochondrial electron transport chain of Leishmania parasites could be targeted similarly for drug development, we investigated the activity of 134 structurally diverse ELQs. A cohort of ELQs was selectively toxic to amastigotes of Leishmania mexicana and L. donovani, with 50% inhibitory concentrations (IC50s) in the low micromolar range, but the structurally similar hydroxynaphthoquinone buparvaquone was by far the most potent inhibitor of electron transport, ATP production, and intracellular amastigote growth. Cytochrome bc1 is thus a promising target for novel antileishmanial drugs, and further improvements on the buparvaquone scaffold are warranted for development of enhanced therapeutics.

Regulation and biological function of a flagellar glucose transporter in Leishmania mexicana: a potential glucose sensor.

In Leishmania mexicana parasites, a unique glucose transporter, LmxGT1, is selectively targeted to the flagellar membrane, suggesting a possible sensory role that is often associated with ciliary membrane proteins. Expression of LmxGT1 is down-regulated ∼20-fold by increasing cell density but is up-regulated ∼50-fold by depleting glucose from the medium, and the permease is strongly down-regulated when flagellated insect-stage promastigotes invade mammalian macrophages and transform into intracellular amastigotes. Regulation of LmxGT1 expression by glucose and during the lifecycle operates at the level of protein stability. Significantly, a ∆lmxgt1 null mutant, grown in abundant glucose, undergoes catastrophic loss of viability when parasites deplete glucose from the medium, a property not exhibited by wild-type or add-back lines. These results suggest that LmxGT1 may function as a glucose sensor that allows parasites to enter the stationary phase when they deplete glucose and that in the absence of this sensor, parasites do not maintain viability when they run out of glucose. However, alternate roles for LmxGT1 in monitoring glucose availability are considered. The absence of known sensory receptors with defined ligands and biologic functions in Leishmania and related kinetoplastid parasites underscores the potential significance of these observations.

Identification of the intracellular gate for a member of the equilibrative nucleoside transporter (ENT) family.

Equilibrative nucleoside transporters of the SLC29 family play important roles in many physiological and pharmacological processes, including import of drugs for treatment of cancer, AIDS, cardiovascular, and parasitic diseases. However, no crystal structure is available for any member of this family. In previous studies we generated a computational model of the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1) that captured this permease in the outward-closed conformation, and we identified the extracellular gate. In the present study we have modeled the inward-closed conformation of LdNT1.1 using the crystal structure of the Escherichia coli fucose transporter FucP and have identified four transmembrane helices whose ends close to form a predicted intracellular gate. We have tested this prediction by site-directed mutagenesis of relevant helix residues and by cross-linking of introduced cysteine pairs. The results are consistent with the predictions of the computational model and suggest that a similarly constituted gate operates in other members of the equilibrative nucleoside transporter family.

Flagellar membrane proteins in kinetoplastid parasites.

All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes.

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

'Transient' genetic suppression facilitates generation of hexose transporter null mutants in Leishmania mexicana.

The genome of Leishmania mexicana encompasses a cluster of three glucose transporter genes designated LmxGT1, LmxGT2 and LmxGT3. Functional and genetic studies of a cluster null mutant (Δlmxgt1-3) have dissected the roles of these proteins in Leishmania metabolism and virulence. However, null mutants were recovered at very low frequency, and comparative genome hybridizations revealed that Δlmxgt1-3 mutants contained a linear extrachromosomal 40 kb amplification of a region on chromosome 29 not amplified in wild type parasites. These data suggested a model where this 29-40k amplicon encoded a second site suppressor contributing to parasite survival in the absence of GT1-3 function. To test this, we quantified the frequency of recovery of knockouts in the presence of individual overexpressed open reading frames covering the 29-40k amplicon. The data mapped the suppressor activity to PIFTC3, encoding a component of the intraflagellar transport pathway. We discuss possible models by which PIFTC3 might act to facilitate loss of GTs specifically. Surprisingly, by plasmid segregation we showed that continued PIFTC3 overexpression was not required for Δlmxgt1-3 viability. These studies provide the first evidence that genetic suppression can occur by providing critical biological functions transiently. This novel form of genetic suppression may extend to other genes, pathways and organisms.

Both sequence and context are important for flagellar targeting of a glucose transporter.

Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins.

Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents.

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product-inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in vitro and in vivo antileishmanial activities, detailed structure-activity relationships, and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family and paves the way for the generation of new oral antileishmanial drugs.

Cysteine cross-linking defines the extracellular gate for the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1).

Equilibrative nucleoside transporters are a unique family of proteins that enable uptake of nucleosides/nucleobases into a wide range of eukaryotes and internalize a myriad of drugs used in the treatment of cancer, heart disease, AIDs, and parasitic infections. In previous work we generated a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani, using ab initio computation. The model suggested that aromatic residues present in transmembrane helices 1, 2, and 7 interact to form an extracellular gate that closes the permeation pathway in the inward-open conformation. Mutation of residues Phe-48(TM1) and Trp-75(TM2) abrogated transport activity, consistent with such prediction. In this study cysteine mutagenesis and oxidative cross-linking were combined to analyze proximity relationships of helices 1, 2, and 7 in LdNT1.1. Disulfide bond formation between introduced paired cysteines at the interface of such helices (A61C(TM1)/F74C(TM2), A61C(TM1)/G350C(TM7), and F74C(TM2)/G350C(TM7)) was analyzed by transport measurement and gel mobility shifts upon oxidation with Cu (II)-(1,10-phenanthroline)(3). In all cases cross-linking inhibited transport. However, if LdNT1.1 ligands were included during cross-linking, inhibition of transport was reduced, suggesting that ligands moved the three gating helices apart. Moreover, all paired cysteine mutants exhibited a mobility shift upon oxidation, corroborating the formation of a disulfide bond. These data support the notion that helices 1, 2, and 7 constitute the extracellular gate of LdNT1.1, thus further validating the computational model and the previously demonstrated importance of F48(TM1) and Trp-75(TM2) in tethering together helices that are part of the gate.

Optimization of Orally Bioavailable Antileishmanial 2,4,5-Trisubstituted Benzamides.

Leishmaniasis, a neglected tropical disease caused by Leishmania species parasites, annually affects over 1 million individuals worldwide. Treatment options for leishmaniasis are limited due to high cost, severe adverse effects, poor efficacy, difficulty of use, and emerging drug resistance to all approved therapies. We discovered 2,4,5-trisubstituted benzamides (4) that possess potent antileishmanial activity but poor aqueous solubility. Herein, we disclose our optimization of the physicochemical and metabolic properties of 2,4,5-trisubstituted benzamide that retains potency. Extensive structure-activity and structure-property relationship studies allowed selection of early leads with suitable potency, microsomal stability, and improved solubility for progression. Early lead 79 exhibited an 80% oral bioavailability and potently blocked proliferation of Leishmania in murine models. These benzamide early leads are suitable for development as orally available antileishmanial drugs.

Nutrient transport and pathogenesis in selected parasitic protozoa.

Parasitic protozoa, such as malaria parasites, trypanosomes, and Leishmania, acquire a plethora of nutrients from their hosts, employing transport proteins located in the plasma membrane of the parasite. Application of molecular genetic approaches and the completion of genome projects have allowed the identification and functional characterization of a cohort of transporters and their genes in these parasites. This review focuses on a subset of these permeases that have been studied in some detail, that import critical nutrients, and that provide examples of approaches being undertaken broadly with these and other parasite transporters. Permeases reviewed include those for hexoses, purines, iron, polyamines, carboxylates, and amino acids. Topics of special emphasis include structure-function approaches, critical roles for transporters in parasite viability and physiology, regulation of transporter expression, and subcellular targeting. Investigations of parasite transporters impact a broad spectrum of basic biological problems in these protozoa.

New Vistas in the Biology of the Flagellum-Leishmania Parasites.

Like other kinetoplastid protozoa, the flagellum in Leishmania parasites plays central roles throughout the life cycle. Discoveries over the past decade have begun to elucidate flagellar functions at the molecular level in both the insect vector stage promastigotes and intra-macrophage amastigotes. This focused review will highlight recent advances that contribute to understanding flagellar function in the various biological contexts encountered by Leishmania parasites.

Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major.

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations.

Adaptive responses to purine starvation in Leishmania donovani.

Starvation of Leishmania donovani parasites for purines leads to a rapid amplification in purine nucleobase and nucleoside transport. Studies with nucleoside transport-deficient L. donovani indicate that this phenomenon is mediated by the nucleoside transporters LdNT1 and LdNT2, as well as by the purine nucleobase transporter LdNT3. The escalation in nucleoside transport cannot be ascribed to an increase in either LdNT1 or LdNT2 mRNA. However, Western analyses on parasites expressing epitope-tagged LdNT2 revealed a marked upregulation in transporter protein at the cell surface. Kinetic investigations of LdNT1 and LdNT2 activities from purine-replete and purine-starved cells indicated that both transporters exhibited significant increases in V(max) for their ligands under conditions of purine-depletion, although neither transporter displayed an altered affinity for its respective ligands. Concomitant with the increase in purine nucleoside and nucleobase transport, the purine salvage enzymes HGPRT, XPRT and APRT were also upregulated, suggesting that under conditions where purines are limiting, Leishmania parasites remodel their purine metabolic pathway to maximize salvage. Moreover, qRT-PCR analyses coupled with cycloheximide inhibition studies suggest that the underlying molecular mechanism for this augmentation in purine salvage occurs post-transcriptionally and is reliant on de novo protein synthesis.

Amino-Substituted 3-Aryl- and 3-Heteroarylquinolines as Potential Antileishmanial Agents.

Leishmaniasis, a disease caused by protozoa of the Leishmania species, afflicts roughly 12 million individuals worldwide. Most existing drugs for leishmaniasis are toxic, expensive, difficult to administer, and subject to drug resistance. We report a new class of antileishmanial leads, the 3-arylquinolines, that potently block proliferation of the intramacrophage amastigote form of Leishmania parasites with good selectivity relative to the host macrophages. Early lead 34 was rapidly acting and possessed good potency against L. mexicana (EC50 = 120 nM), 30-fold selectivity for the parasite relative to the macrophage (EC50 = 3.7 µM), and also blocked proliferation of Leishmania donovani parasites resistant to antimonial drugs. Finally, another early lead, 27, which exhibited reasonable in vivo tolerability, impaired disease progression during the dosing period in a murine model of cutaneous leishmaniasis. These results suggest that the arylquinolines provide a fruitful departure point for the development of new antileishmanial drugs.

Amplification of an alternate transporter gene suppresses the avirulent phenotype of glucose transporter null mutants in Leishmania mexicana.

A glucose transporter null mutant of the parasitic protozoan Leishmania mexicana, in which three linked glucose transporter genes have been deleted by targeted gene replacement, is unable to replicate as amastigote forms within phagolysomes of mammalian host macrophages and is avirulent. Spontaneous suppressors of the null mutant have been isolated that partially restore replication of parasites within macrophages. These suppressor mutants have amplified the gene for an alternative hexose transporter, the LmGT4 permease (previously called the D2 permease), on a circular extrachromosomal element, and they overexpress LmGT4 mRNA and protein. The suppressors have also regained the ability to transport hexoses, and they have reverted other phenotypes of the null mutant exhibiting enhanced resistance to oxidative killing, heat shock and starvation for nutrients, as well as augmented levels of the storage carbohydrate beta-mannan, increased cell size and increased growth as insect stage promastigotes compared with the unsuppressed mutant. Complementation of the null mutant with the LmGT4 gene on a multicopy episomal expression vector also reverted these phenotypes, confirming that suppression results from amplification of the LmGT4 gene. These results underscore the importance of hexose transporters for the infectious stage of the parasite life cycle.