Ferlins and TgDOC2 in Toxoplasma Microneme, Rhoptry and Dense Granule Secretion.

The host cell invasion process of apicomplexan parasites like Toxoplasma gondii is facilitated by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis is facilitated by a double C2 domain (DOC2) protein family. This class of C2 domains is derived from an ancestral calcium (Ca2+) binding archetype, although this feature is optional in extant C2 domains. DOC2 domains provide combinatorial power to the C2 domain, which is further enhanced in ferlins that harbor 5-7 C2 domains. Ca2+ conditionally engages the C2 domain with lipids, membranes, and/or proteins to facilitating vesicular trafficking and membrane fusion. The widely conserved T. gondii ferlins 1 (FER1) and 2 (FER2) are responsible for microneme and rhoptry exocytosis, respectively, whereas an unconventional TgDOC2 is essential for microneme exocytosis. The general role of ferlins in endolysosmal pathways is consistent with the repurposed apicomplexan endosomal pathways in lineage specific secretory organelles. Ferlins can facilitate membrane fusion without SNAREs, again pertinent to the Apicomplexa. How temporal raises in Ca2+ combined with spatiotemporally available membrane lipids and post-translational modifications mesh to facilitate sequential exocytosis events is discussed. In addition, new data on cross-talk between secretion events together with the identification of a new microneme protein, MIC21, is presented.

Targeting a Subpocket in Trypanosoma brucei Phosphodiesterase B1 (TbrPDEB1) Enables the Structure-Based Discovery of Selective Inhibitors with Trypanocidal Activity.

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent ( Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 µM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.

Functional and genetic evidence that nucleoside transport is highly conserved in Leishmania species: Implications for pyrimidine-based chemotherapy.

Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy.

Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase.

BACKGROUND: Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. METHODOLOGY/PRINCIPAL FINDINGS: Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. CONCLUSIONS/SIGNIFICANCE: Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential.

The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference!

Kinetoplastids are unicellular, eukaryotic, flagellated protozoans containing the eponymous kinetoplast. Within this order, the family of trypanosomatids are responsible for some of the most serious human diseases, including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.), and leishmaniasis (Leishmania spp). Although cAMP is produced during the life cycle stages of these parasites, its signaling pathways are very different from those of mammals. The absence of G-protein-coupled receptors, the presence of structurally different adenylyl cyclases, the paucity of known cAMP effector proteins and the stringent need for regulation of cAMP in the small kinetoplastid cells all suggest a significantly different biochemical pathway and likely cell biology. However, each of the main kinetoplastid parasites express four class 1-type cyclic nucleotide-specific phosphodiesterases (PDEA-D), which have highly similar catalytic domains to that of human PDEs. To date, only TbrPDEB, expressed as two slightly different isoforms TbrPDEB1 and B2, has been found to be essential when ablated. Although the genomes contain reasonably well conserved genes for catalytic and regulatory domains of protein kinase A, these have been shown to have varied structural and functional roles in the different species. Recent discovery of a role of cAMP/AMP metabolism in a quorum-sensing signaling pathway in T. brucei, and the identification of downstream cAMP Response Proteins (CARPs) whose expression levels correlate with sensitivity to PDE inhibitors, suggests a complex signaling cascade. The interplay between the roles of these novel CARPs and the quorum-sensing signaling pathway on cell division and differentiation makes for intriguing cell biology and a new paradigm in cAMP signal transduction, as well as potential targets for trypanosomatid-specific cAMP pathway-based therapeutics.

Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei adenosine transporter 1 (TbAT1) and the proposal of a structural model for the protein.

The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1(-/-) T. brucei line. Out of a set of six reported SNPs from a reported 'resistance allele', none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [(3)H]-adenosine and [(3)H]-diminazene, identical to the tbat1(-/-) control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [(3)H]-diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.

Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA.

One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development.

Pyrimidine biosynthesis is not an essential function for Trypanosoma brucei bloodstream forms.

BACKGROUND: African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite. METHODOLOGY/PRINCIPAL FINDINGS: Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2'deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5(-/-) trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line. CONCLUSIONS/SIGNIFICANCE: Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.