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1.  A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence 
PLoS Pathogens  2016;12(1):e1005340.
Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.
Author Summary
Leishmaniasis is a serious parasitic disease that affects 12 million people worldwide, with clinical manifestations ranging from self-healing cutaneous lesions to deadly visceralizing disease. A vaccine is not available, and new and less toxic drugs against this protozoan parasite are urgently needed. Following introduction into vertebrate hosts during a sand fly blood meal, Leishmania parasites undergo extensive changes in morphology and metabolism that are critical for adaptation to life inside host macrophages and replication as amastigotes. Earlier studies identified major events that occur during amastigote differentiation, but the signaling mechanism initiating this process remained poorly understood. Previously we demonstrated a novel role for the reactive oxygen species (ROS) H2O2 in initiating amastigote differentiation, a process proposed to be dependent on iron availability inside the parasite’s mitochondria. In this study we identify LMIT1, a Leishmania transmembrane protein that functions as a mitochondrial iron transporter and is conserved in other trypanosomatid protozoan parasites. Reduced LMIT1 expression impairs mitochondrial function in the infective amastigote stage, abolishing parasite virulence. Our findings identify LMIT1 as a promising new drug target, and support the conclusion that iron-dependent ROS signals generated in the mitochondria regulate differentiation of virulent Leishmania amastigotes.
PMCID: PMC4704735  PMID: 26741360
2.  IRONy OF FATE: Role of iron-mediated ROS in Leishmania differentiation 
Trends in parasitology  2013;29(10):489-496.
The protozoan parasite Leishmania experiences extreme environmental changes as it alternates between insect and mammalian hosts. In some species, differentiation of insect promastigotes into mammalian-infective amastigotes is induced by elevated temperature and low pH, conditions found within macrophage parasitophorous vacuoles (PVs). However, the signaling events controlling amastigote differentiation remain poorly understood. Recent studies revealed a novel role for iron uptake in orchestrating the differentiation of amastigotes, in a process that involves production of reactive oxygen species (ROS) and is independent from pH and temperature changes. ROS are generally thought to be deleterious for pathogens, but it is becoming increasingly apparent that they can also function as signaling molecules regulating Leishmania differentiation, in a process that is tightly controlled by iron availability.
PMCID: PMC3783550  PMID: 23948431
Leishmania; differentiation; iron; virulence; ROS; FeSOD
3.  Heme Uptake Mediated by LHR1 Is Essential for Leishmania amazonensis Virulence 
Infection and Immunity  2013;81(10):3620-3626.
The protozoan parasite Leishmania amazonensis is a heme auxotroph and must acquire this essential factor from the environment. Previous studies showed that L. amazonensis incorporates heme through the transmembrane protein LHR1 (Leishmania Heme Response 1). LHR1-null promastigotes were not viable, suggesting that the transporter is essential for survival. Here, we compared the growth, differentiation, and infectivity for macrophages and mice of wild-type, LHR1-single-knockout (LHR1/Δlhr1), and LHR1-complemented (LHR1/Δlhr1 plus LHR1) L. amazonensis strains. LHR1/Δlhr1 promastigotes replicated poorly in heme-deficient media and had lower intracellular heme content than wild-type parasites. LHR1/Δlhr1 promastigotes were also less effective in reducing ferric iron to ferrous iron, a reaction mediated by the heme-containing parasite enzyme LFR1 (Leishmania Ferric Reductase 1). LHR1/Δlhr1 parasites differentiated normally into aflagellated forms expressing amastigote-specific markers but were not able to replicate intracellularly after infecting macrophages. Importantly, the intracellular growth of LHR1/Δlhr1 amastigotes was fully restored when macrophages were allowed to phagocytose red blood cells prior to infection. LHR1/Δlhr1 parasites were also severely defective in the development of cutaneous lesions in mice. All phenotypes observed in LHR1/Δlhr1 L. amazonensis were rescued by expression of episomal LHR1. Our results reveal the importance of efficient heme uptake for L. amazonensis replication and vertebrate host infectivity, reinforcing the potential usefulness of LHR1 as a target for new antileishmanial drugs.
PMCID: PMC3811768  PMID: 23876801
4.  Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels 
Iron uptake promotes hydrogen peroxide–dependent differentiation of Leishmania promastigotes into infective amastigotes.
During its life cycle, Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. Elevated temperature and decreased pH, conditions encountered after macrophage invasion, can induce axenic differentiation of avirulent promastigotes into virulent amastigotes. Here we show that iron uptake is a major trigger for the differentiation of Leishmania amazonensis amastigotes, independently of temperature and pH changes. We found that iron depletion from the culture medium triggered expression of the ferrous iron transporter LIT1 (Leishmania iron transporter 1), an increase in iron content of the parasites, growth arrest, and differentiation of wild-type (WT) promastigotes into infective amastigotes. In contrast, LIT1-null promastigotes showed reduced intracellular iron content and sustained growth in iron-poor media, followed by cell death. LIT1 up-regulation also increased iron superoxide dismutase (FeSOD) activity in WT but not in LIT1-null parasites. Notably, the superoxide-generating drug menadione or H2O2 was sufficient to trigger differentiation of WT promastigotes into fully infective amastigotes. LIT1-null promastigotes accumulated superoxide radicals and initiated amastigote differentiation after exposure to H2O2 but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species in orchestrating the differentiation of virulent Leishmania amastigotes in a process regulated by iron availability.
PMCID: PMC3570109  PMID: 23382545
5.  2′-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family 
Nucleic Acids Research  2011;39(11):4756-4768.
The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.
PMCID: PMC3113572  PMID: 21310715
6.  Hypermethylated cap 4 maximizes Trypanosoma brucei translation 
Molecular microbiology  2009;72(5):1100-1110.
Through trans-splicing of a 39-nt Spliced Leader (SL) onto each protein-coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5′-cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2′-O-ribose methyltransferases, TbMTr1, TbMTr2, and TbMTr3, reveal distinct roles for four 2′-O-methylated nucleotides. Elimination of individual gene pairs yields viable cells, however attempts at double knockouts resulted in the generation of a TbMTr2−/−/TbMTr3−/− cell line only. Absence of both kinetoplastid-specific enzymes in TbMTr2−/−/TbMTr3−/− lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1−/− translation is comparable to wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1−/− and TbMTr2−/−/TbMTr3−/− lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm-independent and nucleoplasmic Sm-dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.
PMCID: PMC2859698  PMID: 19504740
gene knockout; methyltransferase; ribose 2′-O-methylation; SL RNA; spliced leader; trans-splicing
7.  Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex ▿ ‡  
Molecular and Cellular Biology  2008;29(5):1202-1211.
Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2′-O-ribose methylation of the first four nucleotides and pseudouridine (ψ) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2′-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL ψ28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3′-end maturation, and ψ28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.
PMCID: PMC2643836  PMID: 19103757
8.  The 2′-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei▿ †  
Molecular and Cellular Biology  2007;27(17):6084-6092.
mRNA cap 1 2′-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2′-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2′-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2′-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2′-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3′-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.
PMCID: PMC1952150  PMID: 17606627
9.  Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei†  
Eukaryotic Cell  2006;5(6):905-915.
In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5′ ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m7G cap, ribose 2′-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2′-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A2 and is not required for subsequent steps; TbMT511 methylates C3, without which U4 methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m7G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A2, C3, and U4 methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.
PMCID: PMC1489268  PMID: 16757738
10.  Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II 
Eukaryotic Cell  2004;3(5):1185-1197.
Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.
PMCID: PMC522618  PMID: 15470247
11.  Characterization of the Crithidia fasciculata mRNA Cycling Sequence Binding Proteins 
Molecular and Cellular Biology  2001;21(14):4453-4459.
The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBPA and CSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kDa, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near the C terminus. The latter motif is conserved in other trypanosomatid species. Gel sieving chromatography and glycerol gradient sedimentation results indicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycling sequence and together can be reconstituted to give an RNA gel shift similar to that of purified CSBP. Proteins in cell extracts bind to an RNA probe containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synchrony with target mRNA levels. CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional factors are involved in the cyclic binding to the cycling sequence elements.
PMCID: PMC87105  PMID: 11416125

Results 1-11 (11)