Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania. Characterization of gene deletion mutants

The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and Deltaarg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The Deltaarg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the Deltaarg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the Deltaarg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.     

Generation of Leishmania mutants lacking antibiotic resistance genes using a versatile hit-and-run targeting strategy

The development of a method to create defined mutants of Leishmania parasites lacking foreign genes conferring resistance to antibiotics has both experimental and practical applications. Mutants deficient in specific virulence genes have potential as attenuated live vaccines, but these can only be of clinical relevance if the antibiotic resistance genes used for selection of the mutants are subsequently removed. In addition, the limited number of antibiotic resistance genes that can be used for genetic manipulation of Leishmania means that a system for recycling them for subsequent use would be highly beneficial when multiple genetic modifications are wanted. In the method we report here, a cassette carrying in tandem the hygromycin resistance gene as a positive marker and thymidine kinase gene as a negative marker is first integrated into the locus of interest and then replaced by a null targeting fragment containing no exogenous DNA. The application of this hit-and-run strategy for removal of one allele of the CPB cysteine peptidase gene array of Leishmania infantum is described.     

Metabolic changes in glucose transporter-deficient Leishmania mexicana and parasite virulence

Leishmania mexicana are parasitic protozoa that express a variety of glycoconjugates that play important roles in their biology as well as the storage carbohydrate beta-mannan, which is an essential virulence factor for survival of intracellular amastigote forms in the mammalian host. Glucose transporter null mutants, which are viable as insect form promastigotes but not as amastigotes, do not take up glucose and other hexoses but are still able to synthesize these glycoconjugates and beta-mannan, although at reduced levels. Synthesis of these carbohydrate-containing macromolecules could be accounted for by incorporation of non-carbohydrate precursors into carbohydrates by gluconeogenesis. However, the significantly reduced level of the virulence factor beta-mannan in the glucose transporter null mutants compared with wild-type parasites may contribute to the non-viability of these null mutants in the disease-causing amastigote stage of the life cycle.     

LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.     

Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.     

Linear amplicons as precursors of amplified circles in methotrexate-resistant Leishmania tarentolae.

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.     

Leishmania infantum nicotinamidase is required for late-stage development in its natural sand fly vector, Phlebotomus perniciosus

Leishmania infantum nicotinamidase, encoded by the Lipnc1 gene, converts nicotinamide into nicotinicacid to ensure Nicotinamide–Adenine–Dinucleotide (NAD+) biosynthesis. We were curious to explore the role of this enzyme during L. infantum development in its natural sand fly vector, Phlebotomus perniciosus (Diptera, Phlebotominae), using null mutants with a deleted Lipnc1 gene. The null mutants developed as well as the wild type L. infantum at the early time points post their ingestion within the bloodmeal. In contrast, once the blood meal digestion was completed, the null mutants were unable to develop further and establish late-stage infections. Data highlight the importance of the nicotinamide degradation pathway for Leishmania development in sand flies. They indicate that the endogenous nicotinamidase is essential for Leishmania development in the sand fly after the blood meal has been digested and the remnants defecated.     

Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major

10-Formyl tetrahydrofolate (10-CHO-THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP⁺-dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10-CO-THF ( K _i 105 nM), as was Leishmania growth (EC₅₀ 1.1 μM). Previous studies showed null ftl ^- mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1 ^- null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania , based on segregational loss of episomal complementing genes rather than transfection; analysis of ∼1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed 'genetic metabolite complementation' using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1 ^- null parasites were readily obtained. These data establish a requirement for 10-CHO-THF metabolism in L. major , and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.     

Ploidy changes associated with disruption of two adjacent genes on Leishmania major chromosome 1

Leishmania major Friedlin (LmjF) is a kinetoplastid protozoan whose genomic sequence has been recently elucidated. About 60% of the identified genes do not have a known function, and many are trypanosomatid-specific. Here we characterise two adjacent genes from LmjF chromosome 1 (chr1): LmjF01.0750, which encodes a predicted protein with a serine/threonine protein kinase motif and LmjF01.0760, which encodes a product with no similarity to other known proteins. Orthologues of both genes are present in Trypanosoma cruzi, but neither occur in Trypanosoma brucei. We have mapped polyadenylation and spliced-leader acceptor sites for both genes, and show that they differ between Leishmania species. Attempts to generate null mutants of LmjF01.0750 by homologous recombination were unsuccessful and led to the apparent triploidy of the entire genome, suggesting that it is an essential gene. Interestingly, at least two copies of LmjF01.0750 are required for cell survival. Further evidence of genome plasticity in Leishmania was provided by changes in chr1 copy number that occurred during in vitro growth of wild-type LmjF promastigotes and following replacement of a single copy of LmjF01.0760.     

Lipophosphoglycan is not required for infection of macrophages or mice by Leishmania mexicana

Cell surface lipophosphoglycan (LPG) is commonly regarded as a multifunctional Leishmania virulence factor required for survival and development of these parasites in mammals. In this study, the LPG biosynthesis gene lpg1 was deleted in Leishmania mexicana by targeted gene replacement. The resulting mutants are deficient in LPG synthesis but still display on their surface and secrete phosphoglycan-modified molecules, most likely in the form of proteophosphoglycans, whose expression appears to be up-regulated. LPG-deficient L.mexicana promastigotes show no significant differences to LPG-expressing parasites with respect to attachment to, uptake into and multiplication inside macrophages. Moreover, in Balb/c and C57/BL6 mice, LPG-deficient L.mexicana clones are at least as virulent as the parental wild-type strain and lead to lethal disseminated disease. The results demonstrate that at least L. mexicana does not require LPG for experimental infections of macrophages or mice. Leishmania mexicana LPG is therefore not a virulence factor in the mammalian host.     

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 β-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.     

A mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host.

The parasitic protozoon Leishmania mexicana undergoes two major developmental stages in its life cycle exhibiting profound physiological and morphological differences, the promastigotes in the insect vector and the amastigotes in mammalian macrophages. A deletion mutant, Deltalmsap1/2, for the secreted acid phosphatase (SAP) gene locus, comprising the two SAP genes separated by an intergenic region of approximately 11.5 kb, lost its ability to cause a progressive disease in Balb/c mice. While in vitro growth of promastigotes, invasion of host cells and differentiation from promastigotes to amastigotes was indistinguishable from the wild-type, the mutant parasites ceased to proliferate when transformed to amastigotes in infected macrophages or in a macrophage-free in vitro differentiation system, suggesting a stage-specific growth arrest. This phenotype could be reverted by complementation with 6 kb of the intergenic region of the SAP gene locus. Sequence analysis identified two open reading frames, both encoding single copy genes; one gene product shows high homology to mitogen-activated protein (MAP) kinases. Complementation experiments revealed that the MAP kinase homologue, designated LMPK, is required and is sufficient to restore the infectivity of the Deltalmsap1/2 mutant. Therefore, LMPK is a kinase that is essential for the survival of L.mexicana in the infected host by affecting the cell division of the amastigotes.     

Intrachromosomal tandem duplication and repeat expansion during attempts to inactivate the subtelomeric essential gene GSH1 in Leishmania

Gamma-glutamylcysteine synthetase encoded by GSH1 is the rate-limiting enzyme in the biosynthesis of glutathione and trypanothione in Leishmania. Attempts to generate GSH1 null mutants by gene disruption failed in Leishmania infantum. Removal of even a single allele invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. In the second and even third round of inactivation, the markers integrated at the homologous locus but always preserved two intact copies of GSH1. We probed into the mechanism of GSH1 duplication. GSH1 is subtelomeric on chromosome 18 and Southern blot analysis indicated that a 10-kb fragment flanked by 466-bp direct repeated sequences was duplicated in tandem on the same chromosomal allele each time GSH1 was targeted. Polymerase chain reaction analysis and sequencing confirmed the generation of novel junctions created at the level of the 466-bp repeats consequent to locus duplication. In loss of heterozygosity attempts, the same repeated sequences were utilized for generating extrachromosomal circular amplicons. Our results are consistent with break-induced replication as a mechanism for the generation of this regional polyploidy to compensate for the inactivation of an essential gene. This chromosomal repeat expansion through repeated sequences could be implicated in locus duplication in Leishmania.     

Characterisation of a Leishmania mexicana knockout lacking guanosine diphosphate-mannose pyrophosphorylase

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies. The null mutants attach avidly to mouse macrophages, but unlike the wild type organisms, they do not bind to the complement receptor 3 and are slow to induce phagocytosis. Once internalised, they localise to the phagolysosome, but in contrast to wild type organisms which transform into the intracellular amastigote and establish in the macrophage, they are cleared by 24 h in culture and by 5 h in vivo. The null mutants are hypersensitive to human but not mouse complement and to temperature and acidic pH. Surprisingly, in view of the lack of several known host-protective antigens, injection of the mutant parasites into BALB/c mice confers significant and long lasting protection against infection, suggesting that these temperature sensitive mutants are an attractive candidate for a live attenuated vaccine.     

Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes

The yeast Candida albicans is the most important fungal pathogen of humans and a model organism for studying fungal virulence. Sequencing of the C. albicans genome will soon be completed, allowing systematic approaches to analyse gene function. However, techniques to define and characterize essential genes in this permanently diploid yeast are limited. We have developed an efficient method to create conditional lethal C. albicans null mutants by inducible, FLP-mediated gene deletion. Both wild-type alleles of the CDC42 or the BEM1 gene were deleted in strains that carried an additional copy of the respective gene that could be excised from the genome by the site-specific recombinase FLP. Expression of a C. albicans-adapted FLP gene under the control of an inducible promoter generated cell populations consisting of > or = 99.9% null mutants. Upon plating, these cells were unable to form colonies, demonstrating that CDC42 and BEM1 are essential genes in C. albicans. The cdc42 null mutants failed to produce buds and hyphae and grew as large, round cells instead, suggesting that they lacked the ability to produce polarized cell growth. However, the cells still responded to hyphal inducing signals by aggregating and expressing hypha-specific genes, behaviours typical of the mycelial growth form of C. albicans. Budding cells and germ tubes of bem1 null mutants exhibited morphological abnormalities, demonstrating that BEM1 is essential for normal growth of both yeast and hyphae. Inducible, FLP-mediated gene deletion provides a powerful approach to generate conditional lethal C. albicans mutants and allows the functional analysis of essential genes.     

PFPI-like genes are expressed in Leishmania major but are pseudogenes in other Leishmania species

Pyrococcus furiosus protease I (PFPI) is a multimeric cysteine peptidase from P. furiosus. Genome analyses indicate that orthologues are present in rather few other organisms, including Dictyostelium discoideum and several bacteria, Archaea and plants. An open reading frame (ORF) coding for a PFPI-like protein (PFP1) was identified in Leishmania major and Leishmania mexicana and full-length spliced and polyadenylated PFP1 mRNA detected for both species. Vestiges of a PFPI-like gene could also be identified in Leishmania braziliensis and Leishmania infantum, but no ORF remains owing to the presence of frame-shifts and stop codons. No evidence for a PFPI-like gene could be found in the syntenic region of Trypanosoma brucei or Trypanosoma cruzi, raising the possibility that the PFPI-like genes were acquired by a lateral gene transfer event after the divergence of trypanosomes and Leishmania. The gene may have subsequently degenerated into a pseudogene in some Leishmania species, owing to the loss of relevant biological function. However, antibodies raised against L. mexicana recombinant protein detected PFP1 in promastigote extracts of L. major, but not in L. mexicana promastigote or amastigote extracts. The expression of PFP1 in L. major suggests that PFP1 might contribute to the disease tropism that distinguishes this Leishmania species from others.     

Interacting protein kinases involved in the regulation of flagellar length

A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis.     

The γ-glutamylcysteine synthetase gene of Leishmania is essential and involved in response to oxidants

Gamma-glutamylcysteine synthetase, encoded by the GSH1 gene, is the rate-limiting enzyme in the biosynthesis of glutathione and of trypanothione in Leishmania. The importance of GSH1 was assessed by generating GSH1 null mutants in Leishmania infantum . Removal of even a single wild-type allelic copy of GSH1 invariably led to the generation of an extra copy of GSH1 , maintaining two intact wild-type alleles. However, by first supplementing the parasites with a rescue plasmid, we succeeded in obtaining both a single and null chromosomal GSH1 mutants. Parasites with one intact GSH1 chromosomal allele lost the rescuing plasmid but not the double knockout, when grown in the absence of antibiotic, indicating the essentiality of the GSH1 gene in Leishmania. Heterozygous mutants with one allele-inactivated transcribed less GSH1 mRNA and synthesized less glutathione and trypanothione. These mutants were more susceptible to oxidative stresses in vitro as promastigotes and showed decreased survival inside activated macrophages producing reactive oxygen or nitrogen species. These mutants showed a significant decreased survival in the presence of antimony (SbV) compared with control cells. All phenotypes were reverted in the add-back mutant, thus proving the importance of thiols in dealing with oxidants including the action of antimonials.