Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana

In the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB-deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.     

A developmentally regulated cysteine proteinase gene of Leishmania mexicana

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.     

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.     

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.     

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.     

Leishmania mexicana: amastigote hydrolases in unusual lysosomes

Leishmania mexicana mexicana (M379) amastigotes were found to contain much higher activities than cultured promastigotes of five putative lysosomal enzymes: cysteine proteinase; arylsulfatase (EC 3.1.6.1); beta-glucuronidase (EC 3.2.1.31); DNase (EC 3.1.22.1), and RNase (EC 3.1.27.1). The release profiles of the first three of these enzymes from digitonin-permeabilized amastigotes suggests that they are located within organelles. Cytochemical staining for cysteine proteinase, using gold labeled antibodies and arylsulfatase, showed that both were present in large organelles previously named "megasomes." Comparative studies with L. mexicana amazonensis (LV78), L. donovani donovani (LV9), and L. major (LV39) revealed that L. mexicana amazonensis was similar to L. mexicana mexicana in possessing both high amastigote cysteine proteinase activity and large numbers of megasome organelles in amastigotes, whereas the other two species lacked both these features. The results suggest that the presence of numerous lysosome-like organelles in the amastigote is a characteristic of the L. mexicana group of parasites.     

Evidence that intracellular beta1-2 mannan is a virulence factor in Leishmania parasites

The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.     

Caspar-like gene depletion reduces Leishmania infection in sand fly host Lutzomyia longipalpis

Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.     

Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death

The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L. major SIR2 gene. Further, using L. infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This phenotype was also observed when L. infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L. infantum cosmid library, carrying the L. infantum SIR2 gene (CLHyg-LiSIR2). In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions. However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced. Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon. Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death.     

Leishmania (Viannia) panamensis: cloning of the histone H1 genes by representational difference analysis

We report the use of representational difference analysis to identify genes that have up-regulated expression in the amastigote life-cycle stage of Leishmania (Viannia) panamensis. This simultaneous process of selection and amplification allowed the cloning of several specific DNA fragments. One of them shows a high percentage of similarity with histone H1 genes from other Trypanosomatids and, as expected, is up-regulated in the amastigote life-cycle stage. The gene is present in two copies that are expressed at different levels in promastigotes and also in amastigotes, which seems to be a consequence of their different 3' untranslated regions.     

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.     

Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.     

Gene expression profiling of Leishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to 'freeze' gene expression levels instantly; this is particularly a delicate task for intracellular amastigotes and for specific 'sensory' genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiation.     

Expression of Cysteine Proteinases by Metacyclic Promastigotes of Leishmania mexicana

ABSTRACT. The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana .     

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.