Metacyclogenesis: a basic process in the biology of Leishmania

Metacyclogenesis is a process whereby Leishmania transforms from poorly infective procyclic promastigotes into highly infective metacyclic promastigotes. In nature, metacyclogenesis occurs in the insect vector. This transformation is accompanied by an increased ability to infect and survive in the vertebrate host, where the parasite is attacked by the host's immune system. Metacyclogenesis has also been shown to occur in axenic cultures of promastigotes. Morphological changes in size and shape, and length of flagellum were first associated with differentiation in the insect gut and in different phases of growth in culture. Later, the expression of molecules such as LPG and the surface protease gp63 were associated with this process. These two molecules were observed to undergo qualitative and quantitative modifications as the promastigotes differentiated from procyclic to metacyclic forms. Using cDNA subtractive hybridization-based methods or differential amplification, previously unknown genes tightly linked to metacyclogenesis have been identified. Gene products exclusively expressed in metacyclic promastigotes included a gene B product and Mat-1--a gene associated with metacyclogenesis. Other proteins, Meta-1, SHERP and HASP, were up-regulated during the metacyclic stage. The function and stage-regulated expression of these molecules and their relationship with infectivity are now under investigation.     

Identification of a protein kinase A regulatory subunit from Leishmania having importance in metacyclogenesis through induction of autophagy

cAMP‐mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP‐binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP‐dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP‐dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation‐induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP‐binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.     

Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that “stressful conditions” will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.     

Stage-Specific Differential Gene Expression of Glutathione Peroxidase in Leishmania Major and Leishmania Tropica

Background:Leishmania (L) major and L. tropica are the etiological agents of cutaneous leishmaniosis. Leishmania species cause a board spectrum of phenotypes. A small number of genes are differentially expressed between them that have likely an important role in the disease phenotype. Procyclic and metacyclic are two morphological promastigote forms of Leishmania that express different genes. The glutathione peroxidase is an important antioxidant enzyme that essential in parasite protection against oxidative stress and parasite survival. This study aimed to compare glutathione peroxidase (TDPX) gene expression in procyclic and metacyclic and also interspecies in Iranian isolates of L. major and L. tropica. Methods:The samples were cultured in Novy-Nicolle-Mc Neal medium to obtain the promastigotes and identified using PCR-RFLP technique. They were then grown in RPMI1640 media for mass cultivation. The expression level of TDPX gene was compared by Real-time PCR.Results:By comparison of expression level, up-regulation of TDPX gene was observed (5.37 and 2.29 folds) in L. major and L. tropica metacyclic compared to their procyclic, respectively. Moreover, there was no significant difference between procyclic forms of isolates, while 3.05 folds up-regulation in metacyclic was detected in L. major compared L. tropica.Conclusion:Our data provide a foundation for identifying infectivity and high survival related factors in the Leishmania spp. In addition, the results improve our understanding of the molecular basis of metacyclogenesis and development of new potential targets to control or treatment and also, to the identification of species-specific factors contributing to virulence and pathogenicity in the host cells.Key Words: Glutathione peroxidase, Leishmania, L. major, L. tropica, Quantitative Real-time PCR     

New insights into the developmental biology and transmission mechanisms of Leishmania

Leishmania alternates between two main morphological forms in its life cycle: intracellular amastigotes in the mammalian host and motile promastigotes in the sandfly vector. Several different forms of promastigote can be recognised in sandfly infections. The first promastigote forms, which are found in the sandfly in the bloodmeal phase, are multiplicative procyclic promastigotes. These differentiate into nectomonad promastigotes, which are a non-dividing migratory stage moving from the posterior to the anterior midgut. When nectomonad promastigotes arrive at the anterior midgut they differentiate into leptomonad forms, a newly named life cycle stage, which resume replication. Leptomonad promastigotes, which are found in the anterior midgut, are the developmental precursors of the metacyclic promastigotes, the mammal-infective stages. Leptomonad forms also produce promastigote secretory gel, a substance that plays a key role in transmission by forming a physical obstruction in the gut, forcing the sandfly to regurgitate metacyclic promastigotes during bloodfeeding.     

Involvement of STI1 protein in the differentiation process of Trypanosoma cruzi

The protozoan Trypanosoma cruzi is a parasite exposed to several environmental stressors inside its invertebrate and vertebrate hosts. Although stress conditions are involved in its differentiation processes, little information is available about the stress response proteins engaged in these activities. This work reports the first known association of the stress-inducible protein 1 (STI1) with the cellular differentiation process in a unicellular eukaryote. Albeit STI1 expression is constitutive in epimastigotes and metacyclic trypomastigotes, higher protein levels were observed in late growth phase epimastigotes subjected to nutritional stress. Analysis by indirect immunofluorescence revealed that T. cruzi STI1 (TcSTI1) is located throughout the cell cytoplasm, with some cytoplasmic granules appearing in greater numbers in late growing epimastigotes and late growing epimastigotes subjected to nutritional stress. We observed that part of the fluorescence signal from both TcSTI1 and TcHSP70 colocalized around the nucleus. Gene silencing of sti1 in Trypanosoma brucei did not affect cell growth. Similarly, the growth of T. cruzi mutant parasites with a single allele sti1 gene knockout was not affected. However, the differentiation of epimastigotes in metacyclic trypomastigotes (metacyclogenesis) was compromised. Lower production rates and numbers of metacyclic trypomastigotes were obtained from the mutant parasites compared with the wild-type parasites. These data indicate that reduced levels of TcSTI1 decrease the rate of in vitro metacyclogenesis, suggesting that this protein may participate in the differentiation process of T. cruzi.     

Leishmania (Viannia) braziliensis metacyclic promastigotes purified using Bauhinia purpurea lectin are complement resistant and highly infective for macrophages in vitro and hamsters in vivo

In this study we characterised metacyclogenesis in axenic culture of Leishmania (Viannia) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World. Metacyclogenesis of other species of Leishmania has been shown by morphological changes as well as molecular modifications in the lipophosphoglycan, the major cell surface glycoconjugate of the promastigotes. In order to obtain metacyclic forms of L. braziliensis we tested a panel of different lectins. Our results showed that Bauhinia purpurea lectin facilitated the purification of metacyclic promastigotes from stationary-phase culture by negative selection. The B. purpurea non-agglutinated promastigotes had a slender short cell body and long flagella, typical of metacyclic morphology. The ultrastructural analysis showed that B. purpurea non-agglutinated promastigotes have a dense and thicker glycocalyx. They are resistant to complement lysis, and highly infective for macrophage in vitro and hamsters in vivo. Contrary to procyclic promastigotes, B. purpurea non-agglutinated forms were poorly recognised by sand fly gut epithelial cells. These results suggest that the B. purpurea non-agglutinated promastigotes are the metacyclic forms of L. braziliensis.     

Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes and then differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.     

Flow cytometric assessment of Leishmania spp metacyclic differentiation: validation by morphological features and specific markers

Characterization of infective metacyclic promastigotes of Leishmania spp can be an essential step in several experimental protocols. Metacyclic forms of all Leishmania species display a typical morphology with short, narrow cell body, and an elongated flagellum. This feature suggests that metacyclics can be distinguished from procyclic forms by non-fluorimetric flow cytometric parameters thus enabling the follow-up of their appearance and acquisition of specific properties, during metacyclogenesis in in vitro cultures. Here we describe the flow cytometric parameters of stage-specific promastigotes of Leishmania major, Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis. Our findings were validated by optical microscopy morphology and specific procyclic labeling with FITC-peanut agglutinin. Furthermore, we show that parasite's distribution in the plot during differentiation in culture is not species specific and that the parasites displaying low forward-angle light scatter (FSC(low)) are three times more infective than the FSC(high) ones. The method here described can be applied to the identification of metacyclics of different Leishmania spp within the whole stationary population.     

Leishmania major CorA-like magnesium transporters play a critical role in parasite development and virulence

Establishment of infection by Leishmania depends on the transformation of the invading metacyclic promastigotes into the obligatory intracellular amastigotes, and their subsequent survival in the macrophage phagolysosome, which is low in magnesium. We show that two Leishmania major proteins designated MGT1 and MGT2, which play a critical role in these processes, belong to the two-transmembrane domain (2-TM-GxN) cation transporter family and share homology with the major bacterial magnesium transporter CorA. Although both are present in the endoplasmic reticulum throughout the life cycle of the parasite, MGT1 is more highly expressed in the infectious metacyclic parasites, while MGT2 is enriched in the immature procyclic stages. The two proteins, although predicted to be structurally similar, have features that suggest different regulatory or gating mechanisms. The two proteins may also be functionally distinct, since only MGT1 complements an Escherichia coliΔCorA mutant. In addition, deletion of one mgt1 allele from L. major led to increased virulence, while deletion of one allele of mgt2 resulted in slower growth and total loss of virulence in vitro and in vivo. This loss of virulence may be due to an impaired transformation of the parasites into amastigotes. Deletion of both mgt1 alleles in the hemizygous MGT2 knockdown parasites reversed the growth defect and partially restored virulence. Our data indicate that the MGTs play a critical role in parasite growth, development and virulence.     

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 .     

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.     

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.     

Social Motility of African Trypanosomes Is a Property of a Distinct Life-Cycle Stage That Occurs Early in Tsetse Fly Transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen—designated as early procyclic forms—express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4–7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.     

Genome-wide analysis reveals increased levels of transcripts related with infectivity in peanut lectin non-agglutinated promastigotes of Leishmania infantum

Metacyclic promastigotes are transmitted during bloodmeals after development inside the gut of the sandfly vector. The isolation from axenic cultures of procyclic and metacyclic promastigotes by peanut lectin agglutination followed by differential centrifugation is controversial in Leishmania infantum. The purpose of this study has been to isolate both fractions simultaneously from the same population in stationary phase of axenic culture and compare their expression profiles by whole-genome shotgun DNA microarrays. The 317 genes found with meaningful values of stage-specific regulation demonstrate that negative selection of metacyclic promastigotes by PNA agglutination is feasible in L. infantum and both fractions can be isolated. This subpopulation up-regulates a cysteine peptidase A and several genes involved in lipophosphoglycan, proteophosphoglycan and glycoprotein biosynthesis, all related with infectivity. In fact, we have confirmed the increased infection rate of PNA⁻ promastigotes by U937 human cell line infection experiments. These data support that metacyclic promastigotes are related with infectivity and the lack of agglutination with PNA is a phenotypic marker for this subpopulation.     

Establishment of an in vitro transgene expression system in epimastigotes of Trypanosoma congolense

Trypanosoma congolense epimastigote forms (EMFs) adhere to the tsetse fly proboscis, proliferate, and differentiate into animal-infective metacyclic forms (MCFs). This differentiation step, called metacyclogenesis, is indispensable for the cyclical transmission of the parasite. Although an in vitro metacyclogenesis culture system was established several decades ago, few genetic tools have been utilized to investigate the molecular mechanisms underlying T. congolense metacyclogenesis. This study established a transgene expression system using an in vitro derived EMF of T. congolense IL3000, and the transgenic EMF successfully underwent metacyclogenesis in vitro. The newly constructed expression vector pSAK was designed for integration into the α–β tubulin locus, which is tandemly arranged in the T. congolense genome. The expression cassette of pSAK/enhanced green fluorescent protein (eGFP) was transfected into the EMF by electroporation. An EMF expressing eGFP was successfully generated and differentiated into an MCF that constitutively expressed eGFP. The in vitro metacyclogenesis system in combination with the transgenic EMF technique will be important tools to investigate the molecular mechanisms of metacyclogenesis.     

PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei,Is Required for Efficient Maturation of Infection

The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T₃₀₅), a modification that depends on the presence of TbMAPK4. Mutation of T₃₀₅ to alanine (T₃₀₅A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T₃₀₅D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein''s structure and the effects of mutation of T₃₀₅ on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite''s decision to divide, differentiate or migrate.     

MDL28170, a calpain inhibitor, affects Trypanosoma cruzi metacyclogenesis, ultrastructure and attachment to Rhodnius prolixus midgut

Background

Trypanosoma cruzi is the etiological agent of Chagas'' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host.

Methodology/Principal Findings

In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption.

Conclusions/Significance

The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi.