Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 donovani metacyclic promastigotes: transformation in vitro, lectin agglutination, complement resistance, and infectivity

Freshly transformed Leishmania donovani amastigotes from hamster spleen were used to establish axenic cultures at high density in a modified Grace's medium, which was only partly replenished when cultures were fed. Small, free-swimming, highly active stationary phase promastigotes with a short cell body and long flagellum were induced in this medium. The freshly transformed stationary phase promastigotes so induced were less able to bind peanut agglutinin, had more than 40-fold increased resistance to killing by normal human serum, and 15-fold increased infectivity both in vivo and in vitro when compared to freshly transformed logarithmic phase or long term culture promastigotes. These short form promastigotes may correspond to the metacyclic promastigote forms in the sand fly vector.     

Leishmania braziliensis: protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro

When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin and lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces.     

Comparison of the Protein Kinase and Acid Phosphatase Activities of Five Species of Leishmania1

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, historic-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [γ-³²P]ATP and Mg²⁺ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [γ-³²P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA^-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA⁺). The PNA^- and PNA⁺ forms of L. major both phosphorylated a 50-kDa protein when incubated with [γ-³²P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity. With the exception of L. braziliensis panamensis for which late log phase organisms contained 12-fold more tartrate-resistant acid phosphatase activity than did early log phase cells, stationary and log phase parasites contained approximately the same amount of acid phosphatase activity.     

The oxidative response of rabbit peritoneal neutrophils to leishmanias and other trypanosomatids

Luminometry has been used to measure the respiratory burst of rabbit peritoneal neutrophils that is elicited by different forms and species of Leishmania and Herpetomonas. Mid-log phase and metacyclic promastigotes of L. major evoked large responses; that due to metacyclics was lower and slower, but they also bound in smaller numbers than mid-log phase cells. Promastigotes of L. mexicana mexicana also stimulated a large respiratory burst whereas amastigotes elicited little or none. Leishmania donovani promastigotes and culture forms of H. muscarum muscarum and H. m. ingenoplastis all evoked large responses by neutrophils. There was, however, very little response to L. mexicana mexicana promastigotes, L. donovani promastigotes or H. muscarum muscarum when they were added in large numbers. This 'inhibition' was not apparent with L. major.     

Leishmania chagasi: homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model

Homogenous metacyclic promastigotes of Leishmania chagasi were isolated by buoyant density from in vitro heterogeneous cultures and used for biochemical characterization of isoforms of the major surface protease (MSP). Compared to stationary phase promastigotes, metacyclic cells had three times more MSP, produced 3-fold higher parasite loads in a mouse model in vivo, and were more resistant to complement-mediated lysis in vitro. These metacyclic L. chagasi expressed both the virulence-associated 59-kDa, and the constitutively expressed 63-kDa, isoforms of MSP.     

Leishmania: overexpression and comparative structural analysis of the stage-regulated meta 1 gene.

The meta 1 gene of Leishmania major is upregulated in metacyclic promastigotes and encodes an 11.5-kDa protein with no significant similarities to other proteins in the existing databases. In this paper, we characterize the homologous meta 1 genes in L. amazonensis and L. donovani. Proteins encoded by this gene in all three species present a high degree of identity. The meta 1 gene cannot be replaced by gene targeting in L. major, suggesting an essential role for the protein, at least in promastigotes. Overexpression of the meta 1 protein in L. amazonensis generates parasites that are more virulent than wild-type organisms in vivo.     

Population changes in Leishmania chagasi promastigote developmental stages due to serial passage

Leishmania chagasi causes visceral leishmaniasis, a potentially fatal disease of humans. Within the sand fly vector, L. chagasi replicates as promastigotes which undergo complex changes in morphology as they progress from early stage procyclic promastigotes, to intermediate stage leptomonad and nectomonad promastigotes, and ultimately to terminal stage metacyclic promastigotes that are highly infective to vertebrates. This developmental progression is largely recapitulated in vitro using axenic promastigote cultures that have been passaged only a few times. Within a single passage (which takes about a week), axenic cultures progress from logarithmic to stationary growth phases; parasites within those growth phases progress from stages that do not have metacyclic cell properties to ones that do. Interestingly, repeated serial passage of promastigote cultures will result in cell populations that exhibit perturbations in developmental progression, in expression levels of surface macromolecules (major surface protease, MSP, and promastigote surface antigen, PSA), and in virulence properties, including resistance to serum lysis. Experiments were performed to determine whether there exists a direct relationship between promastigote developmental form and perturbations associated with repeated serial passage. Passage 2 to passage 4 L. chagasi cultures at stationary growth phase were predominately (>85%) comprised of metacyclic promastigotes and exhibited high resistance to serum lysis and high levels of MSP and PSA. Serial passaging 8, or more, times resulted in a stationary phase population that was largely (>85%) comprised of nectomonad promastigotes, almost completely devoid (<2%) of metacyclic promastigotes, and that exhibited low resistance to serum lysis and low levels of MSP and PSA. The study suggests that the loss of particular cell properties seen in cells from serially passaged cultures is principally due to a dramatic reduction in the proportion of metacyclic promastigotes. Additionally, the study suggests that serially passaged cultures may be a highly enriched source of nectomonad-stage promastigotes, a stage that has largely been characterized only in mixtures containing other promastigote forms.     

The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid

A monoclonal antibody, 3F12, was generated which reacted specifically against infective or metacyclic stage Leishmania major promastigotes, but not with noninfective promastigotes obtained from log phase cultures. The antibody recognized a cell surface and released molecule that could be metabolically labeled with [14C]glucose, [3H]mannose, [3H]galactose, and [3H]palmitic acid, but not with [35S]methionine or [3H]leucine. The molecule was the major species surface-labeled by [3H]sodium borohydride after periodate treatment. The glycolipid appeared to be shed primarily as free carbohydrate because 70% of the released material partitioned in the aqueous fraction after phase separation in TX-114. The molecule could be distinguished from the L. major glycolipid which has already been extensively described because its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was of higher relative m.w. However, a close relationship between the two molecules was indicated by the finding that another monoclonal antibody, WIC-79.3, recognized both forms of the glycolipid; one produced and released only by log phase promastigotes, and one produced and released only by metacyclic promastigotes. The loss of agglutination with peanut agglutinin which has been shown to accompany metacyclogenesis was found to be caused by the loss of expression of the log form of the glycolipid which in most cases appeared to be the result of the developmental modification of this molecule. A survey of a number of virulent and avirulent. L. major strains and clones reinforced an absolute association between the ability of these promastigotes to initiate infection in BALB/c mice and their expression and release of the 3F12-binding, developmentally regulated form of the glycolipid. Not only does this glycolipid serve as the first well defined molecular marker for infective stage metacyclic promastigotes, but its unique structure is very likely to contribute to the adaptive changes that allow these parasites to survive within the vertebrate host.     

A sensitive method for assaying chemotaxic responses of Leishmania promastigotes

This article describes a sensitive, cheap, and easy method for assaying chemotaxic responses of Leishmania promastigotes. A gradient of the substance to be assayed was produced inside a series of commercially available capillary tubes submerged in a promastigote suspension. After an incubation period, the attractiveness of the substance under test was measured by counting the number of parasites in the capillaries in a Neubauer chamber. Different responses were detected in two strains of Leishmania amazonensis and one strain of L. chagasi after standardization of the method to assay attraction to carbohydrates. Very different responses were obtained when the test was performed using promastigotes of the same strain in two different physiological states (log and stationary phase). The stationary phase cells showed an enhanced chemotaxic capability, which can be explained by the fact that the metacyclic forms commonest in stationary phase cultures have greater mobility than other promastigotes. This method will permit studies to be made of the attractive response to different substances in Leishmania species and other trypanosomatids and facilitate characterization of the potential receptors involved in the chemotaxic response. An adaptation of the method to assay the response to repellent substances is also provided.     

The effect of buthionine sulfoximine on the growth of Leishmania donovani in culture

Changes in composition of the principal low molecular mass thiols of Leishmania donovani were monitored during the transformation of promastigotes, first to stationary phase metacyclic forms and then to amastigotes. No consistent variation in the thiol composition of the parasite which could account for the known increase in resistance of metacyclic and amastigote lifecycle forms to oxidant stress could be established. Amastigotes cultivated at 37 degrees C also produced ovothiol A, as judged by incorporation of radiolabel from [3-methyl]methionine and [14C]histidine, and the incorporation of radiolabel from [35S]cysteine into ovothiol A represented about 10-15% of the total label recovered in ovothiol A, glutathione and trypanothione. Amastigotes were less susceptible than promastigotes to the effects of the redox cyclers paraquat and menadione and grew in culture in the presence of up to 20 mM buthionine sulfoximine, which completely blocked the synthesis of glutathione and its spermidine conjugates. Glutathione and trypanothione biosynthesis is, therefore, not necessary for the replication of L. donovani amastigotes in culture. Inhibition of the formation of glutathione and trypanothione did not result in an upregulation of ovothiol A production.     

Electrophoretic analysis of the polypeptides of Leishmania amastigotes and promastigotes

The polypeptides of Leishmania mexicana mexicana (M379), L. m. amazonensis (LV78), L. major (LV39) and L. d. donovani (LV39) amastigotes and cultured promastigotes have been analysed by SDS-polyacrylamide gel electrophoresis. The polypeptide banding patterns of the promastigotes of the four species were quite similar, but distinct differences were detected between those of amastigotes. The results suggest that the various species of Leishmania are adapted differently for survival and growth in the mammalian host. The polypeptides of L. m. mexicana amastigotes were very rapidly hydrolysed unless protected by the cysteine proteinase inhibitor leupeptin.     

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.     

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.     

Expression of biopterin transporter (BT1) protein in Leishmania

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite.     

Analysis of Leishmania proteinases reveals developmental changes in species-specific forms and a common 68-kDa activity

Abstract The proteinases of three species of Leishmania have been analysed by electrophoresis. Amastigotes of L. mexicana mexicana have several high-activity, low- M _r cysteine proteinases which are absent from log-phase promastigotes of L. m. mexicana and from all developmental stages of the other species analysed ( L. donovani and L. major ). Low-activity, low- M _r proteinases were present in populations of stationary-phase promastigotes of L. m. mexicana . All three species of Leishmania had higher M _r proteinases, a number of which showed developmental regulation, some of them being stage-specific. Significantly, at all stages of the life cycle in all three species a 68-kDa proteinase was apparent. In its size, sensitivity to inhibitors and ability to bind concanavalin A-agarose, this resembles the major surface protein thought to be present in all Leishmania species and which has recently been reported to possess proteinase activity in L. major promastigotes.     

Leishmania species: mechanisms of complement activation by five strains of promastigotes

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.     

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.     

A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.

At the end of their growth in the sand fly, Leishmania parasites differentiate into the infective metacyclic promastigote stage, which is transmitted to the mammalian host. Thus, in experimental studies of parasite infectivity toward animals or macrophages, the use of purified metacyclics is generally preferred. While metacyclics of several Leishmania species can be efficiently purified with the aid of lectins or monoclonal antibodies, which differentially exploit stage-specific differences in the structure of the abundant surface glycolipid lipophosphoglycan (LPG), such reagents are unavailable for most species and they are unsuitable for studies involving LPG-deficient mutants. Here we describe a simple density gradient centrifugation method, which allows the rapid purification of infective metacyclic parasites from both wild-type and LPG-deficient Leishmania major. The purified metacyclic promastigotes are authentic, as judged by criteria such as their morphology, expression of the metacyclic-specific gene SHERP, and ability to invade and replicate within macrophages in vitro. Preliminary studies suggest that this method is applicable to other Leishmania species including L. donovani.