Log-phase Promastigotes Research Articles

Molecular, structural, and functional characterization of delta subunit of T-complex protein-1 from Leishmania donovani.

Chaperonins/Heat shock protein 60 are ubiquitous multimeric protein complexes that assist in the folding of partially and/or misfolded proteins using metabolic energy into their native stage. The eukaryotic group II chaperonin, also referred as T-complex protein-1 ring complex (TRiC)/T-complex protein-1 (TCP1)/chaperonin containing T-complex protein (CCT), contains 8-9 paralogous subunits, arranged in each of the two rings of hetero-oligomeric complex. In Leishmania, till date, only one subunit, LdTCP1γ, has been well studied. Here, we report the molecular, structural, and functional characterization of TCP1δ subunit of Leishmania donovani (LdTCP1δ), the causative agent of Indian kala-azar. LdTCP1δ gene exhibited only 27.9% identity with LdTCP1γ and clustered in a separate branch in the phylogenic tree of LdTCP1 subunits. The purified recombinant protein formed a high molecular weight complex (0.75 MDa), arranged into 16-mer assembly, and performed in vitro chaperonin activity as assayed by ATP-dependent luciferase folding. LdTCP1δ exhibits 1.8-fold upregulated expression in metabolically active, rapidly dividing log phase promastigotes. Over-expression of LdTCP1δ in promastigotes results in increased infectivity and rate of multiplication of intracellular amastigotes. The study thus establishes the existence of an individual functionally active homo-oligomeric complex of LdTCP1δ chaperonin with its role in parasite infectivity and multiplication.

Clustering of adenosine A2B receptors with ectonucleotidases in caveolin-rich lipid rafts underlies immunomodulation by Leishmania amazonensis.

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

In vitro inhibitory effect of Ambrosia maritima (Asteraceae) extract on the survival of Leishmania donovani

The in vitro susceptibilities of the isolates strain of Leishmania donovani (EMA, EAO, ZAA, HIH, MYIO and HYEA) to Sodium stibogluconate (SSG) and the experimental crude extract of Ambrosia maritime L. (Asteraceae ) were determined for extracellular log-phase promastigotes and interacellular amastigotes in peripheral blood mononuclear cells (PBMCs). Susceptibility to reference drug (Pentostam) with different concentrations (1000, 500, 250, 125, 62.5 and 31.25) showed an increase in growth inhibition with the reduction in the concentration till then decrease in growth inhibition with the more reduction in concentration. While the Ambrosia maritima crude extract showed the decrease of growth inhibition with the increasing in concentration after 24, 48 and 72 hours. Ethanolic crude extract of A. maritime , it caused growth inhibition of L. donovani promastigotes at (1000, 500, 250, 125, 61.5, 31.25 mg/ mL), but the concentration 500ml/ml of the extract showed high biological activity 100% the study indicated that further analysis still to be done on the active crudes; bio-guided fractionation should also be conducted and may lead to the isolation of the major components in the active crude. Higher potency was observed against intercellular amastigots stage (IC 50 is different for all parasites isolates EMA=1.339 to.541 EAO=1.473 to1.719, ZAA=1.447 to 1.719, HIH=1.465 to 1.686, MYIO= 1.280 to 1.559 and HYEA=1.473 to 1.707 mg/mL), a similar was observed for reference drug (SSG) (IC 50 =1.945 to2.788mg/mL).

Therapeutic Efficacy of Stable Analogues of Vasoactive Intestinal Peptide against Pathogens

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide recently identified as a potential antimicrobial peptide. To overcome the metabolic limitations of VIP, we modified the native peptide sequence and generated two stable synthetic analogues (VIP51 and VIP51(6-30)) with better antimicrobial profiles. Herein we investigate the effects of both VIP analogues on cell viability, membrane integrity, and ultrastructure of various bacterial strains and Leishmania species. We found that the two VIP derivatives kill various non-pathogenic and pathogenic Gram-positive and Gram-negative bacteria as well as the parasite Leishmania major through a mechanism that depends on the interaction with certain components of the microbial surface, the formation of pores, and the disruption of the surface membrane. The cytotoxicity of the VIP derivatives is specific for pathogens, because they do not affect the viability of mammalian cells. Docking simulations indicate that the chemical changes made in the analogues are critical to increase their antimicrobial activities. Consequently, we found that the native VIP is less potent as an antibacterial and fails as a leishmanicidal. Noteworthy from a therapeutic point of view is that treatment with both derivatives increases the survival and reduces bacterial load and inflammation in mice with polymicrobial sepsis. Moreover, treatment with VIP51(6-30) is very effective at reducing lesion size and parasite burden in a model of cutaneous leishmaniasis. These results indicate that the VIP analogues emerge as attractive alternatives for treating drug-resistant infectious diseases and provide key insights into a rational design of novel agents against these pathogens.

Myriocin, a Serine Palmitoyltransferase Inhibitor, Blocks Cytokinesis in Leishmania (Viannia) braziliensis Promastigotes

We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log-phase promastigotes treated with 1μM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5μM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5μM myriocin-treated parasites, respectively. High percentage of myriocin-treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5μM myriocin, respectively). Transmission electron microscopy following treatment with 1μM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3-ketodihidrosphingosine to myriocin-treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid-free mutant, where addition of ethanolamine rescue wild-type parasite characteristics.

Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani

T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1γ), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1γ of Leishmania donovani (LdTCP1γ), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1γ revealed the presence of all the characteristic features of TCP1γ. However, leishmanial TCP1γ represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1γ exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1γ as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1γ was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1γ with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite.

Fusion between Leishmania amazonensis and Leishmania major Parasitophorous Vacuoles: Live Imaging of Coinfected Macrophages

Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes - which were destroyed - differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation – a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs.

Role of a differentially expressed cAMP phosphodiesterase in regulating the induction of resistance against oxidative damage in Leishmania donovani

Differentiation-coupled induction of resistance of Leishmania parasites to macrophage oxidative damage was shown to be associated with an increased cAMP response. This study explores the significance of the cAMP response in the parasite by identifying a differentially expressed cAMP phosphodiesterase (LdPDEA) and deciphering its role in regulating antioxidant machineries in the parasite. LdPDEA, a high K M class I cytosolic cAMP phosphodiesterase, was expressed maximally in log-phase promastigotes, but was significantly reduced in stationary-phase promastigotes and amastigotes. Chemical inhibition or silencing of PDEA conferred enhanced resistance to pro-oxidants in these cells and this led to studies on trypanothione biosynthesis and utilization, as trypanothione is one of the major modulators of antioxidant defense in kinetoplastidae. Despite enhanced arginase and ornithine decarboxylase activity, trypanothione biosynthesis seemed to be unaffected by PDEA blockage, whereas significant elevations in the expression of tryparedoxin peroxidase, ascorbate peroxidase, and tryparedoxin were detected, suggesting a definite shift of trypanothione-pool utilization bias toward antioxidant defense. Moreover, parasites that overexpressed PDEA showed reduced resistance to oxidative damage and reduced infectivity toward activated macrophages. This study reveals the significance of a cAMP phosphodiesterase in the infectivity of Leishmania parasites.

In Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences

The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC50], 0.6 to 0.7 microM), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC50, 0.1 to 0.4 microM). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC50, 0.4 to 3.8 microM) and intracellular (IC50, 0.9 to 4.3 microM) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC50, >64 microg SbV/ml) but showed good efficacy against intracellular amastigotes (IC50, 22 to 28 microg SbV/ml); the crystalline substance was about two to three times more potent (IC50, 9 to 11 microg SbV/ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC50, 0.1 microg/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.

The levels and patterns of cytokines produced by CD4 T lymphocytes of BALB/c mice infected with Leishmania major by inoculation into the ear dermis depend on the infectiousness and size of the inoculum.

The production of cytokines by CD4 lymph node T lymphocytes derived from BALB/c mice recently infected in the ear dermis with high (10(6) parasites) or low (10(3) parasites) doses of Leishmania major metacyclic promastigotes (MP) was examined over a 3-week period following inoculation. Results were compared with those obtained when mice were injected with less infectious parasite populations, namely, stationary-phase or log-phase promastigotes (LP). Cells were purified 16 h and 3, 8, and 19 days after inoculation, and the amounts of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) released in response to LACK (Leishmania homolog of receptors for activated C kinase) or total L. major antigens were assessed. We found that LACK-reactive T cells from mice inoculated with a high dose of parasites first produced IFN-gamma and later on IL-4; the level of IFN-gamma produced early by these cells was dependent upon the stage of the promastigotes inoculated, the highest level being reached with cells recovered from mice inoculated with the least infectious parasites, LP; sequential production of IFN-gamma and then of IL-4 also characterized L. major antigen-reactive CD4 T cells, suggesting that the early production of IFN-gamma does not impede the subsequent rise of IL-4 and finally the expansion of the parasites; after low-dose inoculation of MP, cutaneous lesions developed with kinetics similar to that of lesions induced after inoculation of 10(6) LP, but in this case CD4 T lymphocytes did not release IFN-gamma or IL-4 in the presence of LACK and neither cytokine was produced in response to L. major antigens before the onset of lesion signs. These results suggest the existence of a discreet phase in terms of CD4 T-cell reactivity for at least the first 8 days following inoculation, a time period during which parasites are able to grow moderately. In conclusion, the levels and profiles of cytokines produced by Leishmania-specific CD4 T lymphocytes clearly depend on both the stage of differentiation and number of parasites used for inoculation.

Transbilayer translocation of membrane phosphatidylserine and its role in macrophage invasion in Leishmania promastigotes.

Infectivity of Leishmania promastigotes has been shown to be growth cycle-dependent and restricted to the stationary phase. By using annexin V-FITC binding and procoagulant activity measurement assays, we show here that the promastigotes in the stationary phase contain significantly higher amounts of phosphatidylserine (PS) on their surface as compared to the log phase promastigotes. We also demonstrate that the infectivity of the promastigotes is determined by the presence of PS on their surface. In addition, by using NBD-labelled phospholipids, we show that the promastigote plasma membrane contains ATP-dependent out-to-in and ATP-independent in-to-out PS translocases which regulate the PS localisation in two-halves of the membrane bilayer, and that the greater amounts of external PS observed in the stationary phase promastigotes is perhaps due to the slower ATP-dependent out-to-in PS movements in these cells, as compared to the log phase promastigotes.

Sensitivity to Glucantime[registered] of Leishmania viannia Isolated from Patients Prior to Treatment

In vitro sensitivity to pentavalent antimony (SbV) as meglumine antimoniate (Glucantime) of Leishmania of the Viannia subgenus isolated prior to treatment from patients with uncomplicated cutaneous leishmaniasis was evaluated for intracellular amastigotes in the U-937 human monocytic cell line and log phase promastigotes. The 50% effective dose (ED50) of pharmaceutical and additive-free formulations of Glucantime were determined based on the kinetics of the response of Leishmania Viannia to SbV in vitro. ED50 to SbV was inversely related to time of exposure to drug. The potency of the additive-free formulation of Glucantime was significantly greater than that of the pharmaceutical formulation, irrespective of the parasite form. In vitro sensitivity to SbV ranged from < 5.3 micrograms/ml to > 170.0 micrograms/ml. Under the conditions used, 11 (39%) of 28 strains were sensitive to clinically achievable serum concentrations of SbV. No correlation was observed between the total amount of SbV required for healing of lesions and the in vitro response to the pharmaceutical formulation of Glucantime. In contrast, a significant correlation (P = 0.001) was observed between clinical response and the in vitro sensitivity of promastigotes to the additive-free formulation of Glucantime. The greater potency of the additive-free formulation of Glucantime, the correlation of in vitro sensitivity of promastigotes to this formulation and the clinical response to treatment, and the effect of time of exposure to SbV demonstrate the importance of assay conditions on the outcome and interpretation of in vitro evaluation of drug sensitivity.

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.

Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes

This study further explores the effects of hypoxia and acute osmotic stress on intermediary metabolism of Leishmania major and Leishmania donovani. Late log phase promastigotes were washed and incubated with glucose as the sole exogenous carbon source, and rates of glucose consumption and product formation were measured as a function of osmotic strength (610, 305, and 167 mOsm kg 1) and pO 2 (95, 10, and 0% O 2) in the presence of 5% CO 2. Very mild hypoxia dramatically altered flux through the pathways of intermediary metabolism and increased the rates of production of the major metabolites, thus confirming the presence of a low-affinity O 2 sensor which was active under all osmolalities tested. The data also require that as pO 2 is lowered towards anoxia an endogenous carbohydrate source is mobilized. Under aerobic conditions, acute hypo-osmotic stress had little effect on product formation, whereas acute hyperosmotic stress altered metabolism in a manner similar to mild hypoxia, with the exception of decreasing the rates of acetate and succinate production. It was also shown in L. donovani promastigotes that the effects of anoxia and hyperosmolality were not additive. Thus, separate sensors with partially overlapping actions are involved in the metabolic responses to hypoxia and hyperosmolality. There was no apparent species-specificity for the responses to pO 2 and osmotic stress. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhy-drazone caused changes in metabolite flux patterns which differed from the changes caused by either hypoxia or acute osmotic stress, while rotenone and calcium ionophore A23187 had no significant effects. The identity of the sensors responsive to pO 2 and osmolality, and the mechanisms by which they regulate flux through the pathways of intermediary metabolism, require further study.

Oxidation of Leucine by Leishmania donovani

The metabolism of leucine by Leishmania donovani was investigated. Washed promastigotes were incubated with [1-14C]- or [U-14C]leucine or [1-14C]alpha-ketoisocaproate (KIC) and 14CO2 release was measured. The amount of KIC-derived acetyl-CoA oxidized in the citric acid cycle was computed. Promastigotes from mid-stationary phase cultures oxidized each of these labeled substrates less rapidly than cells from late log phase cultures, and significantly less acetyl-CoA derived from KIC oxidation was oxidized in the citric acid cycle. Glucose was a stronger inhibitor than was acetate of CO2 formation in the citric acid cycle in log phase promastigotes, but the reverse was observed in cells from mid-stationary phase. Alanine also inhibited leucine catabolism, but glutamate had little effect. Acute hypo-osmotic stress did not affect leucine catabolism, but hyper-osmotic stress caused appreciable inhibition of leucine oxidation. Cells grown under hypo- or hyper-osmotic conditions showed no changes in the effects of hypo- or hyper-osmotic stress on leucine catabolism, i.e. L. donovani is not an osmoconformer with respect to leucine metabolism. Leucine utilization in L. donovani was insensitive to a number of drugs that affect leucine metabolism in mammalian cells, indicating that the leucine pathway in L. donovani is not regulated in the same manner as in mammalian cells.

Hydrogen peroxide-mediated toxicity for Leishmania donovani chagasi promastigotes. Role of hydroxyl radical and protection by heat shock.

Leishmania must survive despite exposure to the toxic oxidant hydrogen peroxide (H2O2) during phagocytosis by macrophages. We investigated the mechanism of H2O2 toxicity for L. donovani chagasi promastigotes, and factors responsible for their relative H2O2 resistance. There was a dose-dependent toxic effect of H2O2 for promastigotes isolated during logarithmic phase of growth. In contrast, stationary phase promastigotes were less susceptible to H2O2 toxicity, and more infectious for BALB/c mice. By spin trapping we found that hydroxyl radical (.OH) was generated after exposure of promastigotes to H2O2, and the amount of .OH was greater with log-phase than with stationary-phase promastigotes. .OH was generated after the addition of H2O2 to the cytosol but not the membranes of fractionated promastigotes, and the magnitude of .OH was greater in log than in stationary promastigote cytosol. Deferoxamine inhibition suggested that intracellular promastigote iron catalyzes .OH formation via the Fenton reaction. Furthermore, exposure of log-phase promastigotes to heat shock induced a relative H2O2-resistant state, which was not associated with a decrease in .OH formation but which required ongoing transcription. Thus, growth to stationary phase and heat shock both induce a state of relative H2O2 resistance, but these are probably due to different resistance mechanisms.

Effects of oxygen concentration on the intermediary metabolism of Leishmania major promastigotes

Leishmania major promastigotes grown in late log phase were incubated with glucose as sole exogenous carbon source in the presence of 5% CO 2 and the amounts of glucose consumed and of the major products formed — succinate, pyruvate, alanine, acetate, glycerol, and d-lactate — were measured as a function of pO 2. Glucose consumption increased as pO 2 was lowered to 6% (a positive Pasteur effect) and then declined to the same level at 95% N 2 as at 95% O 2. The production of d-lactate and of glycerol increased as pO 2 dropped from 95%, reaching a maximum at about 2% O 2. Succinate production, however, increased dramatically when pO 2 was reduced to 6% and remained at that level with further reduction of pO 2. The amount of succinate produced relative to the amount of glucose carbon consumed suggests utilization of an endogenous carbon source. Acetate production did not change between 95% O 2 and 6% O 2 and then declined with decreasing pO 2. These observations suggest the presence of two sensors, one with a high and one with a low affinity for oxygen. When glycerol or alanine were the only exogenous sources of carbon, the primary products released were acetate and succinate. Acetate production from alanine declined slightly as pO 2 was reduced to 2%, and then dropped markedly when pO 2 was reduced to 0%. Acetate production from glycerol increased over 4-fold when the pO 2 was reduced from 95% to 4%, and then declined with further reduction in pO 2. No succinate was formed from either substrate until complete anaerobiosis. This pattern of response, while differing from that when glucose was sole exogenous carbon source, is also consistent with the regulation of metabolism by a high and a low affinity O 2 sensor. Cells from cultures in early stationary phase, before the appearance of metacyclic forms, consumed glucose at about the same rate as log phase promastigotes, but did not show a Pasteur effect. Stationary cells also consumed glycerol at the same rate as did log phase promastigotes, but consumed alanine at a much lower rate. Reduction of pO 2 affected product formation from each of these substrates differently than for log phase promastigotes, demonstrating the sensitivity of several pathways of intermediary metabolism to regulation by pO 2 during the transition from log to stationary phase.

Membrane glycoprotein M-2 protects against Leishmania amazonensis infection.

Previous passive antibody transfer experiments have indicated that immunity to a 46-kilodalton membrane glycoprotein (M-2) of Leishmania amazonensis may protect against infection with this parasite. In the studies described in this paper, we investigated the ability of the purified M-2 molecule to elicit a protective immune response in conjunction with Freund incomplete and complete adjuvants, saponin, and Corynebacterium parvum. Both relatively susceptible (BALB/c and CBA) and resistant (C57BL/6) strains of mice were examined. C. parvum appeared to be the most effective adjuvant in the three mouse strains tested. The level of protection varied with the mouse strain, although all animals received identical preparations of antigen and adjuvant. Immunization of CBA mice with the M-2 glycoprotein and C. parvum resulted in complete protection against a challenge infection of 10(4) and 10(6) late log-phase promastigotes of L. amazonensis. In the BALB/c strain, complete protection was observed in some of the immunized animals (28 to 50%); in the rest of the mice the onset of infection was significantly delayed. Protective immunity for C57BL/6 mice was observed only at the low infecting dose (10(4) L. amazonensis organisms). The level of protection observed is reflected by increased antibody response (immunoglobulins G1 and G2) developed to the M-2 molecule. The relationship of pure T-cell (nonantibody) immunity to this protection remains to be elucidated.

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

The proteinases of three species of Leishmania have been analysed by electrophoresis. Amastigotes of L. mexicana mexicana have several high-activity, low-Mr 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-Mr proteinases were present in populations of stationary-phase promastigotes of L. m. mexicana. All three species of Leishmania had higher Mr 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.

Analysis ofLeishmaniaproteinases reveals developmental changes in species-specific forms and a common 68-kDa activity

The proteinases of three species of Leishmania have been analysed by electrophoresis. Amastigotes of L. mexicana mexicana have several high-activity, low-Mr 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-Mr proteinases were present in populations of stationary-phase promastigotes of L. m. mexicana. All three species of Leishmania had higher Mr 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.