Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.     

Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes.

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.     

Properties of an acid phosphatase from Legionella micdadei which blocks superoxide anion production by human neutrophils

The high-speed supernatant (100,000 g, 1 h) obtained after centrifuging a suspension of Legionella micdadei that had been freeze-thawed and sonicated contained (i) considerable acid phosphatase activity when assayed using 4-methylumbelliferyl phosphate (MUP) as the substrate, and a factor that blocked superoxide anion production by human neutrophils stimulated with f-Met-Leu-Phe. Chromatography of the extract on a hydroxylapatite column resolved two acids phosphatases (designated ACP1 and ACP2). Subsequent chromatography of ACP2 on a Sephadex G-150 column revealed coincident elution of phosphatase activity and neutrophil blocking activity. When heated at 45 degrees C for various periods of time, the phosphatase activity of the acid phosphatase preparation was lost at the same rate as the ability of the preparation to block superoxide anion production by neutrophils. Furthermore, preincubation of neutrophils and acid phosphatase together in the presence of a heteropolymolybdate complex that inhibits the phosphatase eliminated the effect of the L. micdadei phosphatase on neutrophil superoxide anion production. ACP2 had the following properties: pH optimum, 6.0; Km for MUP, 3.8 mM; isoelectric point, 4.5; substrate specificity, MUP greater than ADP greater than phosphoenolpyruvate greater than phosphothreonine greater than phosphoserine greater than phosphotyrosine; molecular weight (estimated by sucrose density gradient centrifugation and gel filtration chromatography), 71,000-86,000. These results indicate that a cell-associated phosphatase may play a role in the virulence of L. micdadei.     

Leishmania donovani: immunochemical localization and secretory mechanism of soluble acid phosphatase

Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.     

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.     

Demonstration of various acid hydrolases and preliminary characterization of acid phosphatase in Naegleria fowleri

Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include beta-glucosidase, beta-galactosidase, beta-fucosidase, alpha-mannosidase, hexosaminidase, arylsulfatase A, and beta-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly "acid" hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: pH optima, 5.5; Km (4-methylumbelliferyl phosphate), 0.60 mM; molecular weight (estimated by gel filtration chromatography), 92,000; inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?     

Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics

In this report, we describe an in vitro culture system for the generation and propagation of axenic amastigotes from the well characterised 1S-CL2D line of Leishmania donovani. Fine structure analyses of these in vitro-grown amastigotes demonstrated that they possessed morphological features characteristic of L. donovani tissue-derived amastigotes. Further, these axenic amastigotes (LdAxAm) were shown to synthesise and release a secretory acid phosphatase isoform similar to that produced by intracellular amastigotes. Such LdAxAm also expressed surface membrane 3'-nucleotidase enzyme activity similar to that of tissue-derived amastigotes. Moreover, LdAxAm, in contrast to promastigotes, expressed significant levels of the amastigote-specific A2 proteins. In addition, LdAxAm, derived from long term cultures of Ld 1S-CL2D promastigotes, had significant infectivity for both human macrophages in vitro and for hamsters in vivo. Thus, the in vitro culture system described herein provides a useful tool for the generation of large quantities of uniform populations of axenic amastigotes of the L. donovani 1S-CL2D line. The availability of such material should greatly facilitate studies concerning the cell and molecular biology of this parasite developmental stage.     

Leishmania donovani: long-term culture of axenic amastigotes at 37 degrees C

L. donovani promastigotes were subjected to heat treatment yielding an axenic amastigote stage which was long-term cultured at 37 degrees C. No differences were observed between the growth rates of axenic amastigotes and promastigotes. Flow cytometry-derived DNA histograms of axenic amastigotes and promastigotes were typical of exponentially growing cell populations. Moreover, axenic amastigotes were metabolically active as evidenced by the release of an immunoprecipitable extracellular acid phosphatase (SAcP) into their culture supernatant. Cell transformation was confirmed by transmission electronmicroscopic examination of thin sections and extended by fracture-flip survey which allowed differentiation of cell membranes. The ultrastructure and nanoanatomy of axenic amastigotes was identical to that of intracellular amastigotes. The production of large amounts of heat-shock axenic amastigotes suitable for biochemical and biological studies of differentiation in Leishmania donovani may have important implications in the development of prevention and/or treatment strategies.     

Metacyclogenesis of Leishmania spp: species-specific in vitro transformation, complement resistance, and cell surface carbohydrate and protein profiles

Metacyclic (stationary) and logarithmic (log) forms of promastigotes of Leishmania donovani and Leishmania major were characterized in several ways. The highly active metacyclic forms were larger with more protein and less carbohydrate. The flagellum increased in length 2.4 times in L. major as compared to 1.8 times in L. donovani. Resistance to complement-mediated lysis by normal human serum of in vitro grown Leishmania promastigotes was related to the species, the growth phase in culture, and also the temperature. Metacyclic forms of both species had a much increased resistance to killing by normal serum at different temperatures. Differences in membrane-exposed carbohydrates were detected by fluorescein-conjugated lectins. Peanut agglutinin and Ulex agglutinin I differentiated log and stationary phase promastigotes of L. major. Higher amounts of acid phosphatase were demonstrated in the metacyclic phase. Differences in polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides of approximately 51 and 114 kDa were found exclusively in metacyclic promastigotes of both species, whereas 38- and 23-kDa polypeptides were lost or reduced during transformation from log to metacyclic phase promastigotes of L. donovani. In addition, a 75-kDa polypeptide was expressed only in metacyclic promastigotes of L. major.     

Decrease of acid phosphatase activity in murine peritoneal macrophages infected with Leishmania donovani

The effect of infection with Leishmania donovani on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with L. donovani were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages were decreased significantly by infection with L. donovani. An inverse relation existed between concentration of acid phosphatase activity and the number of intracellular L. donovani. Reduced concentrations of acid phosphatase activity were also observed in macrophages uninfected but exposed to L. donovani. The isoenzyme composition in macrophages did not change during the course of infection with L. donovani. These results demonstrate that L. donovani reduces the acid phosphatase activity of macrophages.     

Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis

The present study intends to evaluate the role of radio-attenuated leishmania parasites as immunoprophylactic agents for experimental murine visceral leishmaniasis. BALB/c mice were immunized with gamma (γ)-irradiated Leishmania donovani. A second immunization was given after 15 days of first immunization. After two immunizations, mice were infected with virulent L. donovani promastigotes. Protection against Kala-azar (KA) was estimated from spleen and liver parasitic burden along with the measurement of nitrite and superoxide anion generation by isolation of splenocytes and also by T-lymphocyte helper 1(Th1) and T-lymphocyte helper 2(Th2) cytokines release from the experimental groups. It was observed that BALB/c mice having prior immunization with radio-attenuated parasites showed protection against L. donovani infection through higher expression of Th1 cytokines and suppression of Th2 cytokines along with the generation of protective free radicals. The group of mice without prior priming with radio-attenuated parasites surrendered to the disease. Thus it can be concluded that radio-attenuated L. donovani may be used for.     

Leishmania donovani lipophosphoglycan blocks NADPH oxidase assembly at the phagosome membrane

Phagocytosis of Leishmania donovani promastigotes is characterized by an inhibition of phagolysosome biogenesis mediated by the surface glycolipid lipophosphoglycan (LPG). However, the consequences of this inhibition on macrophage function remain to be determined. In this study, we investigated the impact of LPG-mediated phagosome remodelling on the assembly and function of the NADPH oxidase complex. Phagocytosis of both wild-type and LPG-defective L. donovani promastigotes triggered the release of similar levels of superoxide. However, wild-type promastigotes, but not LPG-defective mutants, inhibited generation of superoxide at the phagosome. Confocal microscopy imaging revealed that the membrane component gp91(phox) and the Rho-family GTPase Rac1 were present on phagosomes containing either wild-type or LPG-defective promastigotes. In contrast, the NADPH oxidase cytosolic components p47(phox) and p67(phox) were excluded from phagosomes in a LPG-dependent fashion. This inhibition is not the consequence of a general defect in the initiation of the NADPH oxidase activation process because both wild-type and LPG-defective promastigotes induced p47(phox) phosphorylation and the formation of complexes containing p47(phox) and p67(phox). Thus, by remodelling their intracellular habitat, L. donovani promastigotes prevent the assembly of a functional phagosomal NADPH oxidase complex, thereby evading an important host innate defence mechanism.     

Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.     

Trafficking of Leishmania donovani promastigotes in non-lytic compartments in neutrophils enables the subsequent transfer of parasites to macrophages

Inoculation of Leishmania ( L.) spp. promastigotes in the dermis of mammals by blood-feeding sand flies can be accompanied by the rapid recruitment of neutrophils, inflammatory monocytes and dendritic cells. Despite the presence of these lytic leucocytes, parasitism is efficiently established. We show here that Leishmania donovani promastigotes are targeted to two different compartments in neutrophils. The compartments harbouring either damaged or non-damaged parasites were characterized at the electron microscopy (EM) level using the glucose 6-phosphatase cytochemistry and endosome–phagosome fusion assays. One involves the contribution of lysosomes leading to the formation of highly lytic compartments where parasites are rapidly degraded. The other is lysosome-independent and involves the contribution of a compartment displaying some features of the endoplasmic reticulum (ER) where parasites are protected from degradation. Using genetically modified parasites, we show that the promastigote surface lipophosphoglycan (LPG) is required to inhibit lysosome fusion and maintain parasites in neutrophil compartments displaying ER features. L. donovani -harbouring neutrophils that eventually enter apoptosis can be phagocytosed by macrophages enabling the stealth entry of parasites into their final replicative host cells. Thus, the ability of L. donovani to avoid trafficking into lysosomes-derived compartments in short-lived neutrophils constitutes a key process for the subsequent establishment of long-term parasitism.     

Leishmania major and L. donovani: effects on proteolytic enzymes of Phlebotomus papatasi (Diptera, Psychodidae)

Phlebotomus papatasi is susceptible to Leishmania major which it transmits in nature, but is resistant to L. donovani. The present study compares the effect of L. major and L. donovani on the proteolytic activity of P. papatasi gut enzymes. The experiments measured digestion of C14-labeled globin by gut homogenates of flies. Homogenates were prepared from flies fed on serum only (controls) or from flies fed serum containing promastigotes or their dried culture overlayer. In other experiments, the promastigotes or dried culture overlayers were added in vitro to the gut homogenate of control flies. Proteolytic activity of gut homogenate from flies infected with L. major was about one-third less than that of controls, while that from flies infected with L. donovani was one-third greater. Ingestion of L. major dried culture overlayer had an effect on flies similar to that of the promastigotes, while L. donovani dried culture overlayer produced no significant effect. When added to gut homogenate in vitro, promastigotes of both species promoted proteolysis as did dried culture overlayer of L. major. Dried culture overlayer of L. donovani, however, had an opposite effect. It is suggested that the observed reduction in proteolytic activity caused by L. major infection may result from inhibition of enzyme production.     

Structurally conserved soluble acid phosphatases are synthesized and released by Leishmania major promastigotes

Previously it was reported that promastigotes of virtually all pathogenic Leishmania species, except Leishmania major, release a structurally conserved soluble acid phosphatase (AcP) activity during their growth in vitro (P. S. Doyle and D. M. Dwyer, Exp. Parasitol. 77, 435-444 1993). In the current study we used a highly sensitive fluorogenic detection method to demonstrate that soluble AcPs were in fact produced by promastigotes of several different strains of L. major. These L. major AcP activities were readily immunoprecipitated with a rabbit antibody previously generated against the L. donovani AcP. Results of metabolic labeling and immunoprecipitations demonstrated that AcPs produced by the L. majors strains examined had an apparent molecular mass of approximately 77 kDa. Results of Southern hybridization analysis with an L. donovani AcP gene probe showed that the AcP gene loci were conserved in the L. major strains examined. Taken together, these results indicate that the AcP enzyme has been structurally and functionally conserved throughout the evolution of pathogenic species of Leishmania. Such conservation suggests that the AcPs play a functional role in the growth and survival of this group of important human pathogens.     

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)     

Prostaglandin E1 and prostaglandin I2 modulation of superoxide production by human neutrophils

15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 rapidly and reversibly inhibit formyl-methionyl-leucyl-phenylalanine induced superoxide production by human neutrophils. In contrast, 15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 did not alter the rate or the total amount of superoxide production by human neutrophils stimulated with either phorbol myristate acetate or arachidonic acid. These data suggest that the production of superoxide anion by human neutrophils may be mediated by at least two mechanisms, one regulated by prostaglandins and intracellular cyclic adenosine monophosphate levels and a second independent of prostaglandin modulation.     

Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.     

Effect of preservation at low temperature (4 °C) on polymorphonuclear neutrophil integrity as determined by biochemical analysis

When polymorphonuclear neutrophils were stored at 4 °C for up to 2 weeks, the maintenance of the integrity of PMNs was examined by determining changes in enzyme activity, enzyme release, stimulated superoxide anion generation, and sensitivity to hypotonicity. Until at least 3-day storage, no changes were observed in enzyme activity, enzyme release, and stimulated superoxide anion generagion. After 1-week storage, the ability of PMNs to generate superoxide anions decreased considerably and the extracellular release of lactate dehydrogenase (LDH) was observed. After 2 weeks of storage, this LDH release and inhibition of O₂^?-generating ability of PMNs increased further, although enzyme activities were only slightly affected except for acid p-nitrophenyl phosphatase. The resistance of PMNs to hypotonic solutions decreased with increasing preservation time at 4 °C.