Histone H1 regulates chromatin condensation in Leishmania parasites

We investigated the functional role of the Leishmania histone H1 and demonstrate for the first time that addition of histone H1 has a strong effect on microccocal digestion, chromatin condensation of parasite nuclei and that its overexpression can modulate parasite infectivity in vivo.     

Infectivity and attenuation of Leishmania donovani promastigotes: association of galactosyl transferase with loss of parasite virulence

During the course of long-term in vitro cultivation of Leishmania donovani parasites, the promastigotes were found to lose their infectivity. Lectin agglutination studies revealed that there is an up-regulation of terminally exposed galactose residues on the noninfective promastigotes. The enzyme galactosyl transferase was absent in the infective form but present in the non-infective form of the parasites. An association between galactosylation and loss of infectivity was also observed. We propose that the enzyme galactosyl transferase is developmentally regulated and induces galactosylation of surface carbohydrates.     

Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major

Macrophages are the major target cell population of the obligate intracellular parasites LEISHMANIA: Although polymorphonuclear neutrophil granulocytes (PMN) are able to internalize Leishmania promastigotes, these cells have not been considered to date as host cells for the parasites, primarily due to their short life span. In vitro coincubation experiments were conducted to investigate whether Leishmania can modify the spontaneous apoptosis of human PMN. Coincubation of PMN with Leishmania major promastigotes resulted in a significant decrease in the ratio of apoptotic neutrophils as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low m.w. DNA fragments, and annexin V staining. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in PMN. The inhibition of PMN apoptosis depended on viable parasites because killed Leishmania or a lysate of the parasites did not have antiapoptotic effect. L. major did not block, but rather delayed the programmed cell death of neutrophils by approximately 24 h. The antiapoptotic effect of the parasites could not be transferred by the supernatants, despite secretion of IL-8 by PMN upon coculture with L. major. In vivo, intact parasites were found intracellularly in PMN collected from the skin of mice 3 days after s.c. infection. This finding strongly suggests that infection with Leishmania prolongs the survival time of neutrophils also in vivo. These data indicate that Leishmania induce an increased survival of neutrophil granulocytes both in vitro and in vivo.     

Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5-15) in G0/G1- and G2/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G0/G1-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G0/G1 into the S and G2/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.     

Down-regulation of gp63 level in Leishmania amazonensis promastigotes reduces their infectivity in BALB/c mice

Episomal expression of the major surface glycoprotein (gp63) sense and antisense mRNAs in Leishmania amazonensis was found previously to modulate the expression of this molecule as well as its infection of macrophages in vitro. Here, we evaluated the in vivo infectivity of these transfectants in BALB/c mice. Antisense downregulation of gp63 renders this parasite sensitive to complement-mediated lysis and less infective to mice, as indicated by a delay in lesion development and a significant reduction in lesion size and parasite loads at the site of inoculation and in the draining lymph nodes (DLNs). CD4+ cells at the site of inoculation decreased in number more rapidly and were 2-fold less numerous than those in controls by week 4. The number of IFN-gamma-positive cells was higher, while IL-10 positive cells were undetectable. In DLNs, CD4+ cells were higher in number, and the profile of cytokine-positive cells followed essentially the same patterns--found at the site of inoculation. These results suggest that the downregulation of gp63 increases extracellular lysis of the mutants by complement, in the in vivo environment, and reduces their infection of macrophages, resulting in a type 1 immune response seen at the site of inoculation and DLNs.     

Cell-mediated responses and protection elicited by a carbohydrate-lipid-containing fraction extracted from Leishmania major promastigotes

Carbohydrate-lipid-containing fractions (CLF) extracted from Leishmania major promastigotes and recognized by sera from immune but not from normal human donors were evaluated for their capacity to elicit cell-mediated responses. It was found that one of these fractions, CLF-1, stimulated the in vitro response of lymphocytes from immune but not from normal human donors. A similarly extracted fraction from L. donovani parasites also elicited an in vitro response by cells from donors immune to L. major. The response was mediated by antigen-presenting cells, and specific Leu 3+ Leu 2- T cells from a human T-cell line responded to the antigen. In vivo, the CLF-1 elicited delayed-type hypersensitivity (DTH) response in L. major-immunized C3H mice, which was comparable to the DTH response elicited by freeze-thawed and sonicated L. major promastigotes. C3H mice were vaccinated with CLF-1 prior to challenge with live L. major promastigotes. Mice vaccinated with CLF-1-containing liposomes showed a significant degree of protection to challenge. These results suggest that the carbohydrate-lipid-containing fraction described here may represent a functional antigenic entity from Leishmania parasites.     

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.     

Leishmania amazonensis arginase compartmentalization in the glycosome is important for parasite infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg(-) L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg(-) mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.     

Role of intracellular cAMP in differentiation-coupled induction of resistance against oxidative damage in Leishmania donovani

Even though the human parasite Leishmania donovani encounters tremendous oxidative burst during macrophage invasion, a set of parasites survives and proliferates intracellularly, leading to transformation from promastigote to amastigote form and disease manifestation. The striking shifts in temperature (from 22 degrees C in the insect gut to 37 degrees C in the mammalian host) and pH (7.2 in the insect gut to 5.5 in the parasitophorous vacuole of macrophages) are the key environmental triggers for differentiation as these cause an arrest in the G1 stage of the cell cycle and initiate transformation. Using an established in vitro culture and differentiation system our study demonstrates that the differentiation-triggering environment induces resistance to oxidative damage and consequently enhances infectivity. Differentiation conditions caused a three- to fourfold elevation in cAMP level as well as cAMP-dependent protein kinase activity. Similar to stress exposure, positive modulation of intracellular cAMP resulted in blockage of cell cycle progression and induction of resistance against oxidative damage. Resistance against pro-oxidants from either stress or cAMP may be associated with upregulation of the expression of three major antioxidant genes, peroxidoxin 1, trypanothione reductase, and superoxide dismutase A. Positive modulation of the intracellular cAMP response enables cells to resist the cytotoxic effects of pro-oxidants. In contrast, downregulation of intracellular cAMP by overexpression of cAMP phosphodiesterase A resulted in a decrease in resistance against oxidative damage and reduced infectivity toward activated macrophages. This study for the first time reveals the importance of cAMP response in the life cycle and infectivity of the Leishmania parasite.     

Interleukin 1alpha activity of peritoneal and bone marrow macrophages infected with Leishmania major and Leishmania donovani in vitro

In this study, the pattern of interleukin-1alpha (IL-1alpha) production by both peritoneal (PM) and bone marrow macrophages (BMM) from resistant (C3H/HeJ) and susceptible (BALB/c) mice was investigated, using a bioassay and an IL-1alpha-specific ELISA kit. PM from normal uninfected mice showed either an initial high (C3H/HeJ) or a neglected (BALB/c) level of IL-1alpha activity, respectively, probably due to thioglycollate stimulation. Infection with Leishmania major induced only a marginal effect on IL-1 production by both cells. Normal, uninfected and unstimulated BMM from both mice did not produce IL-1alpha over a 7-day period of cultivation in vitro. Upon stimulation with either lipopolysaccharide (LPS) (BALB/c) or concanavalin A (Con A) (C3H/HeJ), both cell types produced IL-1alpha that peaked within the first 12-24 h following stimulation. BMM from C3H/HeJ and BALB/c mice failed to produce IL-1alpha when infected in vitro with L. major or L. donovani promastigotes. However, infection with these two parasites did not interfere with the capability of the host cell to produce IL-1alpha when stimulated with LPS or Con A. The level of IL-1alpha production was independent of the degree of parasitization of the macrophages. Similar results were observed with IL-1beta and IL-6 production by BMM, even though their levels were generally slightly higher than those obtained with IL-1alpha.     

Adenine aminohydrolase from Leishmania donovani: unique enzyme in parasite purine metabolism

Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent K(m) of 15.4 μM, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Δaah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Δaah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Δaah lesion was also introduced into a conditionally lethal Δhgprt/Δxprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2'-deoxycoformycin. The Δaah/Δhgprt/Δxprt triple knock-out no longer required 2'-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani.     

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.     

Structure and regulation of histone H2B mRNAs from Leishmania enriettii.

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).     

Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning

Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.     

Control of infection with Leishmania major in susceptible BALB/c mice lacking the common gamma-chain for FcR is associated with reduced production of IL-10 and TGF-beta by parasitized cells

Previous studies have shown that the in vitro ligation of FcgammaRs with IgG-opsonized Leishmania amastigotes promotes IL-10 production by macrophages. In addition, infection of either BALB/c mice lacking the common gamma-chain of Fc receptors (FcgammaR(-/-)) or mice genetically altered to lack circulating Ab (J(H)D) with Leishmania pifanoi results in reduced and delayed lesion development and a deficit in the recruitment of inflammatory cells into infected lesions. We show in this study that FcgammaR(-/-) mice can control infection with Leishmania major and totally resolve cutaneous lesions. The ability to eventually control infection is not associated with a reduction in lesion inflammation or a reduction in the ability of Leishmania to parasitize cells through week 6 of infection. The immune response in healing FcgammaR(-/-) mice is associated with a reduction in numbers of cells producing Th2-type cytokines, including IL-4 and IL-10, but not an increase in numbers of IFN-gamma-producing cells characteristic of a dominant Th1-type response. Instead, we observe a reduction in levels of IL-10 and TGF-beta within infected lesions, including reduced levels of these cytokines within parasitized macrophages. Together, these results suggest that uptake of opsonized parasites via FcgammaRs may be a strong in vivo stimulus for the production of anti-inflammatory cytokines that play a role in susceptibility to infection.     

The human cytokine response to Leishmania major early after exposure to the parasite in vitro

Leishmaniasis is caused by the protozoan parasite Leishmania spp. In murine leishmaniasis, a T helper cell type-I (Th1) response, characterized by the secretion of interferon (IFN)-gamma is necessary for clearing the infection. whereas a Th2 response, accompanied by the production of interleukin (IL)-5, can exacerbate the disease. Moreover, the early cytokine milieu is thought to play an important role in determining the outcome of infection. In human leishmaniasis little is known about this early cytokine response. Because of this, we cocultured human peripheral blood mononuclear cells (PBMC) with Leishmania major in vitro and measured the production of IFN-gamma, IL-5, and IL-10. We also treated PBMC cultures with various cytokines and neutralizing anticytokines. We found that the principal cytokine produced was IFN-gamma and that its production was regulated by IL-10 and IL-12. In contrast, only low levels of Th2 cytokines such as IL-5 were produced. Therefore, the Th1-Th2 dichotomy that exists in inbred strains of mice does not appear to apply to the response of humans to L. major. Rather, Th2 cytokines may play a role in regulating IFN-gamma production.