Programmed cell death in the unicellular protozoan parasite Leishmania.

In the present study we have demonstrated some features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral Leishmaniasis. We report that PCD is initiated in stationary phase cultures of promastigotes and both in actively growing cultures of axenic amastigotes and promastigotes upon treatment with anti Leishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antileishmanial drugs differently. The features of PCD in L. donovani promastigotes are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, increase in plasma membrane permeability, decrease in the mitochondrial membrane potential (DeltaPsi m) and induction of a PhiPhiLux (PPL)-cleavage activity. PCD in both stationary phase culture and upon induction by amphotericin B resulted first in the decrease of mitochondrial membrane potential followed by simultaneous change in plasma membrane permeability and induction of PPL-cleavage activity. Of the total PPL-cleavage activity, several caspase inhibitors inhibited a significant amount (21-34%). Inhibitors of cathepsin or calpain did not inhibit PPL-cleavage activity. Taken together this study demonstrates that the characteristic features of PCD exist in unicellular protozoan Leishmania donovani. The implication of PCD on the Leishmania pathogenesis is discussed.     

Dispersal, eviction, and conflict in meerkats (Suricata suricatta): an evolutionarily stable strategy model

Decisions regarding immigration and emigration are crucial to understanding group dynamics in social animals, but dispersal is rarely treated in models of optimal behavior. We developed a model of evolutionarily stable dispersal and eviction strategies for a cooperative mammal, the meerkat Suricata suricatta. Using rank and group size as state variables, we determined state-specific probabilities that subordinate females would disperse and contrasted these with probabilities of eviction by the dominant female, based on the long-term fitness consequences of these behaviors but incorporating the potential for error. We examined whether long-term fitness considerations explain group size regulation in meerkats; whether long-term fitness considerations can lead to conflict between dominant and subordinate female group members; and under what circumstances those conflicts were likely to lead to stability, dispersal, or eviction. Our results indicated that long-term fitness considerations can explain group size regulation in meerkats. Group size distributions expected from predicted dispersal and eviction strategies matched empirical distributions most closely when emigrant survival was approximately that determined from the field study. Long-term fitness considerations may lead to conflicts between dominant and subordinate female meerkats, and eviction is the most likely result of these conflicts. Our model is computationally intensive but provides a general framework for incorporating future changes in the size of multimember cooperative breeding groups.     

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.     

Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania.

In this study we investigated the role of several parameters governing the efficiency of gene targeting mediated by homologous recombination in the protozoan parasite Leishmania. We evaluated the relative targeting frequencies of different replacement vectors designed to target several sequences within the parasite genome. We found that a decrease in the length of homologous sequences <1 kb on one arm of the vector linearly influences the targeting frequency. No homologous recombination was detected, however, when the flanking homologous regions were <180 bp. A requirement for a very high degree of homology between donor and target sequences was found necessary for efficient gene targeting in Leishmania , as targeted recombination was strongly affected by base pair mismatches. Targeting frequency increased proportionally with copy number of the target only when the target was part of a linear amplicon, but remained unchanged when it was present on circles. Different chromosomal locations were found to be targeted with significantly variable levels of efficiency. Finally, different strains of the same species showed differences in gene targeting frequency. Overall, gene targeting mediated by homologous recombination in Leishmania shares similarities to both the yeast and the mammalian recombination systems.     

Comparative physiology of two protozoan parasites, Leishmania donovani and Trypanosoma brucei, grown in chemostats.

Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate. L. donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T. brucei is 1.06 day-1 and the Ks is 0.06 mM. At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity. L. donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP). Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium. L. donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted. T. brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates. Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism. The Ks is constant as long as glucose is the rate-limiting substrate. The results of this study demonstrate that L. donovani and T. brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts.     

Activation of human immunodeficiency virus type 1 in monocytoid cells by the protozoan parasite Leishmania donovani.

In this study, we demonstrated that the protozoan parasite Leishmania donovani and one of its major surface molecules, the lipophosphoglycan (LPG), can induce human immunodeficiency virus type 1 (HIV-1) expression in U1 and OM-10.1, two cell lines of monocytoid origin latently infected with HIV-1. Treatment of U1 cells with various concentrations of LPG (1, 5, and 10 microM) resulted in a dose-dependent secretion of tumor necrosis factor alpha (TNF-alpha). Suppression of LPG-induced HIV-1 expression by polyclonal anti-TNF-alpha antibodies further confirmed the involvement of this cytokine. Results from these studies indicate that the protozoan parasite L. donovani can induce the secretion of TNF-alpha that will function in an autocrine or paracrine manner to upregulate HIV-1 expression. Our data suggest for the first time that this protozoan parasite can be viewed as a potential cofactor in the pathogenesis of AIDS.     

Flow cytometry in the study of the interaction between murine macrophages and the protozoan parasite Leishmania amazonensis.

A flow cytometry method was adapted to study interaction between murine macrophages and Leishmania amazonensis. Using this method it was possible to detect internalization of parasites through an increase in macrophage granularity (side scatter), with the latter indicating the presence of parasites inside parasitophorus vacuoles. A quenching technique was used to confirm the feasibility of the method and to distinguish between internalized and externally attached parasites. Experiments using fixed-labeled and killed-unlabeled parasites gave similar results, demonstrating that granularity was an adequate parameter in the study of parasite-macrophage interaction, when compared with labeling methods. Experiments that measured internalization using only the increase in macrophage granularity as an indicator showed that living L. amazonensis was internalized to a greater extent than killed-unlabeled parasites. This finding suggests that the parasite has an active role in the process of internalization. The 2 methods in combination, flow cytometry and labeling, can be used to study murine peritoneal cell-L. amazonensis interactions and to sort phagocytosing and nonphagocytosing subpopulations of macrophages for further studies.     

Theory of spontaneous cell fusion. Sexuality in cell populations as an evolutionarily stable strategy. Applications to immunology and cancer.

It is argued that spontaneous fusion of cells in culture can be understood as a strategy of individual cells to counteract disadvantageous mutations. This explanation generates several predictions, suggested experiments, and a possible mutagen-carcinogen assay. The theory also implies that the standard method of measuring fusion rates (by means of media in which only hybrids can grow) will stimulate fusion and thus may grossly distort spontaneous fusion rates. Fusion and subsequent ploidy reducing divisions are the equivalent of sexual recombination. An appendix discusses implications for a general theory of sex. A second appendix discusses implications for the generation of millions of lymphocyte clones with different antigenic specificities from a small number of germ line genes. Cell fusions in vivo could function as a genetic repair mechanism and may play a role in cancer.     

Paleopathology in an Iroquoian ossuary, with special reference to tuberculosis

Pathological skeletal remains from the Uxbridge Ossuary (1490 +/- 80 A.D., N = 457) are classified into four broad categories: trauma, congenital disability, tumor, and infection. Traumatic injuries are relatively common (fractures in 5-9.4% of total), considering the date and subsistence pattern of the population. Congenital disabilities and tumors are rare, affecting approximately 2% of the population. Nonspecific periosteitis and osteitis affect 5% of the sample. By far the most common pathological skeletal changes are lytic lesions leading to cavitation of cancellous bone, especially in the lower vertebral and sacro-iliac regions. It is argued that the changes seen and their distribution are most consistent with a diagnosis of tuberculosis. Applying clinical observations regarding bone involvement, it is estimated that a minimum of 26 skeletons were affected. This in turn indicates a very high population tuberculosis incidence. The Uxbridge sample is neither the only nor the earliest Iroquoian ossuary to display apparent tuberculosis (Hartney 1981). The common presence of this disease in some communities and its low incidence in others are discussed in the context of the epidemic wave phenomenon. There is strong evidence for warfare at Uxbridge, and this warfare may have produced crowding, poor hygiene and diet, such that the disease could flourish.     

Regulation of guanylyl cyclase by intracellular Ca2+ in relation to the infectivity of the protozoan parasite, Leishmania donovani

A neuronal type Ca2+ stimulated nitric oxide synthase was earlier reported by us to be present in the protozoan parasite Leishmania donovani. As part of nitric oxide-cyclic GMP transduction signaling operative in higher eukaryotes and involved in the long-term potentiation, a soluble guanylyl cyclase has also been detected in this lower eukaryote. However, detailed biochemical characterization revealed the enzyme to be Ca2+ modulated and unstimulated by nitric oxide donors as opposed to higher eukaryotes. The possible role of intracellular Ca2+ level in the regulation of guanylyl cyclase activity as well as L. donovani infectivity was explored by measuring the intracellular survival of the parasites in mammalian macrophages after treatments, which decrease or elevate the intracellular Ca2+. Parasites loaded with intracellular Ca2+ chelators displayed significantly decreased infectivity and cyclic GMP level. In contrast, pretreatment with Ca2+ ionophores, which elevated Ca2+ levels in L. donovani, significantly enhanced the cyclic GMP level as well as the infectivity of the parasites. Moreover, treatment with selective inhibitors of soluble guanylyl cyclase also reduced infectivity, even in cases of calcium ionophore-treated parasites. The gene encoding the soluble guanylyl cyclase was cloned, sequenced and over expressed in bacterial system. The recombinant protein showed enzyme characteristics similar to that obtained in L. donovani promastigote cytosol. Together these results suggest a possible link between guanylyl cyclase, intracellular Ca2+ content and parasite infectivity.     

Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii

A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.     

The role of reduced pterins in resistance to reactive oxygen and nitrogen intermediates in the protozoan parasite Leishmania

During its life cycle, the protozoan parasite Leishmania experiences oxidative stress when interacting with macrophages. Reduced pterins are known scavengers of reactive oxygen and nitrogen intermediates. Leishmania has a pteridine reductase, PTR1, whose main function is to provide reduced pterins. We investigated the role of PTR1 in resistance to oxidative and nitrosative stress in Leishmania tarentolae, Leishmania infantum, and Leishmania major PTR1^?/? mutants. The PTR1^?/? cells of the three species were more sensitive to H₂O₂- and NO-induced stress. Using a fluorescent probe allowing ROI quantification, we demonstrated an increase in intracellular oxidant molecules in the PTR1^?/? mutants. The disruption of PTR1 increased metacyclogenesis in L. infantum and L. major. We purified metacyclic parasites from PTR1^?/? mutants and control cells and tested their intracellular survival in the J774 mouse cell line and in human monocyte-derived macrophages. Our results showed that PTR1^?/? null mutants survived less in both macrophage models compared to control cells and this decrease was more pronounced in macrophages activated for oxidant production. This study demonstrates that one physiological role of reduced pterins in Leishmania is to deal with oxidative and nitrosative species, and a decreased ability to provide reduced pterins leads to decreased intracellular survival.     

Seed predators and the evolutionarily stable flowering strategy in the invasive plant, Carduus nutans

In plants where reproduction is fatal, seed-feeding insects may have a major impact on the evolutionarily stable reproductive strategy by altering fecundity schedules in a size-dependent manner. We explored this in Carduus nutans, a facultative biennial native to Europe, using two years of data from the South of France. An integral projection model based on detailed statistical models of the demography of Carduus nutans and characteristics of herbivore attack showed that seed predators select for smaller flowering size. An elasticity analysis showed that changes in the slope relating seed herbivore attack rates to plant total receptacle area had a large effect on lifetime reproductive success relative to most other plant demographic rates. Together, these two results indicate that in the absence of seed predators, as is the case in the exotic range of this invasive species, flowering size could evolve to be larger. Further analysis also showed that subsequent introduction of different species of seed-feeders as biocontrol agents could lead to different evolutionary outcomes dependent on the ecological characteristics of the seed-feeders, allowing the direction and magnitude of evolutionary change in flowering size to be predicted based on what seed predators have been introduced where and when. Such data would allow us to distinguish between the effect of seed predators and other hypotheses for size increase in the invasive habitat.     

Studies on the fine structure of the protozoan Cyclidium,with special reference to the mitochondria,pellicle and surface-associated bacteria

Summary The ultrastructure of Cyclidium, including the cilia, kinetosomes, pellicle, microtubules and kinetodesmal fibers is similar to that recorded for other ciliates. Of special interest is the attachment of rod-shaped bacteria within the longitudinally directed shallow surface folds of the protozoan. Both the bacteria and the surface of Cyclidium seem to possess an outer coating of a sticky substance which upon contact holds the bacteria to the protozoan. The bacteria appear to be attached by only a relatively small area of their surfaces. A dense substance appears within the alveolus of the pellicle at the regions of the attachment of the bacteria. The association of the organisms is probably a temporary one, and it is unknown whether either organism is benefited or harmed by the association. The position of the mitochondria in Cyclidium is unusual in that they all lie flattened against the inner membrane of the pellicle, usually in a position directly opposite to that of the attachment of the bacteria to the surface, thus being separated from the bacteria by only the outer cell membrane and the pellicle. Whether or not this close topographical relationship is of significance is unknown.We are indebted to Dr. Dorothy Pitelka for help in determining the identity of certain membranes and microtubular structures in Cyclidium.     

IgE response in Strongyloides ratti-infected rats with special reference to the life cycle of the parasite

Parasite-specific IgE antibody response was examined in Strongyloides ratti-infected rats. The results showed that the parasite-specific IgE antibody response was generated after a primary infection. However, repeated infections rather depressed the level of parasite-specific IgE antibody in the serum. Immunization limited to specific stages of the parasite revealed that stimulation of parasite-specific IgE antibody was related to the intestinal adult stage. On the other hand, depression of IgE titers was related to the tissue-migrating larval stage. The capacity of the each stage of the parasite to induce specific IgE response may be related to the variable results of the IgE responses in human strongyloidiasis.     

Characterization of metacaspases with trypsin-like activity and their putative role in programmed cell death in the protozoan parasite Leishmania

In this report, we have characterized two metacaspases of Leishmania donovani, L. donovani metacaspase-1 (LdMC1) and LdMC2. These two proteins show 98% homology with each other, and both contain a characteristic C-terminal proline-rich domain. Both genes are transcribed in promastigotes and axenic amastigotes of L. donovani; however, LdMC1 shows increased mRNA levels in axenic amastigotes. An anti-LdMC antibody was obtained and showed reactivity with a single approximately 42-kDa protein band in both promastigote and axenic amastigote parasite whole-cell lysates by Western blotting. Pulse-chase experiments suggest that LdMCs are not synthesized as proenzymes, and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs efficiently cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, LdMC activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as leupeptin, antipain, and N(alpha)-tosyl-L-lysine-chloromethyl ketone (TLCK). In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death (PCD) in Leishmania and that parasites overexpressing metacaspases are more sensitive to hydrogen peroxide-induced PCD. These findings suggest that Leishmania metacaspases are not responsible for the caspase-like activities reported in this organism and suggest a possible role for LdMCs as effector molecules in Leishmania PCD.