Influence of erythrocyte membrane components on malaria merozoite invasion.

Chymotrypsin- and Pronase-treated human erythrocytes were refractory to invasion by P. knowlesi merozoites; invasion was not inhibited by trypsin or neurammidase treatment. These data implicate a surface protein other than sialoglycoprotein as the receptor site for merozoites. Invasion of rhesus erythrocytes was unaffected by pretreatment with these enzymes. Differences in membrane structure of erythrocytes from various species may explain the absence of an enzyme effect on rhesus erythrocytes.

A developmental defect in Plasmodium falciparum male gametogenesis.

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha-tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.

Vertebrate cells express protozoan antigen after hybridization.

Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

Vertebrate cell cycle modulates infection by protozoan parasites.

Synchronized HeLa cell populations were exposed to Trypanosoma cruzi or Toxoplasma gondii, obligate intracellular protozoan parasites that cause Chagas' disease and toxoplasmosis, respectively, in humans. The ability of the two parasites to infect HeLa cells increased as the HeLa cells proceeded from the G1 phase to the S phase of their growth cycle and decreased as the cells entered G2-M. Characterization of the S-phase cell surface components responsible for this phenomenon could be beneficial in the development of vaccines against these parasitic diseases.

Invasion of erythrocytes by malaria merozoites.

An electro-optical system was developed to record microscope images with high resolution at low light intensities. The system was used to study the invasion of erythrocytes by malaria merozoites. Invasion consists of attachment of the anterior end of the parasite to the erythrocyte, deformation of the erythrocyte, and the entry of the parasite by erythrocyte membrane invagination.

Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants.

Duffy blood group negative human erythrocytes (FyFy) are resistant to infection by Plasmodium knowlesi, a simian malaria that infects Duffy positive human erythrocytes. The P. knowlesi resistance factor, Duffy negative erythrocytes, occurs in high frequency in West Africa, where the people are resistant to vivax malaria. This suggests that Duffy blood group determinants (Fya or Fyb) may be erythrocyte receptors for P. vivax.

Cytometric analysis of neoplastic transformation of vertebrate cell populations.

Two parameters of neoplastic transformation in spontaneously transforming fibroblasts were examined. One parameter, the predilection for rounding of cells at metaphase, was studied using time-lapse cinephotomicrography and fixed, stained preparations. Ten different cell lines were assayed, including established lines of hamster, rat, and mouse cells, and six rodent cultures of known neoplastic potential as determined by animal injection and tumor production. There was a close correlation between the assumption by cells of a spherical shape at metaphase and their ability to form tumors on injection in syngeneic hosts. The projected area of adherent cells 24 hr after plating over a coverglass was used to assay the transition of these rodent cell populations in culture from nonneoplastic to neoplastic. As the cell population became neoplastic, there was a significant decrease in the mean projected area of the cells. Furthermore, as the cell cultures became capable of producing tumors, the projected area profile of the population shifted proportionally to smaller area classes.

A biochemical comparison of glucosephosphate isomerase isozymes from Trypanosoma cruzi.

The glucosephosphate isomerase (D-glucose-6-phosphate Ketol-isomerase, EC 5.3.1.9) isozymes of Trypanosoma cruzi were characterized with respect to their native and subunit molecular size, isoelectric point and in vitro thermostability. The molecular weight data are consistent with a dimeric enzyme structure. The apparent native and subunit size homogeneity and differences in pI values imply that the electrophoretic mobility differences of isozymes in native gels are determined by their molecular charge. Minor differences in peptide maps indicate the existence of some heterogeneity in the primary structure of the isozymes. The stability of triple-banded glucosephosphate isomerase electrophoretic profiles was confirmed, supporting the view that these phenotypes represent non-interconvertible enzyme species.

Identification, isolation, and characterization of naturally-occurring Trypanosoma cruzi variants.

Naturally occurring DNA variants of the single-cell-derived Y-02 stock of Trypanosoma cruzi were discovered during a routine assay of the stock. Three DNA variant types were isolated. One type was indistinguishable from the parental Y-02 stock on the basis of total DNA cell-1. The other two types contained approximately 30% and 70% more DNA cell-1 than the parental Y-02 stock. Both the nucleus and kinetoplast were involved in the DNA content differences. The increase in DNA cell-1 was not G-C- or A-T-specific and was unrelated to the developmental stage of the parasite. Epimastigote population doubling times, isoenzymes, and schizodeme analyses could not differentiate the variant stocks. However, marked karyotype polymorphisms were observed by pulse-field gel electrophoresis, and restriction-fragment-length-polymorphisms were detected in hybridizations of some endonuclease-restricted samples to the spliced leader probe. We postulate that the Y-02 variants are genetic homologs. The ability to form viable hybrids or aneuploids provides T. cruzi with a mechanism to survive environmental stress, promote intra-specific heterogeneity and generate the diversity observed in the presentation and course of Chagas' disease.

Kinetoplastidae display naturally occurring ancillary DNA-containing structures.

Kinetoplast-derived, DNA-containing structures were found in several members of the order Kinetoplastida. The structures, for which we propose the name ancillary DNA-containing structures (aDNA), were discovered during the course of low-light-level video fluorescence microscopy studies using several nucleic acid-specific fluorescent reagents. DNase treatment and supravital stain with Höechst 33342 confirmed that aDNA is not an artifact of specimen preparation. Fluorescent in situ hybridization using either a 122-bp kinetoplast DNA-specific probe derived from a conserved region of minicircle DNA or a 188-bp nuclear DNA-specific probe derived from highly repetitive nuclear DNA demonstrated that aDNA is derived from the kinetoplast and not the nucleus. However, the structures do not contain minicircle DNA replication intermediates. Immunofluorescence assays using an anti-mitochondrial protein antibody, anti-mtp70, demonstrated that the structures contain mitochondrial protein and confirmed their kinetoplast origin. The frequency of occurrence of aDNA varies markedly between members of the Kinetoplastida. In the case of Trypanosoma cruzi stocks, the percentage of cells with aDNA was positively correlated to the population doubling time of the stock. However, there is no statistically significant relationship between the developmental or replicative stage of the parasite and the frequency of aDNA. An inhibitor of DNA topoisomerase I had no effect upon the frequency of aDNA. An inhibitor of DNA topoisomerase II gave equivocal results depending upon the parasite stock used. We speculate that aDNA may be the morphological consequence of a yet-to-be-determined biological process intrinsic to but variable within the Kinetoplastida.

Influence of monosaccharides on the infection of vertebrate cells by Trypanosoma cruzi and Toxoplasma gondii.

The effect of 9 monosaccharides which constitute cell surface carbohydrates on the infection of bovine embryonic skin and muscle (BESM) cells by Trypanosoma cruzi trypomastigotes and Toxoplasma gondii tachyzoites was assayed. Most of the monosaccharides tested stimulated the infection of BESM cells by T. gondii; none of the monosaccharides were inhibitory. In contrast (at a concentration of 50 mM or greater) the monosaccharides inhibited non-specifically the infection of BESM cells by T. cruzi trypomastigotes whereas the other 8 monosaccharides were ineffective. The inhibition was due to an effect on the trypomastigotes and not on the vertebrate cells. It is proposed that there is a wheat-germ agglutinin-like lectin on the T. cruzi trypomastigote surface which recognizes and attaches to an N-acetylglucosamine-containing receptor on the vertebrate cell surface prior to infection. Infection of vertebrate cells by T. gondii tachyzoites appears to be mediated by other cell surface components. If monosaccharides are involved in infection by tachyzoites, they are ones not commonly found on animal cell surfaces. Alternatively, infection of vertebrate cells by T. cruzi and T. gondii is effected by different mechanisms.

Temperature modulation of growth rates and glucosephosphate isomerase isozyme activity in Trypanosoma cruzi.

In an attempt to understand the conditions which might influence the geographical distribution of Trypanosoma cruzi isozyme profiles (zymodemes), the thermal response of different glucosephosphate isomerase (GPI) phenotypes was studied. T. cruzi clones with single- and triple-banded GPI phenotype showed a similar response to temperature with respect to both growth rates and GPI kinetic parameters. However, the relative activity of each GPI isozyme was dependent on parasite incubation temperature. In view of the similar kinetic properties of the isozymes, enzyme regulation is not a consequence of an adaptative response to thermal conditions and the suggestion of a phenotype distribution determined selectively by temperature is not supported by the present study.

Identification and analysis of epimastigote surface and metabolic proteins in Trypanosoma cruzi.

Metabolic and surface membrane proteins of four epimastigote-stage Trypanosoma cruzi clones were analyzed by one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No major inter-clonal differences were observed in the metabolic protein patterns, indicating that these proteins are highly conserved. However, marked quantitative and qualitative differences were observed in surface-labeled protein patterns following both one and two-dimensional electrophoretic analyses. Inter and intra-clonal differences in antigenic properties also were demonstrated by immunoprecipitation of the surface proteins with sera from animals immunized or infected with various T. cruzi stocks. Thus, a wide spectrum of both phenotypic and antigenic diversity exists in T. cruzi which may be relevant to problems of the diagnosis and immunotherapy of Chagas' disease.

Isolate-dependent differences in the oxidative metabolism of Trypanosoma cruzi epimastigotes.

Epimastigote cultures of the two cloned Trypanosoma cruzi stocks, CA-I/72 and HO-3/15, grown under identical conditions, differ both qualitatively and quantitatively in their cytochrome content. The CA-I/72 stock has a four-fold higher cytochrome b content (19.2 nM (mg protein)-1) than the HO-3/15 stock (4.9 nM (mg protein)-1). Cytochrome o is present at 29 nM (mg protein)-1 in the CA-I/72 stock but is below detectable limits in the HO-3/15 stock. There is no inter-stock difference in oxygen utilization (12-15 nM O2 min-1 (mg protein)-1) during exponential growth. However, stationary phase CA-I/72 epimastigotes utilize twice as much oxygen as HO-3/15 epimastigotes. Oxygen utilization by HO-3/15 epimastigotes incubated in Dulbecco's phosphate buffer solution (starvation conditions), was stimulated earlier and to higher levels by the addition of glucose than by CA-I/72 epimastigotes under identical conditions. Under starvation conditions and with the cytochrome chain partially inhibited by antimycin A,(anti-A) the addition addition of glucose also increased oxygen utilization by CA-I/72 epimastigotes. In contrast, anti-A did not influence glucose-stimulated oxygen utilization by HO-3/15 epimastigotes. Following partial inhibition with anti-A, salicylhydroxamic acid produced an additional 50% inhibition in oxygen utilization in both stocks irrespective of the growth phase of the organisms. These data indicate that marked intra-specific differences in oxidative metabolism exist within the T. cruzi population and that an alpha-glycerophosphate oxidase or similar salicylhydroxamic acid-inhibitable compound may be present in the organism.

Nucleoside uptake in Trypanosoma cruzi: analysis of a mutant resistant to tubercidin.

Nucleoside salvage pathways are vital to the parasitic protozoan Trypanosoma cruzi, and have become important targets in the development of new chemotherapeutic agents against this organism. We produced a mutant T. cruzi clone with a defect in the uptake of the adenosine analogue tubercidin which allowed us to hypothesize that there are at least two distinct nucleoside transport pathways in this parasite. The mutant shows a marked defect in the uptake of tubercidin and thymidine, whereas the uptake of adenosine and inosine are normal. Inhibition and metabolic studies suggest that the defect is related to transport and that there are two transport processes relatively specific for purines and pyrimidines, respectively, although tubercidin is transported via the latter. This is similar to the reported dual nucleoside transport pathways in Leishmania donovani and may be a common system in the Trypanosomatidae. These transport processes are markedly different from those which have been described for mammalian cells and may play an important role in the design of strategies for the chemotherapy of human infection with these pathogenic parasites.

Strains and clones of Trypanosoma cruzi differ in their expression of a surface antigen identified by a monoclonal antibody.

Four Trypanosoma cruzi strains and 50 clones derived from a total of 10 strains were assayed with a monoclonal antibody, WIC 29.26 Ab, for expression of an epitope previously demonstrated to be on the carbohydrate portion of a 72 000 Da surface glycoprotein of Y strain epimastigotes. WIC 29.26 Ab bound to only 2 of the 4 strains and 23 of the 50 clones tested. A group of 10 cloned isolates from one strain contained both reactive and non-reactive clones. Competitive inhibition studies with soluble extracts of the reactive and non-reactive isolates suggested that in addition to being absent from the surface membrane, the antigenic determinant is not synthesized by the non-reactive parasites. These data indicate that the epitope recognized by WIC 29.26 Ab is not present on all T. cruzi epimastigotes, and provide the first demonstration of clone-specific differences in a parasite antigen detected by a monoclonal antibody. No correlation was found between the reactivity of isolates with WIC 29.26 Ab and the previously investigated parameters of growth rate and modal volume.

Trypanosoma cruzi: elemental composition heterogeneity of cloned stocks.

Concentrates of the epimastigote stage of Trypanosoma cruzi stocks derived from single cell clones and cultured under identical conditions display a spectrum of 'colors' varying from dark brown to milk white. The color of the concentrate is reproducible for a parasite stock. An essential component of the culture medium for epimastigote growth is hemin, an iron-containing compound. Consequently, it seemed possible that the color spectrum of the epimastigote stocks reflected differences in the uptake, concentration or utilization of iron. This report describes the quantitative studies utilizing electron probe X-ray elemental mapping, energy dispersive X-ray microanalysis, and energy dispersive X-ray fluorescence spectrometry of the epimastigote stage of two T. cruzi stocks (CA-I/72 and HO-3/15) which display extreme color differences. Striking and statistically significant quantitative differences were found in the levels of Fe, Zn, and K between the two stocks. On the basis of atomic ratios, the CA-I/72 stock contains approximately two-fold more Fe per cell than the HO-3/15 stock. However, in the case of Zn the ratio is reversed; the HO-3/15 stock contains approximately two-fold more Zn per cell than the CA-I/72 stock. The marked inter-stock differences which exist in the levels of several elements could modulate the pathogenicity, survival, or adaptability of T. cruzi and, consequently, be important factors in our understanding of the complex problem of Chagas' disease.

Trypanosoma cruzi: antigenic analysis of cloned stocks.

The antigenic constitution of two Trypanosoma cruzi strains and six cloned stocks was determined immunoelectrophoretically. Five to seven antigens common to all of the T. cruzi stocks were found. However, each stock also contained "strain-specific antigens," and five of the six cloned stocks presented "clone-specific antigens." These data demonstrate that a T. cruzi strain is composed of an antigenically heterogeneous population of organisms.

The effects of Lampit (Bayer 2502) on the interaction of Trypanosoma cruzi with vertebrate cells in vitro.

The effects of 3-methyl-4-(5'-nitrofurfurylidene-amino)-tetrahydro-4H-1,4-thiazine-1,1-dioxide [Lampit, Bayer 2502] on the intracellular cycle of Trypanosoma cruzi were studied with an in vitro steady-state culture system that permitted the continuous analysis of individual host cell-parasite interactions. Lampit concentrations of 10(-4) and 10(-5) M significantly affected the ability of trypomastigotes to penetrate vertebrate cells. Lampit concentrations above 10(-4) M were toxic to the host cell population and Lampit concentrations below 10(-5) M did not affect the ability of trypomastigotes to penetrate vertebrate cells. Lampit concentrations of 10(-5) M or greater completely inhibited the intracellular cycle of T. cruzi whereas Lampit concentrations of 10(-7) M or less had no effect on the intracellular cycle. Continuous perfusion with 10(-6) M Lampit resulted in a linear increase in intracellular parasite reproduction time. A stable strain of T. cruzi was produced in vitro that was resistant to a 100-fold greater concentration of Lampit that the parent strain.

Growth and development of two Trypanosoma cruzi clones in the arthropod Dipetalogaster maximus.

Trypomastigotes of single-cell-isolate Trypanosoma cruzi clones WA-250/1 and Esmereldo/2 were used to infect third-instar Dipetalogaster maximus. The level of infection was directly proportional to the concentration of trypomastigotes present in the blood meal. The minimum infectious dose for D. maximus was calculated as 5 organisms/ml. Clone WA-250/1 underwent epimastigote-to-metacylic trypomastigote transformation over the 30-day course of the experiments; clone Esmeraldo/2 did not undergo transformation to metacylic trypomastigotes. These data extend our knowledge of the biological diversity of T. cruzi stocks.