Stumpy Forms Research Articles

Altered intracellular polyamines in bloodstream form Trypanosoma brucei brucei: transformation to procyclic trypomastigotes

A monomorphic strain of Trypanosoma brucei brucei (EATRO 110) was cultured as long slender bloodstream forms in vitro for 24 h with 100 μM Eflornithine HCl. This resulted in depletion of intracellular putrescine, a greater than 50% decrease in spermidine, and a cessation of cell division. These Eflornithine treated trypanosomes were stimulated to synchronously transform to procyclic trypomastigotes by transfer into SDM-79 medium containing the citric acid cycle intermediates citrate and cis-aconitate (CCA). Within 48 h morphological transformation was complete and occurred without any increase in cell numbers. The coadministration of 100 μM putrescine with Eflornithine prevented the depletion of putrescine and abrogated the cytostatic effect of Eflornithine alone, but did not prevent the transformation to procyclic trypomastigotes. Eflornithine-induced short stumpy form trypanosomes resulting from 4 days of culture with 100 μM Eflornithine did not transform to procyclic trypomastigotes in SDM-79 medium unless it contained CCA. We conclude that the high concentration of CCA can trigger transformation in all morphological types of bloodstream form trypanosomes, regardless of their threshold of sensitivity for the stimulus. Furthermore, the CCA-stimulated transformation is not dependent on putrescine and can occur independently of cell division.

Comparison of the effects of immune killing mechanisms on Trypanosoma brucei parasites of slender and stumpy morphology

Trypanosoma brucei slender forms predominate over stumpy forms as the parasite population grows but at the peak of a parasitaemic wave and during remission of infection stumpy forms predominate. To determine whether this change in predominance might be caused by selective killing of slender forms, the fates of slender and stumpy form trypanosomes in two in vitro assays of immune-mediated killing were compared. Parasite populations in which > 90% of cells were of slender morphology were observed to be killed by antibody-dependent complement-mediated lysis approximately five times faster than populations in which < 15% of cells were slender and most were of intermediate or stumpy morphology. Quantification of the relationship between the proportion of slender forms in the population and the rate of lysis indicated that slender forms were killed approximately 7.3 times faster than other forms. In an opsonization assay, no differences were observed between slender and stumpy forms in the extent to which they attached to macrophages in an antibody-dependent manner. These results suggest that the change in proportions of slender and stumpy forms at the peak of a parasitaemic wave results from slender forms being more susceptible to complement-mediated killing as the antibody response develops.

Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei.

The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has been suggested that glycolysis is the sole source of ATP in all bloodstream forms. However, earlier results indicated that in the mitochondria of the transitional intermediate/short stumpy bloodstream forms, the biochemical pathways are present that could allow intra-mitochondrial production of ATP. Using a high mannitol buffer to enhance permeability, we confirm previous observations showing that transitional forms maintain motility and respiratory activity with 2-oxoglutarate as the sole substrate. Using a luminometer to measure intracellular ATP levels via the luciferin/luciferase chemiluminescence assay, we show that these same transitional forms, but not long slender forms, maintain high levels of intracellular ATP in the presence of 2-oxoglutarate. Further, in the presence of bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, ATP levels are reduced with subsequent death and lysis of the cells when 2-oxoglutarate, but not glucose, is used as sole substrate. These data are direct evidence of ATP production by transitional bloodstream form mitochondria.

Life Cycle of Leishmania major (Kinetoplastida: Trypanosomatidae) in the Neotropical Sand Fly Lutzomyia longipalpis (Diptera: Psychodidae)

The development of Leishmania major Yakimoff & Schokhor in the New World sand fly Lutzomyia longipalpis (Lutz & Neiva) was examined by light and electron microscopy. In this unnatural host, parasites differentiated into 10 typical morphological forms, multiplied at three sites, migrated anteriorly and established in the foregut, and attached to gut surfaces. In the blood meal, amastigotes divided and transformed into two successive dividing, stumpy promastigote stages. Elongate nectomonad promastigotes developed from stumpy forms and subsequently rounded up in some flies into paramastigotes and opisthomastigotes. Differentiation into round opisthomastigotes and the apparent fusion of paramastigotes in the blood meal were novel observations in this study. Three nectomonad promastigotes--elongate, short, and metacyclic--were free-swimming in the midgut lumen. Elongate nectomonad promastigotes were highly oriented in the midgut, with their flagella embedded between the epithelial microvilli. Short haptomonad promastigotes were the predominant form attached to the intima of the stomodeal valve, whereas pear-shaped haptomonad promastigotes and paramastigotes colonized surfaces of the esophagus and pharynx. Peripylarian attachment of promastigotes and paramastigotes in the pylorus, ileum, and colon was noted in 21% of flies, suggesting that suprapylarian leishmanias have not lost the ability to colonize the hindgut. L. longipalpis was a successful biological host for L. major, allowing complete development of the parasite.

Stage-specific differential polyadenylation of mini-exon derived RNA in African trypanosomes

The differentiation of African trypanosomes through several distinct stages in their mammalian host and insect vector correlates with differential expression of some of the parasites' genes. In the search for genes from Trypanosoma brucei brucei involved in switching from the actively dividing long slender to the non-dividing short stumpy bloodstream forms, we have isolated cDNA clones which hybridise specifically to mRNA from short stumpy forms. All clones that were characterised contained similar sequences. Northern blot analysis showed that: (i) RNA transcripts which hybridise to these clones are barely detectable in the poly(A) + fraction of RNA from longslender bloodstream forms and absent in procyclic culture forms, but are abundant in the poly(A) + fraction of RNA from short stumpy forms; (ii) the RNA transcripts are abundant in the poly(A) − fraction of RNA from all life cycle stages of the trypanosomes, without significant differences and, (iii) three transcripts of 160, 280 and 400 nucleotides in size are detected in the poly(A) + fraction of RNA, whereas only a single size-class of transcript of between 140 and 160 nucleotides is detectable in the poly(A) − fraction. Sequence analysis revealed that these clones correspond to mini-exon derived RNA containing a poly(A) tail at their 3′ end. The polyadenylation of the transcript is a post-transcriptional event since sequences from genomic DNA could not be amplified in the polymerase chain reaction when mini-exon and oligo(dT) nucleotide sequences were used as primers. The differences in size of the transcripts detected can be accounted for by variations in the poly(A) tail length.

Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor.

Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press).

Differential expression of the oligomycin-sensitive ATPase in bloodstream forms of Trypanosoma brucei brucei

We report the differential expression of the oligomycin-sensitive mitochondrial ATPase in pleomorphic bloodstream forms of Trypanosoma brucei brucei as observed with enzymatic assays and electron microscope histochemistry. As the cells differentiate from long slender to short stumpy forms, total specific activity of the mitochondrial ATPase in a crude mitochondrial fraction doubles and the oligomycin-sensitive specific activity increases 5-fold. Upon in vitro differentiation to procyclic forms, there is a further doubling of total specific activity and a further tripling of oligomycin-sensitive specific activity. The oligomycin-insensitive ATPase activity remained essentially constant throughout differentiation. We have attempted to characterize this oligomycin-insensitive activity utilizing inhibitors of several other ATPases.

Phosphorylation differences among proteins of bloodstream developmental stages of Trypanosoma brucei brucei

Early in an infection the bloodstream forms of the African trypanosome Trypanosoma brucei brucei are long, slender and rapidly dividing. Later, non-dividing, short, stumpy forms may be found. In this report we described biochemical differences between the two parasite populations in the phosphorylation of their proteins in vitro. Compared with the slender populations, the non-dividing stumpy forms of the parasites exhibit decreased phosphorylation of an 80 kDa protein and enhanced phosphorylation of 37 kDa and 42 kDa proteins (pp37 and pp42). These changes occurred regardless of whether the stumpy trypanosomes were generated naturally during the course of the infection or induced by difluoromethylornithine treatment. The phosphorylation of pp37 and pp42 occurs on serine and threonine residues and is totally dependent upon the presence of Mn2+ or Mg2+. However, excess Mn2+ or Mg2+ inhibits phosphorylation. Maximal phosphorylation of pp42 occurs with 1 mm-Mn2+ or 10 mm-Mg2+, whereas that of pp37 occurs with 50 mM-Mn2+ or greater than 100 mm-Mg2+. The phosphorylation of pp37 is greatly enhanced by KCl, whereas that of pp42 is only slightly increased by this salt. Ca2+, calmodulin, phospholipids and cyclic AMP have no discernible effect upon the phosphorylation of pp42 or pp37 in vitro, whereas heparin, suramin, polylysine, polyarginine and polyamines all inhibit phosphorylation. Thus the enzymes that phosphorylate pp42 and pp37 have properties similar to, but distinct from, those of mammalian casein kinase II. Since the casein-kinase-like activity is higher in the slender than in the stumpy forms, the enhanced phosphorylation of pp42 and pp37 in the non-dividing parasites is probably a result of the enhanced synthesis of these acidic proteins.

Mitochondrial development in Trypanosoma brucei brucei transitional bloodstream forms

Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F 1F 0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production.

Etude de la capacité vectorielle de &lt;em&gt;Glossina palpalis gambiensis&lt;/em&gt; (Bobo Dioulasso) vis-à-vis de &lt;em&gt;Trypanosoma brucei brucei&lt;/em&gt; EATRO 1125

A total of 440 teneral Glossina palpalis gambiensis received one single bloodmeal on a guinea pig infected chronically with Trypanosoma brucei brucei EATRO 1125. Metacyclic infections were present in 11.29% of the flies, in 2.32% infections were limited to procyclical stages. No significant difference in vectorial capacity was observed between male and female flies, the level of metacyclic infections being 13.19% in the former and 9.55% in the latter. The parasitaemia level, the percentage of stumpy forms at the moment of the blood meal and the maintenance conditions of the flies seemed to influence the infection of the flies.

Expression and deletion analysis of the Trypanosoma brucei rhodesiense cysteine protease in Escherichia coli

Trypanosoma brucei, the cause of African sleeping sickness, differentiates in the mammalian bloodstream from a long, slender trypanosome into a short, stumpy trypanosome. This event is necessary for infection of the tsetse fly and maintenance of the life cycle. We have previously shown that the stumpy form contains 10- to 15-fold-greater cysteine protease activity than either the slender form or the insect midgut procyclic, and we have isolated a cDNA encoding the protease. In order to determine whether the cDNA encodes the developmentally regulated cysteine protease, we have purified the protease from trypanosomes and have made a polyclonal antiserum against it. The trypanosomal protease gene was then expressed in Escherichia coli with three different methionines within the pre- and propeptides acting as initiation sites. In each case, a protein was synthesized that was recognized by an antiserum specific for the developmentally regulated trypanosomal cysteine protease. The protein synthesized from the more upstream initiation site within the propeptide was proteolytically active. The recombinant protease and the trypanosomal enzyme were identical with respect to peptide substrates and protease inhibitors. The protein remained active when synthesized in a truncated form lacking the nine consecutive prolines and carboxy-terminus extension, indicating that the terminal 108 amino acids are not necessary for proteolytic activity.

Multinuclear forms in a dyskinetoplastic strain ofTrypanosoma evansiin mice

The production of short stumpy and multinuclear trypanosomes in a Chinese strain of dyskinetoplastic Trypanosoma evansi maintained in rabbits and mice is described. Production of multinuclear trypanosomes was increased following passage through a reptile (gecko), in which the trypanosomes did not multiply, and transfer back to mice. The multinuclear trypanosomes showed more nuclei than flagella indicating that disruption of the normal cell cycle had taken place and not simply inhibition of cleavage. A Chinese kinetoplastic T. evansi treated similarly rarely produced stumpy or multinuclear forms.

A comparison of multiplication rates in primary and challenge infections ofTrypanosoma bruceibloodstream forms

The hypothesis that division of Trypanosoma brucei slender bloodstream forms is dependent upon the availability of a host-derived growth factor has been tested by superimposing challenge doses of slender-form trypanosomes onto preexisting infections at a time during the primary infection when stumpy forms predominated. The challenge populations grew in the doubly-infected mice indicating that depletion of a putative growth factor by the expanding population of the primary infection had not prevented division of the trypanosomes although slight reductions in multiplication rates were observed. This effect was independent of the variable antigen type (VAT) of the trypanosomes and of their stock of origin.

Differentiation of Trypanosoma brucei bloodstream trypomastigotes from long slender to short stumpy-like forms in axenic culture

An axenic cultivation system [10] was used to study the differentiation of Trypanosoma brucei bloodstream forms from long slender to short stumpy-like forms. Trypanosomes in the logarithmic phase are similar to long slender bloodstream forms freshly isolated from infected mice, differing only in the rate of oxygen uptake. In contrast, trypanosomes in the stationary phase show a decreased level of glucose oxidation, express pyrroline-5-carboxylate reductase (proline oxidase), are inhibited in oxygen uptake to about 44% by KCN, undergo considerable morphological changes on the cellular and subcellular level, and have a significantly smaller cell volume. These results are comparable to those observed during the differentiation of long slender to short stumpy forms in infected animals, suggesting that the differentiation process towards insect procyclic forms can be initiated in culture at 37°C. As judged from immunofluorescence and electron microscopy analysis, the surface coat remains intact.

Serum lipoprotein and Trypanosoma brucei brucei interactions in vitro

Trypanosoma brucei brucei IL3201 and IL3202, which are dependent on serum high or low-density lipoproteins to multiply under axenic culture conditions, acquired lipoprotein-associated 3H-lipids without binding, accumulating or degrading apolipoproteins. Uptake by the T. b. brucei of lipoprotein-associated [1α, 2α(n)- 3H]cholesterol, [1α, 2α(n)- 3H]cholesteryl linoleate, [1α, 2α(n)- 3H]cholesteryl oleoyl ether and l-3-phosphatidyl [ N-methyl- 3H]choline, 1,2-dipalmitoyl, occurred at 37°C but not at 0°C, and tended towards saturation with increasing concentrations of 3H-lipid-labelled lipoproteins in the incubation mixture. The uptake processes did not discriminate between high- or low-density lipoproteins, did not require exogenous divalent ions and were not inhibited by the presence of acidotropic agents (chloroquine, ammonium chloride) in the incubation mixture. Uptake by T. b. brucei of lipoprotein cholesterol was likely to result mainly from desorption and diffusion processes, whereas specific binding sites were probably involved in the uptake by T. b. brucei of lipoprotein cholesteryl linoleate, cholesteryl oleoyl ether and possibly phosphatidylcholine. Exponentially growing T. b. brucei hydrolysed cholesteryl linoleate to cholesterol and had only a small capacity to re-esterify cholesterol, whereas committed non-dividing stumpy form T. b. brucei had a large capacity to esterify cholesterol. Conversion products of phosphatidylcholine were generated during or after uptake of this phospholipid by exponentially growing T. b. brucei.

Physiological Activation of the Mitochondrion and the Transformation Capacity of DFMO Induced Intermediate and Short Stumpy Bloodstream form Trypanosomes

Bloodstream forms of Trypanosoma brucei brucei (EATRO 110) were cultured with 100 microM difluoromethylornithine (DFMO). After 48 hr, intracellular putrescine was depleted and cells were positive when histochemically stained for the mitochondrial marker enzyme, NAD diaphorase, and exhibited mitochondrial proliferation and cristae development when examined by electron microscopy. This suggested that the mitochondrion was undergoing the physiological transformation necessary for successful transmission of the bloodstream form to the vector, namely the initiation of development of a TCA cycle and cytochrome system. The short stumpy forms that appeared by day 4 of culture, although physiologically transformed, were not viable in so far as they were not capable of transforming to procyclic trypomastigotes when introduced into SDM-79 medium. When rats infected with T. b. brucei were given 4% (w/v) DFMO in their drinking water, they were cured within 72 hr. Trypanosomes removed from animals and stained for NAD diaphorase showed mitochondrial transformation, as well as an intermediate and short stumpy morphology, at 36 and 60 hr, respectively. Data from this study on the growth and transformation characteristics of the DFMO induced intermediate and short stumpy form trypanosomes supports the observation that the intermediate form, and not the short stumpy form, is able to successfully transform to procyclic trypomastigotes.

Elevated phosphoglycerate kinase mRNA but not protein in monomorphic Trypanosoma brucei: implications for stage-regulation and post-transcriptional control

Phosphoglycerate kinase (PGK) is present in high levels in the glycosomes of bloodstream stage Trypanosoma brucei, but is virtually absent in procyclic stage glycosomes. Glycosomes isolated from slender and slumpy stage bloodforms show similar levels of PGK, although levels are slightly lower in stumpy forms. Lower levels of glycosomal PGK transcripts are observed in stumpy form RNA, paralleling the decrease in glycosomal PGK activity. Monomorphic strains and pleiomorphic strains show similar glycosomal PGK activity, but monomorphic strains have much higher levels of the glycosomal PGK transcript. In three separate cases, predominantly monomorphic strains derived from highly pleiomorphic strains showed increased levels of glycosomal PGK (gPGK) mRNA. gPGK synthesis rates in monomorphic and pleiomorphic strains were similar, and no significant differences in turnover were observed. These data suggest the possibility of translational control of gPGK protein levels in trypanosome bloodforms. The data also indicate that the metabolism of gPGK mRNA in highly passaged laboratory strains is altered, and counsel caution when attributing differences in transcript levels to stage-specific regulation.

Trypanosome sociology and antigenic variation.

Survival of the trypanosome (Trypanosoma brucei) population in the mammalian body depends upon paced stimulation of the host's humoral immune response by different antigenic variants and serial sacrifice of the dominant variant (homotype) so that minority variants (heterotypes) can continue the infection and each become a homotype in its turn. New variants are generated by a spontaneous switch in gene expression so that the trypanosome puts on a surface coat of a glycoprotein differing in antigenic specificity from its predecessor. Homotypes appear in a characteristic order for a given trypanosome clone but what determines this order and the pacing of homotype generation so that the trypanosome does not quickly exhaust its repertoire of variable antigens, is not clear. The tendency of some genes to be expressed more frequently than others may reflect the location within the genome and mode of expression of the genes concerned and may influence homotype succession. Differences in the doubling time of different variants or in the rate at which trypanosomes belonging to a particular variant differentiate into non-dividing (vector infective) stumpy forms have also been invoked to explain how a heterotype's growth characteristics may determine when it becomes a homotype. Recent estimations of the frequency of variable antigen switching in trypanosome populations after transmission through the tsetse fly vector, however, suggest a much higher figure (0.97-2.2 x 10(-3) switches per cell per generation) than that obtained for syringe-passed infections (10(-5)-10(-7) switches per cell per generation) and it seems probable that most of the variable antigen genes are expressed as minority variable antigen types very early in the infection. Instability of expression is a feature of trypanosome clones derived from infective tsetse salivary gland (metacyclic) trypanosomes and it is suggested that high switching rates in tsetse-transmitted infections may delay the growth of certain variants to homotype status until later in the infection.

Growth of Pleomorphic Trypanosoma brucei rhodesiense in Irradiated Inbred Mice

It was shown that irradiation (650 rad) of 7 inbred strains of mice did not block the ability of Trypanosoma brucei rhodesiense to transform from the long slender (LS) to the short stumpy (SS) form or alter the plateau in parasitemia. In addition, it was observed that significant differences in parasitemia levels, in the rate of transformation from the LS to the SS form, as well as in the survival times occurred between the irradiated C3HeB/FeJ and several of the other strains. These differences in the nonspecific ability to control parasitemia appeared to be characteristic for each inbred strain of mice. The resistant strains generally had lower parasitemia than the susceptible strains. However, it was also shown that there is not a one-to-one correlation between the innate ability of a mouse strain to control its initial parasitemia, and the strain's ability to clear the parasitemia or increase its survival time. It was therefore concluded that the hypothesis which states that the ability of an animal to increase nonspecifically the rate of transformation, and therefore to lower the parasitemia, allowing intact animals to respond immunologically and survive longer is either incorrect or incomplete. The results further show that the ability of mice to clear their initial parasitemia by an antibody response is not necessarily correlated with their survival time. Therefore, this study suggests that factors other than an antibody response and the nonspecific control of parasitemia are important in resistance.

Developmental aspects of uridine addition within mitochondrial transcripts of Trypanosoma brucei.

The mitochondrial respiratory system is absent in slender bloodstream forms of Trypanosoma brucei, incomplete in stumpy bloodstream forms, and complete in procyclic (insect) forms. The steady-state abundance of transcripts of some mitochondrially encoded components of the respiratory system correlates with its differential expression in different life cycle stages. Recently, it was reported that uridines which are not encoded in the genome are added to cytochrome b and cytochrome oxidase II transcripts. We now report that the (U)+ transcripts of both genes are found in procyclic forms and to some degree in stumpy forms but are absent in slender forms. The uridine additions to cytochrome oxidase II correct a frameshift in the gene and presumably allow production of a full-length protein, whereas those added to cytochrome b create an in-frame AUG which extends the N terminus of the predicted protein by 20 amino acids. The stage specificity of uridine additions to these transcripts thus reflects the life cycle stage during which the protein products would be used. Transcripts of MURF2, a gene of unknown function, have additional uridines in both slender and procyclic forms which create two in-frame AUGs. MURF2 transcripts additionally differ from the DNA sequence in ways which cannot be explained by uridine addition alone, implying that other processes alter these transcripts.