Short Stumpy Research Articles

The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study.

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clinical symptoms manifested by the disease in the two environments are different. Information on whether blood stream are pathologically different from CNS trypanosomes is lacking. This study undertook to compare the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense ( Tbr) isolates in Swiss white mice. Methods: Donor mice infected with each of the five isolates were euthanized at 21 days post infection (DPI) for recovery of BSF trypanosomes in heart blood and CNS trypanosomes in brain supernatants. Groups of Swiss white mice (n = 10) were then infected with BSF or CNS forms of each isolate and monitored for parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics. Results: Amplification of SRA gene prior to trypanosome morphology and pathogenicity studies confirmed all isolates as T. b. rhodesiense. At 21 DPI, CNS trypanosomes were predominantly long slender (LS) while BSF were a mixture of short stumpy and intermediate forms. The density of BSF trypanosomes was on average 2-3 log-scales greater than that of CNS trypanosomes with isolate KETRI 2656 having the highest CNS trypanosome density. Conclusions: The pathogenicity study revealed clear differences in the virulence/pathogenicity of the five (5) isolates but no distinct and consistent differences between CNS and BSF forms of the same isolate. We also identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies.

The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clinical symptoms manifested by the disease in the two environments are different. Information on whether blood stream are pathologically different from CNS trypanosomes is lacking. This study undertook to compare the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense (Tbr) isolates in Swiss white mice. Methods: Donor mice infected with each of the five isolates were euthanized at 21 days post infection (DPI) for recovery of BSF trypanosomes in heart blood and CNS trypanosomes in brain supernatants. Groups of Swiss white mice (n = 10) were then infected with BSF or CNS forms of each isolate and monitored for parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics. Results: Amplification of SRA gene prior to trypanosome morphology and pathogenicity studies confirmed all isolates as T. b. rhodesiense. At 21 DPI, CNS trypanosomes were predominantly long slender (LS) while BSF were a mixture of short stumpy and intermediate forms. The density of BSF trypanosomes was on average 2-3 log-scales greater than that of CNS trypanosomes with isolate KETRI 2656 having the highest CNS trypanosome density. Conclusions: The pathogenicity study revealed clear differences in the virulence/pathogenicity of the five (5) isolates but no distinct and consistent differences between CNS and BSF forms of the same isolate. We also identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies.

Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry

BackgroundTrypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology.ResultsUsing Stable Isotope Labelling of Amino acids in Cell culture (SILAC) in combination with mass spectrometry we determined the abundance of >1600 proteins in the long slender (LS), short stumpy (SS) mammalian bloodstream form stages relative to the procyclic (PC) insect-form stage. In total we identified 2645 proteins, corresponding to ~30% of the total proteome and for the first time present a comprehensive overview of relative protein levels in three life stages of the parasite.ConclusionsWe can show the extent of pre-adaptation in the SS cells, especially at the level of the mitochondrial proteome. The comparison to a previously published report on monomorphic in vitro grown bloodstream and procyclic T. brucei indicates a loss of stringent regulation particularly of mitochondrial proteins in these cells when compared to the pleomorphic in vivo situation. In order to better understand the different levels of gene expression regulation in this organism we compared mRNA steady state abundance with the relative protein abundance-changes and detected moderate but significant correlation indicating that trypanosomes possess a significant repertoire of translational and posttranslational mechanisms to regulate protein abundance.

Cellular and Molecular Remodeling of the Endocytic Pathway during Differentiation of Trypanosoma brucei Bloodstream Forms

During the course of mammalian infection, African trypanosomes undergo extensive cellular differentiation, as actively dividing long slender (SL) forms progressively transform into intermediate (I) forms and finally quiescent G(1)/G(0)-locked short stumpy (ST) forms. ST forms maintain adaptations compatible with their survival in the mammalian bloodstream, such as high endocytic activity, but they already show preadaptations to the insect midgut conditions. The nutritional requirements of ST forms must differ from those of SL forms because the ST forms stop multiplying. We report that the uptake of several ligands was reduced in ST forms compared with that in SL forms. In particular, the haptoglobin-hemoglobin (Hp-Hb) complex was no longer taken up due to dramatic downregulation of its cognate receptor, TbHpHbR. As this receptor also allows uptake of trypanolytic particles from human serum, ST forms were resistant to trypanolysis by human serum lipoproteins. These observations allowed both flow cytometry analysis of SL-to-ST differentiation and the generation of homogeneous ST populations after positive selection upon exposure to trypanolytic particles. In addition, we observed that in ST forms the lysosome relocates anterior to the nucleus. Altogether, we identified novel morphological and molecular features that characterize SL-to-ST differentiation.

CpG Oligodeoxynucleotide Treatment Enhances Innate Resistance and Acquired Immunity to African Trypanosomes

Relative resistance to African trypanosomiasis is based on the development of a type I cytokine response, which is partially dependent on innate immune responses generated through MyD88 and Toll-like receptor 9 (TLR9). Therefore, we asked whether enhancement of the immune response by artificial stimulation with CpG oligodeoxynucleotide (ODN), a TLR9 agonist, would result in enhanced protection against trypanosomes. In susceptible BALB/c mice, relative resistance to infection was significantly enhanced by CpG ODN treatment and was associated with decreased parasite burden, increased cytokine production, and elevated parasite-specific B- and T-cell responses. In relatively resistant C57BL/6 mice, survival was not enhanced but early parasitemia levels were reduced 100-fold and the majority of the parasites were nondividing, short stumpy (SS) forms. CpG ODN treatment of lymphocyte-deficient C57BL/6-scid and BALB/cByJ-scid mice also enhanced survival and reduced parasitemia, indicating that innate resistance to trypanosome infection can be enhanced. In C57BL/6-scid and BALB/cByJ-scid mice, the parasites were also predominantly SS forms during the outgrowth of parasitemia. However, the effect of CpG ODN treatment on parasite morphology was not as marked in gamma interferon (IFN-gamma)-knockout mice, suggesting that downstream effects of IFN-gamma production may play a discrete role in parasite cell differentiation. Overall, these studies provide the first evidence that enhancement of resistance to African trypanosomes can be induced in susceptible animals in a TLR9-dependent manner and that CpG ODN treatment may influence the developmental life cycle of the parasites.

A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle.

Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of approximately 1 microM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.

A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle

Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of ∼1 μM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.

Loss of the mono-allelic control of the VSG expression sites during the development of Trypanosoma brucei in the bloodstream.

Transcription of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei occurs in a single of multiple polycistronic expression sites (ESs). Analysis of RNA from proliferative long slender (LS) bloodstream forms demonstrated that initiation of transcription occurs in different ESs, but inefficient RNA processing and elongation is linked to RNA polymerase arrest in all except one unit at a time. The pattern of ES transcripts was analysed during the transformation of dividing LS forms into quiescent short stumpy (SS) forms. The results demonstrated that the mono-allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady-state ES transcripts, particularly the VSG mRNA, were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run-on transcription analysis indicated that RNA polymerase was still fully associated with the promoter-proximal half of the 'active' ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the 'active' ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ. This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.

Role of the long slender to short stumpy transition in the life cycle of the african trypanosomes

It is shown using mouse models that the African trypanosomes exert a significant drain upon their host's carbohydrate (energy) resources; and that the higher the parasitemia, the greater the energy demand. It is, therefore, hypothesized that the long slender (LS) to short stumpy (SS) transition evolved, in part, to help control the parasitemia and to increase host survival time. It is also suggested that the SS population is heterogeneous. One part of the population is tsetse infective, while a second older SS population is undergoing apoptotic-like events, which leads to their cell death and their stimulation of the host's immune response. This immune stimulation by the old dying SS forms would eliminate the major LS and SS variant antigen population, and produce the chronic relapsing infection. It is concluded that the SS stages during the apoptosis-like process are acting altruistically. They give their lives to insure the long-term survival of the host, and to insure renewed growth of the minor LS variants and new infective SS forms. This process is predicted to increase the probability for the successful transmission of the trypanosomes to a new host.

Life after death: are trypanosomatids programmed to die for the survival of their partners?

In the year 1315, on the battle field of Morgarten, a Swiss hero named Winkelried, stabbed himself with lances he grabbed from the wall of armed Austrian soldiers. His sacrifice, was thus essential to open the way for the victory of the Swiss army. In his mind, there was a clear suicide plan under which action was taken towards a goal, that is, the survival of his colleagues. Such a suicide plan is found at the cellular level in metazoa, namely in their cell death program. Programmed cell death (PCD), is a suicidal pathway that has clear benefits for multicellular organisms, whereby, cells that are damaged, infected, or no longer required are eliminated. Considering the importance of PCD in metazoa, it is reasonable to ask if such a predetermined mechanism of behavior, or operations, exists in phylogenetically ancient, single cell organisms, such as, pathogenic trypanosomatids. In this issue, Seed and Wenck make comments relating to the role of the long slender (LS) to short stumpy (SS) transition in the life cycle of the African trypanosomes. The authors quote several different reports describing the presence of PCD in trypanosomatids. There is strong evidence to suggest that cell death in Trypanosoma exhibits some morphological features of PCD common to higher eukaryotes. However, these morphological changes are not yet definitive proof of a program or coded set of instructions. Thus far, genes, that are involved in metazoan PCD, have not been functionally characterized in trypanosomatids. Furthermore, it is likely that the Kinetoplastida genomes do not encode caspases, suggesting that, if programmed cell death does exist, it is not an apoptotic process but an apoptotic-like mechanism. Such apoptotic-like programs have already been described in higher eukaryotes. The observations that these death features are visible under stress or during differentiation of parasites into the infectious stages are, however, interesting. In the infectious stage, the parasite will encounter its host and be exposed to the hosts' immune system. From this point onwards, the parasite may either, survive by controlling or escaping the immune system of the host, or alternatively, be killed. In the latter case, it is advantageous for the parasite to expose phosphatidylserine and be phagocytized as for any metazoan cell undergoing death. In Leishmania mexicana, it has been shown that antigens can be presented to T cells by macrophages harbouring dead organisms but not by cells harbouring live parasites [1]. Such observations, however, do not imply, de facto, that the parasite is acting altruistically. In their analysis on the role of LS and SS forms of T. brucei, Seed and Wenck conclude that some parasites of >...and also >. According to the authors' conclusion, one ends up with the feeling that some short stumpy (SS) forms of the African trypanosomes are programmed to die and that this program is altruistic. We do not contest the authors' comments concerning the different roles of the LS and SS forms, however, we feel that interpretation of the data concerning the altruistic role of the SS should be taken with more caution. There is strong evidence suggesting that quorum sensing exists in bacteria, therefore, cross-talk between protozoan parasites may also take place, and thereby controlling the virulence and genetic competence of the parasite. These processes are, however, designed for the survival of the pathogen and not for its programmed elimination. We therefore feel that the authors may have gone one step too far by asserting that the death of some SS parasites is due to an altruistic program. The question concerning the role of cell death in trypanosomatids is still pending. Some recent reports suggest that promastigotes of Leishmania parasites may undergo autophagy when exposed to specific peptides [2]. This process differs from the cell death program observed when parasites are exposed to stress conditions such as exposure to H2O2, suggesting that parasites could die using varying mechanisms. It is likely that the immune response of the host will then differ depending on which pathway is used. In conclusion, most of the ingredients are presently available to better understand the mechanism(s) of death in trypanosomatids. However, the pathways of cell death in these parasites require much further dissection. Once this has been achieved we may have some ideas to the definitive role of a cell death program(s) in trypanosomatids and its potential relevance to the immune response of the host.

Identification of a developmentally regulated iron superoxide dismutase of Trypanosoma brucei.

An iron superoxide dismutase (FeSOD) gene of the protozoan parasite Trypanosoma brucei has been cloned and its gene product functionally characterized. The gene encodes a protein of 198 residues which shows 80% identity with FeSODs from other trypanosomatids. Inhibitor studies with purified recombinant FeSOD expressed in Escherichia coli confirmed that the enzyme is an iron-containing SOD. The FeSOD is developmentally regulated in the parasite, expression being lowest in the cell-cycle-arrested, short stumpy bloodstream forms. Differential expression of the FeSOD protein contrasts with only minor quantitative changes in the FeSOD mRNA, indicating post-transcriptional regulation of the enzyme. As the level of FeSOD increases during differentiation of cell-cycle-arrested short stumpy into dividing procyclic forms, it is suggested that the enzyme is only required in proliferating stages of the parasite for the elimination of superoxide radicals which are released during the generation of the iron-tyrosyl free-radical centre in the small subunit of ribonucleotide reductase.

Identification of a developmentally regulated iron superoxide dismutase of Trypanosoma brucei

An iron superoxide dismutase (FeSOD) gene of the protozoan parasite Trypanosoma brucei has been cloned and its gene product functionally characterized. The gene encodes a protein of 198 residues which shows 80% identity with FeSODs from other trypanosomatids. Inhibitor studies with purified recombinant FeSOD expressed in Escherichia coli confirmed that the enzyme is an iron-containing SOD. The FeSOD is developmentally regulated in the parasite, expression being lowest in the cell-cycle-arrested, short stumpy bloodstream forms. Differential expression of the FeSOD protein contrasts with only minor quantitative changes in the FeSOD mRNA, indicating post-transcriptional regulation of the enzyme. As the level of FeSOD increases during differentiation of cell-cycle-arrested short stumpy into dividing procyclic forms, it is suggested that the enzyme is only required in proliferating stages of the parasite for the elimination of superoxide radicals which are released during the generation of the iron-tyrosyl free-radical centre in the small subunit of ribonucleotide reductase.

Trypanosoma brucei:Analysis of Cytoplasmic Ca2+during Differentiation of Bloodstream Stagesin Vitro

The highly regulated intracellular concentration of calcium (Ca2+) is a well-described regulator of diverse cellular events, including cell cycle control. In the present study we have addressed the regulation of cytosolic Ca2+in differentiation events in the life cycle of the protozoan parasiteTrypanosoma brucei.Bloodstream form (BSF) trypanosomes include the mitotically active long slender forms (LS) which differentiate to two nondividing stages—intermediate (INT) which transform into short stumpy (SS) forms. An axenicin vitroculture system was used to cultivate LS to a density greater than 1.0 × 106cells/ml/day. Populations of the intermediate BSF (INT) and SS were derived from cultured LS by treatment with difluoromethyl ornithine (DFMO, 100 μM) for 2 and 4 days, respectively. A semiquantitative reverse transcriptase-coupled polymerase chain reaction protocol (SQ-RT–PCR) was developed to objectively distinguish the three BSF by monitoring the relative levels of stage-specific mRNAs—cytochrome oxidase II (COXII), variant surface glycoprotein, and procyclin during the differentiation of LS to SS, showing an increase in COXII and procyclin mRNA expression during this process of differentiation. Basal cytosolic Ca2+levels [Ca2+]iof populations of LS, INT, and SS were studied using Indo-1 dual emission fluorometry. [Ca2+]iwas maximal in dividing LS cells and was shown to decrease coincidentally with early events in the process of differentiation to INT and SS. Thapsigargin (1 μM), reported to cause the release of Ca2+from the endoplasmic reticulum, elevated [Ca2+]iby about 30–60 nMin all BSF; however, the total thapsigargin-releasable stores decreased in parallel with the decrease in basal [Ca2+]i. Control treatments verified that elevations in [Ca2+]iin response to thapsigargin were intracellular in origin. These results may reflect the cessation of cytosolic Ca2+transients involved in the regulation of mitosis as the parasite exits from the cell cycle and differentiates from rapidly dividing LS to the nondividing SS.

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.

Mechanism of long slender (LS) to short stumpy (SS) transformation in the African trypanosomes.

The transformation of the long slender to the short stumpy stages of the African trypanosomes is an essential part of the trypanosome life cycle. Four possible mechanisms which could control this event have been investigated. It has been shown that (a) the dividing long slender to non-dividing short stumpy transition is not a programmed event in the trypanosome life cycle; nor (b) would it appear to be initiated by some form of cell to cell contact inhibition of growth. In addition, evidence is presented which would suggest that (c) the transition is not started by the depletion of a critical growth nutrient from the environment during the growth of the trypanosomes. The last possibility (d) considered is that during trypanosome growth, a growth inhibitor-short stumpy inducer accumulates in the trypanosomes' environment. Evidence is presented which shows that plasma from infected animals can inhibit the incorporation of thymidine by the trypanosomes. These data are consistent with the suggestion of an exogenous growth inhibitor accumulating during the infection.

Identification of a developmentally regulated cysteine protease of Trypanosoma brucei

Trypanosoma brucei undergoes dramatic metabolic changes during differentiation from the mammalian bloodstream form into the procyclic form of the insect midgut. Because modulation of protein degradation is likely to be important during this process we studied T. brucei for life cycle mediated proteolysis. We detected an increase in the activity of a 28 kDa protease as pleomorphic GUTat 3.1 trypanosomes differentiate in the mammalian bloodstream from long slenders into short stumpies. Short stumpy trypanosomes hydrolyse z-Phe-Arg-AMC 12 fold more actively than either long slenders or procyclics. The 28 kDa protease is activated by dithiothreitol and is inhibited by trans-epoxysuccinyl- l-leucyl-amido(4-guanidino) butane (E-64), indicating that it is a cysteine protease. The proteolytic activity of monomorphic ILTat 1.4 trypanosomes does not increase during mammalian parasitemia. If monomorphic ILTat 1.4 trypanosomes are induced to differentiate into short stumpies by exposure to difluoromethylornithine, however, the activity of the 28 kDa cysteine protease increases 8 fold. This suggests that polyamine depletion induces the 28 kDa cysteine protease and that is expression may be regulated by mechanisms not previously described in protozoa.

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.

Polyamine depletion following exposure to DL-alpha-difluoromethylornithine both in vivo and in vitro initiates morphological alterations and mitochondrial activation in a monomorphic strain of Trypanosoma brucei brucei.

DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.

Metabolic transformation of Trypanosoma rhodesiense in culture.

THE pathogenic African trypanosome, Trypanosoma rhodesiense, has three morphological cell types in the mammalian bloodstream—long slender (LS), intermediate short stumpy (ISS) and short stumpy (SS). The terminal respiration of the LS forms is independent of cytochrome pigments1 and is effected by L-glycerol-3-phosphate oxidase2 located in discrete extramitochondrial particles3. A functional citric acid cycle is absent4 and there is a promitochondrion in the form of a single canal with no cristae5. Vickerman5,6 has shown that in the transition of LS to SS forms, cristae and NADH-tetrazolium reductase activity develop within the mitochondrial tubule which may be related to the acquired ability of these stages to oxidize a-oxoglutarate5,7,8. These results suggested that a respiratory switch to intramitochondrial oxidation had occurred. Bowman et al.9, however, demonstrated that this switch is unlikely to be complete, for the SS forms do not possess all the enzymes of the citric acid cycle nor were cytochromes detectable. In contrast the culture (epimastigote) form derived from the SS congener has developed a functional citric acid cycle10, a cytochrome mediated electron transport system11 and a single complex mitochondrion6. It seems that mitochondrial activation is initiated in the mammalian bloodstream but is not completed until transfer into culture. This report describes the sequence of metabolic changes associated with the acquisition of mitochondrial mediated metabolism during transformation into culture forms.