An essential role for the Leishmania major metacaspase in cell cycle progression

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.     

Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.     

Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.     

The forms of the intercellular contacts of Leishmania in culture]

Two kinds of intercellular interactions have been observed in cultures of 12 investigated Leishmania species (L. major, L. tropica, L. donovani, L. infantum, L. sp. ZMA, L. mexicana, L. hertigi, L. braziliensis, L. tarentolae, L. adleri, L. gymnodactyli, L. gulickae). The first kind looks as adhesion of two specimens with their fore-ends. This way is characteristic of promastigotes of different morphotypes as well as of the interphase and dividing organisms to be most frequently seen in L. mexicana and L. gymnodactyli, and in both dark and lucid forms. The second kind of intercellular interactions involves a coupled adhesion of morphologically similar promastigotes with free ends of flagella. It is most characteristic of L. gymnodactyli and specially of dark promastigotes. Proves are provided that both the kinds of cell interactions are not associated with cell division, that they may only partially be connected with the phenomenon of rosette formation, and that they represent different phenomena. It is supposed that the intercellular contacts with the fore-ends may reflect gene exchanges in two partners, with a possible involvement of the kinetoplast DNA.     

Anisomorphic cell division by African trypanosomes

In the bloodstream of a mammalian host, African trypanosomes are pleomorphic; the shorter, non-proliferative, stumpy forms arise from longer, proliferative, slender forms with differentiation occurring via a range of morphological intermediates. In order to investigate how the onset of morphological change is co-ordinated with exit from the cell cycle we first characterized slender form cell division. Outgrowth of the new flagellum was found to occur at a linear rate, so by using outgrowth of the new flagellum as a temporal marker of the cell cycle we were able determine the order in which single copy organelles (nucleus, kinetoplast and mitochondrion) were segregated. We also found that flagellar length was an effective marker of the slender to stumpy differentiation and were, therefore, able to study both cell division and differentiation. When these differentiating cells were compared to cells undergoing proliferative cell division, they were found to be anisomorphic – showing discernible differences not only in the length of their new flagella but also in the shape and size of the cells and their nuclei.     

A simple procedure for the preparation of pure kinetoplast DNA network free of nuclear DNA from the kinetoplast hemoflagellate Leishmania donovani

A simple, inexpensive procedure for preparing pure kinetoplast DNA network from Leishmania donovani is described. L. donovani promastigotes were lysed by incubating with pronase in presence of sodium dodecylsulfate. Crude kinetoplast DNA networks were obtained by centrifugation of the lysate through a 20% sucrose solution. The pellet containing kinetoplast DNA was deproteinized by phenol extraction. Contaminating nuclear DNAs were removed by denaturation with alkali, neutralization, and addition of polyethylene glycol-8000 to a concentration of 10% to facilitate precipitation of kinetoplast DNA. kDNA isolated after centrifugation was deproteinized several times with phenol and finally precipitated with ethanol. The average yield by this procedure is 30-50 micrograms of kDNA per gram of wet cells. By slot-blot hybridization with a nuclear DNA probe, no nuclear DNA contamination of the kDNA networks could be detected.     

Introductory Remarks: Symposium on Differentiation of Trypanosomatids

ABSTRACT. Leishmania differentiation in the gut of phlebotomine sand flies was evaluated based on five light and electron microscopic studies of natural (Leishmania panamensis/Lutzomyia gomezi, Leishmania chagasi/Lutzomyia longipalpis) and unnatural (Leishmania mexicana/Lutzomyia abonnenci, Leishmania panamensis/Phlebotomus papatasi, Leishmania major/Lutzomyia longipalpis) life cycles. In the bloodmeal, transformation of amastigotes into stumpy promastigotes occurred before or during division. Further division in pairs or rosettes resulted in the development of spatulate and/or elongate nectomonad (free-swimming) promastigotes. Elongate, short, and metacyclic nectomonad promastigotes, and nectomonad paramastigotes were present in the midgut lumen. Dividing short promastigotes predominated in the cardia, and appeared to generate metacyclic forms which were observed in three life cycles. Haptomonad (attached) forms of Leishmania panamensis in the hindgut were primarily spatulate promastigotes (natural host) or pear-shaped promastigotes (unnatural host); paramastigotes and dividing forms were rare. At the stomodeal valve, short haptomonad promastigotes predominated in unnatural hosts, while both short and pear-shaped haptomonads were abundant, along with paramastigotes in natural hosts. Haptomonad paramastigotes and pear-shaped promastigotes colonized the esophagus, while paramastigotes predominated in the pharynx. Metacyclics were free-swimming in the lumen of the foregut.     

A PHO80-like cyclin and a B-type cyclin control the cell cycle of the procyclic form of Trypanosoma brucei

Cyclins bind and activate cyclin-dependent kinases that regulate cell cycle progression in eukaryotes. Cell cycle control in Trypanosoma brucei was analyzed in the present study. Genes encoding four PHO80 cyclin homologues and three B-type cyclin homologues but no G1 cyclin homologues were identified in this organism. Through knocking down expression of the seven cyclin genes with the RNA interference technique in the procyclic form of T. brucei, we demonstrated that one PHO80 homologue (CycE1/CYC2) and a B-type cyclin homologue (CycB2) are the essential cyclins regulating G1/S and G2/M transitions, respectively. This lack of overlapping cyclin function differs significantly from that observed in the other eukaryotes. Also, PHO80 cyclin is known for its involvement only in phosphate signaling in yeast with no known function in cell cycle control. Both observations thus suggest the presence of simple and novel cell cycle regulators in trypanosomes. T. brucei cells deficient in CycE1/CYC2 displayed a long slender morphology, whereas those lacking CycB2 assumed a fat stumpy form. These cells apparently still can undergo cytokinesis generating small numbers of anucleated daughter cells, each containing a single kinetoplast known as a zoid. Two different types of zoids were identified, the slender zoid derived from reduced CycE1/CYC2 expression and the stumpy zoid from CycB2 deficiency. This observation indicates an uncoupling between the kinetoplast and the nuclear cycle, resulting in cell division driven by kinetoplast segregation with neither a priori S phase nor mitosis in the trypanosome.     

Myriocin,a Serine Palmitoyltransferase Inhibitor,Blocks Cytokinesis in Leishmania (Viannia) braziliensis Promastigotes

We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log‐phase promastigotes treated with 1 μM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 μM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 μM myriocin‐treated parasites, respectively. High percentage of myriocin‐treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 μM myriocin, respectively). Transmission electron microscopy following treatment with 1 μM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3‐ketodihidrosphingosine to myriocin‐treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid‐free mutant, where addition of ethanolamine rescue wild‐type parasite characteristics.     

Fine Structure and Cytochemistry of the Nucleus and the Kinetoplast of Epimastigotes of Trypanosoma cruzi1

ABSTRACT. Ultrastructural cytochemical techniques were used to analyze the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi. With the use of ethanolic phosphotungstic acid, which detects basic proteins, reaction product was seen in the chromatin and at the periphery of the kinetoplast. Thallium alcoholate, which interacts with DNA, stained strongly the whole kinetoplast and the chromatin. With the use of a silver impregnation method that detects acidic nucleolar proteins, silver granules were seen preferentially located in the central region of the nucleolus. With the EDTA method, which reveals the presence of ribonucleoproteins, staining was observed in the nuclear pores. Also 6–8 nm fibrils, 25 nm and 40 nm granules, which correspond to the perichromatin fibers, interchromatin granules and the perichromatin granules, respectively, were identified in the nucleus. The EDTA method also revealed the presence of 40 nm granules in the kinetoplast. These granules were seen mainly at the two extremities of the kinetoplast. Freeze-fracture images indicate that the nuclear membrane contains ca. 9 pores/μm² of nuclear surface area. The mean diameter of the pores was 80 nm. All these results suggest that epimastigotes of T. cruzi have a very active nucleus and a high rate of nucleocytoplasmic interchange.     

The Cell Cycle in Crithidia fasciculata. Temporal Relationships between Synthesis of Deoxyribonucleic Acid in the Nucleus and in the Kinetoplast1, 2

SYNOPSIS. Autoradiographic technics with tritium-labeled thymidine have been used to determine G1, S, G2 and D for the kinetoplast and the nucleus of Crithidia fasciculata at 15, 25 and 32 C. The kinetoplast completes division before the nucleus at all 3 temperatures. The S phases of both organelles occur in approximate synchrony and are approximately equal in length but the nucleus begins and completes S before the kinetoplast at the 2 lower temperatures. This relationship is reversed at 32 C. Most of the effect of temperature on generation time is due to its effect on the length of S. The results are compared with similar studies on C. luciliae, Trypanosoma mega, other protozoa and tissue cells in culture. The role of the approximate synchrony of nuclear and kinetoplastic cycles in maintenance of the kinetoplastic condition is discussed and the hypothesis is proposed that this synchrony results from the sharing by nucleus and kinetoplast of the same mechanism for the production of the deoxyribonucleotides used in replication of their respective DNAs.     

Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes.

In situ hybridization on cultured promastigotes and sandfly smears were performed with nonradioactively labeled total DNA and recombinant DNA probes containing minicircle kinetoplast DNA (kDNA) or nuclear DNA inserts. Total DNA probes lack specificity whereas recombinant nuclear DNA probes work only if they contain repetitive sequences. Minicircle kDNAs of five Leishmania isolates, representative of five Leishmania taxa found in Kenya, were sequenced. Comparison of the sequences showed a 150-bp region with around 80% homology, whereas the rest of the minicircles had about 50% homology. Nevertheless, application of these probes in in situ hybridization assays as tested on Leishmania promastigotes in the vector gave good specificity and hybridization signal. Two types of labeling were tested: incorporation of biotin-labeled dUTP or directly horseradish peroxidase (HRP)-labeled nucleotides. Both techniques provided good sensitivity and signal-to-noise ratio on cultured promastigotes. Hybridization with HRP-labeled kDNA probes gave a superior signal-to-noise ratio if tested on sandfly preparations. This method provided a reliable and fast identification and facilitated the detection of promastigotes in sandflies. The technique presented here may be helpful in rapid identification of Leishmania promastigotes, and thus make epidemiological studies easier and less time consuming.     

Interacting protein kinases involved in the regulation of flagellar length

A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis.     

Cytochemical study of nucleic acids in relation to the polymorphism of the trypomastigote forms of Trypanosoma cruzi

Four morphological types of T. cruzi trypomastigotes are distinguished in mouse blood. These differ in RNA contents, in the distribution pattern of RNA in the cytoplasm, in the intensity of the Feulgen reaction and the topography of DNA in the nucleus, and in the contents and distribution of both the nucleic acids in the kinetoplast. Among the trypomastigotes examined, forms C and S differ at a lesser degree, than their slender and middle variants differing much stronger. The early slender trypomastigotes are characterized by poor and diffuse RNA in the cytoplasm, by a homogeneous distribution of DNA in the nucleus and by a low content of DNA (sometimes RNA) in the kinetoplast. The middle trypomastigotes, dominating at the final step of the infection, are rich in granular RNA, differ (despite their inability to divide) in their nuclear organization mostly characterized by a large karyosome and uneven distribution of chromatin at the periphery; the kinetoplast is rich in DNA and often contains RNA. The peak of trypomastigotes with the kinetoplast deprived of obviously stained RNA precedes the impetuous increase of parasitemia. It coincides with the decrease in the number of destroyed parasites, and with the active substitution of slender variants by the middle ones within both C- and S-forms. Thus, changes in the nucleus and kinetoplast are involved in the trypomastigote transformation and in the development of infection.     

Isolation of the 66-kilodalton polypeptide from promastigotes of Leishmania donovani as a ligand molecule for binding to macrophages.

A 66-kDa major plasma membrane-associated molecule of promastigotes of Leishmania donovani (UR6) was purified by affinity chromatography. The immunoreactivity of the 66-kDa molecule was lost upon exposure to heat or treatment with trypsin. The metaperiodate oxidation significantly reduced its immunoreactivity. The 66-kDa molecule is, therefore, glycoprotein in nature. With a fluorescent probe, the 66-kDa molecule was found to be located on the tip of flagellum and on the kinetoplast. The exposure of promastigotes of L. donovani to monospecific anti-66-kDa antibodies significantly reduced the percentage of macrophages with attached promastigotes in the cultured cell line (J774G8). The data suggested that promastigotes of L. donovani utilize the 66-kDa molecule in recognizing and as ligand for binding to macrophages.     

Effect of Berenil on Growth,Mitochondrial DNA and Respiration of Leishmania tarentolae Promastigotes*

SYNOPSIS Incubation of Leishmania tarentolae promastigotes in 1.0 μg/ml Berenil for 96 hr resulted in 33% inhibition of cell growth and 42.5% dyskinetoplasty in the cell population. The buoyant density of kinetoplast DNA (kDNA), ρ= 1.703 g/ml, remained unchanged after 96-hr exposure to the drug. Endogenous respiration as well as proline- and glucose-induced respiration dropped markedly after 36-hr exposure to Berenil. This drop occurred 12 hr before the onset of dyskinetoplasty, a result which suggests that this drug adversely affects mitochondrial respiratory activity of the promastigotes.     

Two New Subspecies of Herpetomonas muscarum (Leidy, 1856) Kent, 1880

SYNOPSIS. Herpetomonas muscarum muscarum n. subsp. was isolated from Musca domestica L. In culture at 20 C it assumed the opisthomastigote (up to 15%), double-flagellate and flagellate promastigote forms. At 30 C or with 4% urea added to cultures at 20 C, the proportion of opisthomastigotes was greater (up to 40%). In experimentally infected flies only transient infections, which included both opisthomastigotes and promastigotes, occurred. The promastigotes were 15–30 μ long and the kinetoplast was small and subspherical or transversely elongate. H. muscarum ingenoplastis n. subsp. was isolated from Phormia regina (Meigen). In culture at 20 C almost all individuals were double-flagellate promastigotes 20–40 μ long and less than 1% were opisthomastigotes. At 30 C or with added urea there was no increase in the proportion of opisthomastigotes and the cultures were not vigorous. In experimentally infected flies opisthomastigotes were 5–39% of the population depending on the part of the gut sampled. In all stages the kinetoplast was large (1.5–2.5 μ long) and tear-drop-shaped with the point directed posteriorly.
In artificially mixed cultures of H. m. muscarum and H. m. ingenoplastis the former predominated after a short time and eventually survived alone. A mixed culture that was about 98% H. m. muscarum was fed to Phormia regina and produced heavy pure infections of H. m. ingenoplastis , which lasted for 22 days with no indication of decline. No evidence of cyst formation was found in either subspecies.     

Cytochemical study of free-living flagellates of suborder Bodonina. I. Morphology and nucleic acids]

Nucleic acids (DNA and RNA) have been discovered in the kinetoplast of free-living Bodonina: Bodo caudatus, Pleuromonas jaculans, Rhynchomonas nasuta--by means of cytochemical methods. The kinetoplast has variable contents of nucleic acids whose chemoarchitectonics is due to their non-homogeneous distribution within the kinetoplast. The Feulgen reaction in the kinetoplast is more intensive than in the nucleus. Kinetoplast is closely connected with the cytoplasmic RNA metabolism. Many individuals of R. nasuta were found to have two kinetoplasts, no other signs of cell division being observed. P. jaculans has up to 45% of dyskinetoplastic forms.