Reduced Growth of Blastocrithidia culicis and Crithidia oncopelti Freed of Intracellular Symbiotes by Chloramphenicol*

The intracellular symbiotes of Blastocrithidia culicis and Crithidia oncopelti can be eliminated from cultures of the flagellates by a single chloramphenicol (CAP) treatment. Effective dosages were determined to be 0.01–0.08% (w/v) CAP after a treatment for 2 weeks or more for B. culicis and 0.08% (w/v) after 1 month for C. oncopelti in most cases. Ineffective dosages only lowered the numbers of symbiote-bearing flagellates. Growth of both species of flagellates in the presence of CAP was reduced in proportion to the drug concentration. Repeated subcultures at effective dosages yielded symbiote-free flagellates, which maintained a low level of growth rate. After repeated subcultures at ineffective dosages, the growth rate rose and the symbiote-bearing cells, initially very few, increased in number. The lowest effective dosages proved to be marginal, often producing symbiote-free cultures, but occasionally cultures with a few symbiote-bearing cells. After repeated subcultures at these drug concentrations, symbiote-containing cultures grew faster than the symbiote-free cultures. Hence, the symbiotic bacteria benefit the growth of their hosts, perhaps by supplying essential factors that are inadequate even in a rich blood medium.     

Symbiote-Free Hemoflagellates,Blastocrithidia culicis and Crithidia oncopelti: their Liver Factor Requirement and Serologic Identity*

SYNOPSIS. Several aposymbiotic strains of Blastocrithidia culicis and Crithidia oncopelti were cultivated in Trager's chemically defined medium as well as in a blood broth, both supplemented with 0.25% (v/v) liver extract concentrate. For all such strains, the liver extract was found to serve as an essential growth factor in the defined medium and as growth promoting additive in the blood broth. The active molecules were found to be water-soluble, heat stable, dialyzable, and probably nonlipid fractions. Antisera were developed in rabbit against all the available aposymbiotic strains. An almost total cross-reactivity at very high titers was observed in reciprocal agglutination test using strains with and without the bacterial symbiotes. These results indicate that the loss of the symbiotes does not affect the antigenic identity of B. culicis and C. oncopelti.     

Ultrastructural visualization of lipids in trypanosomatids

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis. T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.     

Ultrastructure of Symbiotic Bacteria in Normal and Antibiotic-Treated Blastocrithidia culicis and Crithidia oncopelti*

SYNOPSIS. An electron microscope study of diplosomes in Blastocrithidia culicis and bipolar bodies in Crithidia oncopelti has shown that both entities appear to be intracellular symbiotes and have a similar fine structure. They are enclosed by 2 unit membranes which are separated by a large space of very low density. The outer membrane is derived probably from the host cell. The matrix of the symbiotes is composed of dense ribosome-like particles and of areas of low density containing fine fibrillae. The particles are of the same size as ribosomes in bacteria and the fibrils have the characteristics of bacterial DNA. Thus, the lucid areas with fibrillae correspond to the nucleoids in bacteria. These observations suggest that the symbiotes are bacteria. The effect of chloramphenicol (CAP) and penicillin G (PCL) on these symbiotic bacteria was studied by culturing the host flagellates in media containing the antibiotics. The effect was analyzed at different intervals after the treatment by electron microscopy. After single treatment in the blood broth containing 0.08% (w/v) CAP, symbiotes appeared to have enlarged nucleoids, became deformed and eventually degenerated. In Grace's medium (supplemented with 10% fetal bovine serum) containing 0.6 or 2.4% (w/v) PCL, symbiotes of C. oncopelti remained unaltered, whereas some symbiotes of B. culicis became pleomorphic. Symbiotes of both species persisted after repeated transfers in PCL media and reverted to normal forms when transferred to PCL-free media. Sensitivity of symbiotes to CAP provides further evidence of their bacterial nature. The effect of PCL on the symbiotes of B. culicis suggests the presence in their cell envelopes of mucopeptide, which probably provides rigidity for maintaining the bacterial shape of the symbiotes.     

Ein Beitrag zur axenischen Kultur von zwei insektenbewohnenden Trichomyceten

Zusammenfassung Die TrichomycetenSmittium inopinatum undS. culicis lassen sich erfolgreich auf bei 80°C koagulierten Hühnereidotter kultivieren. Wenn die bei Zimmertemperatur 7 Tage gewachsenen Kulturen anschliessend bei 4–6°C aufgehoben werden, genügt es, Weiterimpfungen nur alle 4 bis 8 Wochen vorzunchmen.S. culicis l?sst sich unter diesen Bedingungen auch auf einem Malzextrakt-Peton-Agar halten.

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Summary Hen's egg yolk coagulated at 80°C proved to be a very suitable culture medium for the TrichomycetesSmittium inopinatum andS. culicis. After growing these cultures for 7 days at room temperature they are subsequently stored at 4–6°C. By this method its is sufficient to make subcultures only at intervals of 4–8 weeks. Under such conditionsS. culicis can be cultivated also on a malt extract peptone agar. — It is possible to storeS. inopinatum for several months at 4° C in soil cultures or in an aqueous solution of 0.675% NaCl.

     

Conserved repeats in the kinetoplast maxicircle divergent region of Leishmania sp. and Leptomonas seymouri

The maxicircle control region [also termed divergent region (DR)] composed of various repeat elements remains the most poorly studied part of the kinetoplast genome. Only three extensive DR sequences demonstrating no significant similarity were available for trypanosomatids (Leishmania tarentolae, Crithidia oncopelti, Trypanosoma brucei). Recently, extensive DR sequences have been obtained for Leishmania major and Trypanosoma cruzi. In this work we have sequenced DR fragments of Leishmania turanica, Leishmania mexicana, Leishmania chagasi and two monogenetic trypanosomatids Leptomonas seymouri and Leptomonas collosoma. With the emergence of the additional extensive sequences some conserved features of DR structure become evident. A conserved palindromic sequence has been revealed in the DRs of the studied Leishmania species, L. seymouri, and T. cruzi. The overall DR structure appears to be similar in all the Leishmania species, their relative L. seymouri, and T. brucei: long relatively GC-rich repeats are interspersed with clusters of short AT-rich repeats. C. oncopelti, L. collosoma, and T. cruzi have a completely different DR structure. Identification of conserved sequences and invariable structural features of the DR may further our understanding of the functioning of this important genome fragment.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession numbers DQ107351, DQ107352, DQ107354-DQ107358, DQ239759-DQ239765, DQ492251-DQ492256.     

Use of glycoconjugates for trypanosomatid taxonomy

Glycoconjugates from five trypanosomatid genera—Crithidia, Herpetomonas, Endotrypanum, Leishmania, and Trypanosoma—were extracted with Triton X-114 and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by periodic acid-Schiff staining. Most of the glycoconjugates were detected in the hydrophobic phase, indicating the presence of anchored glycoconjugates. All the trypanosomatids expressed a glycoconjugate with a low molecular weigh (below 20 kDa) in this phase. In each species, however, a characteristic and specific pattern of glycoconjugates was also observed in both phases. In the hydrophobic phase: 14–29 kDa lycoconjugates in C. guilhermei; 24–70 kDa in C. fasciculata, C. luciliae, E. schaudinni, and T. cruzi Y and G strains; 45–66 kDa in C. oncopelti and H. samuelpessoai; above 36 kDa in T. dionisii; 20–24 kDa, 36–45 kDa, and 70 kDa in L. tarentolae and T. mega. In the hydrophilic phase, typical glycoproteins were observed in some trypanosomatids: 60 kDa in T. mega and T. cruzi Y strain; 70 kDa in H. samuelpessoai; 66 kDa in C. oncopelti; 20–70 kDa in C. luciliae. These findings suggest that Triton X-114-extracted glycoconjugates could be useful markers for trypanosomatid taxonomy.     

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy

Summary The usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.     

Lipid droplets as ubiquitous fat storage organelles in <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

Lipid droplets are a class of eukaryotic cell organelles for storage of neutral fat such as triacylglycerol (TAG) and cholesterol ester (CE). We and others have recently reported that lysosome-related organelles (LROs) are not fat storage structures in the nematode C. elegans. We also reported the formation of enlarged lipid droplets in a class of peroxisomal fatty acid β-oxidation mutants. In the present study, we seek to provide further evidence on the organelle nature and biophysical properties of fat storage structures in wild-type and mutant C. elegans.     

The perilipin‐like PPE15 protein in Mycobacterium tuberculosis is required for triacylglycerol accumulation under dormancy‐inducing conditions

Mycobacterium tuberculosis (Mtb) causes latent tuberculosis infection in one‐third of the world population and remains quiescent in the human body for decades. The dormant pathogen accumulates lipid droplets containing triacylglycerol (TAG). In mammals, perilipin regulates lipid droplet homeostasis but no such protein has been identified in Mtb. We identified an Mtb protein (PPE15) that showed weak amino acid sequence identities with mammalian perilipin‐1 and was upregulated in Mtb dormancy. We generated a ppe15 gene‐disrupted mutant of Mtb and examined its ability to metabolically incorporate radiolabeled oleic acid into TAG, accumulate lipid droplets containing TAG and develop phenotypic tolerance to rifampicin in two in vitro models of dormancy including a three‐dimensional human granuloma model. The mutant showed a significant decrease in the biosynthesis and accumulation of lipid droplets containing TAG and in its tolerance of rifampicin. Complementation of the mutant with a wild‐type copy of the ppe15 gene restored the lost phenotypes. We designate PPE15 as mycobacterial perilipin‐1 (MPER1). Our findings suggest that the MPER1 protein plays a critical role in the homeostasis of TAG ‐containing lipid droplets in Mtb and influences the entry of the pathogen into a dormant state.     

Identification and partial characterization of plasma membrane polypeptides of Crithidia guilhermei,crithidia deanei and Crithidia oncopelti

• 1.1. Components of the cell surface of Crithidia guilhermei, Crithidia deanei and Crithidia oncopelti were radioiodinated by the iodogen technique. The distribution of proteins in the detergent-poor (DPP) and detergent-enriched phase (DRP) were studied using a phase separation technique in Triton X-114 and one- and two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulphate (1D and 2D SDS-PAGE).
• 2.2. Significant differences were noted in the proteins present in the DRP when the three species were compared.
• 3.3. Two major bands with mol. wt 28,000 and 56,000 and motility in the pH gradient of 7.4 and 6.3, respectively, were observed in C. guilhermei, but not discernible in C. deanei and C. oncopelti.
• 4.4. One polypeptide with mol. wt 50,000 and p1 4.9 was identified in the DRP of C. deanei.
• 5.5. A broad band with mol. wt 68,000–140,000 and pI 4.7–5.5 was clearly observed in the DRP of C. deanei and one or two polypeptides only present in the DPP were observed in the three Crithidia species analyzed.
• 6.6. Our observations show that C. guilhermei has characteristic surface polypeptides not found in C. deanei and C. oncopelti.
• 7.7. Our results, in association with those reported by others, show that the phase separation using Triton X-114 offers a simple approach to the separation and further analysis of a select group of proteins from the bulk of the cellular proteins.

     

Autophagy genes are required for normal lipid levels in C. elegans

Autophagy is a cellular catabolic process in which various cytosolic components are degraded. For example, autophagy can mediate lipolysis of neutral lipid droplets. In contrast, we here report that autophagy is required to facilitate normal levels of neutral lipids in C. elegans. Specifically, by using multiple methods to detect lipid droplets including CARS microscopy, we observed that mutants in the gene bec-1 (VPS30/ATG6/BECN1), a key regulator of autophagy, failed to store substantial neutral lipids in their intestines during development. Moreover, loss of bec-1 resulted in a decline in lipid levels in daf-2 [insulin/IGF-1 receptor (IIR) ortholog] mutants and in germline-less glp-1/Notch animals, both previously recognized to accumulate neutral lipids and have increased autophagy levels. Similarly, inhibition of additional autophagy genes, including unc-51/ULK1/ATG1 and lgg-1/ATG8/MAP1LC3A/LC3 during development, led to a reduction in lipid content. Importantly, the decrease in fat accumulation observed in animals with reduced autophagy did not appear to be due to a change in food uptake or defecation. Taken together, these observations suggest a broader role for autophagy in lipid remodeling in C. elegans.     

Morphology of female reproductive system of Mediterranean flatheaded peachborer,Capnodis tenebrionis (Linnaeus, 1761) (Coleoptera: Buprestidae)

Capnodis tenebrionis causes damage in many species of Rosaceae. The present study investigates on the morphology of the female reproductive system of C. tenebrionis. The female reproductive system of C. tenebrionis has a pair of ovaries, lateral oviducts, a common oviduct, spermatheca, and bursa copulatrix. Each ovary in C. tenebrionis consists of approximately 24 telotrophic meroistic type ovarioles. The ovarioles of C. tenebrionis have four regions (terminal filament, tropharium, vitellarium, and pedicel). Tropharium have trophocytes, young oocytes, and prefollicular cells. Vitellarium consists of previtellogenic, vitellogenic, and choriogenic oocytes. Previtellogenic oocyte is surrounded by cylindrical epithelial cells. Its ooplasm is homogeneous and basophilic. In vitellogenic oocyte, there are intercellular spaces between monolayered follicle cells. Its ooplasm has yolk granules and lipid droplets. Choriogenic oocyte are surrounded by chorion and single-layered cylindrical cells. There are yolk granules and lipid droplets in its ooplasm which is asidophilic. In C. tenebrionis female, spermatheca and bursa copulatrix wall is surrounded by thin cuticular intima, monolayer epithelial, glandular cells, and muscle layer. Spermatheca lumen contains a large number of spermatozoa. Bursa copulatrix lumen is filled with secretory material. This study may be useful in terms of the morphology of mature female reproductive organs of Buprestidae and other coleopteran species.     

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P < 0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol + vitrification, 69.98%) and Taxol pretreatment significantly (P < 0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 μm). The number of lipid droplets (5–10 μm in diameter) in vitrified oocytes pretreated with Taxol was higher (P < 0.05) than that in the oocytes without Taxol pretreatment (81.87 ± 13.63 vs. 64.27 ± 13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group.In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.     

Cloning and characterization of a new gene from the PAT protein family,in a marsupial,the stripe‐faced dunnart (Sminthopsis macroura)

Recent studies of PAT proteins in Drosophila and Xenopus have revealed significant roles for this family of proteins in the polarized transport of lipid droplets and maternal determinants during early embryogenesis. In mammals, PAT proteins are known to function mainly in lipid metabolism, yet research has yet to establish a role for PAT proteins in mammalian embryogenesis. Oocytes and early cleavage stages in Sminthopsis macroura show obvious polarized cytoplasmic distribution of organelles, somewhat similar to Drosophila and Xenopus, suggesting that a PAT protein may also be involved in S. macroura embryonic development. In the present study, we identified a new marsupial gene for PAT family proteins, DPAT, from S. macroura. Expression analyses by RT‐PCR and whole mount fluorescent in situ hybridization revealed that DPAT expression was specific to oocytes and cleavage stage conceptuses. Analysis of the localization of lipid droplets during S. macroura early embryonic development found a polarized distribution of lipid droplets at the two‐ and four‐cell stage, and an asymmetric enrichment in blastomeres on one side of conceptuses from two‐ to eight‐cell stage. Lipid droplets largely segregate to pluriblast cells at the 16‐cell stage, suggesting a role in pluriblast lineage allocation. Mol. Reprod. Dev. 77: 373–383, 2010. © 2010 Wiley‐Liss, Inc.     

OSMIUM STAINING OF ENDOPLASMIC RETICULUM AND MITOCHONDRIA IN THE RAT ADRENAL CORTEX

The zona fasciculata of the rat adrenal cortex synthesizes and secretes glucocorticoids. As observed after aldehyde fixation, the cells in this zone contain an extensive endoplasmic reticulum (ER), a small Golgi apparatus, a moderate number of lipid droplets, and abundant mitochondria with tubulovesicular cristae. Numerous areas within the endoplasmic reticulum and mitochondrial cristae appear clear. In addition, a small percentage of mitochondria encompasses large, clear areas. After immersion of finely minced adrenal cortex in unbuffered 2% OsO₄ (40–48 hr at 40°C), deposits of osmium are seen within the Golgi apparatus, the entirety of the ER, and occasionally within mitochondria. In some mitochondria, the deposits are within cristae; in others, within vacuoles; in still others, in both cristae and vacuoles. These localizations correspond best to the clear areas found in aldehyde-fixed tissue. Osmium is not deposited in lipid droplets, in bar-containing inclusions, in mitochondrial matrix inclusions, or in the peripheral, outer mitochondrial spaces. Addition of zinc-iodide to OsO₄ increases the amount of Golgi apparatus and mitochondrial staining. Adrenocorticotropin (ACTH) does not affect the localization of deposits; hypophysectomy decreases mitochondrial staining. This study (a) emphasizes the necessity for electron microscopic confirmation of osmium localization when this technique is used as a Golgi apparatus stain; and (b) suggests that the ER-staining pattern may be consistent in cells actively synthesizing steroids or steroid-like compounds.     

The formation and consumption of lipid droplets in the zygote and germinated cells ofClosterium ehrenbergii

The formation and consumption of lipid droplets was observed with an electron microscope in the zygote and the germinated cells of the green alga,Closterium ehrenbergii. The lipid droplets were formed in lysosomal vesicles during zygote maturation following conjugation. In the germinated cells, they were enclosed in ERs and gradually consumed in them. This consumption occurred in the cells at the early stages of expansion. The derivative substances may possibly be used for cell surface expansion.     

Tobacco pollen tubes – a fast and easy tool for studying lipid droplet association of plant proteins

In recent years, lipid droplets have emerged as dynamic organelles rather than inactive storage sites for triacylglycerol. The number of proteins known to be associated with lipid droplets has increased, but remains small in comparison with those found with other organelles. Also the mechanisms of how lipid droplets are recognized and bound by proteins need deeper investigation. Here, we present a fast, simple and inexpensive approach to assay proteins for their association with lipid droplets in vivo that can help to screen protein candidates or mutated variants of proteins for their association in an efficient manner. For this, a system to transiently transform Nicotiana tabacum pollen grains was used because these naturally contain lipid droplets. We designed vectors for fast cloning of genes as fusions with either mVenus or mCherry. This allowed us to assay colocalization with lipid droplets stained with Nile Red and Bodipy 505/515, respectively. We successfully tested our system not only for proteins from Arabidopsis thaliana, but also for proteins from the moss Physcomitrella patens and the alga Chlamydomonas reinhardtii. The small size of the vector used allows easy exchange of codons by site‐directed mutagenesis. We used this to show that two proline residues in the proline knot of a caleosin are not essential for the binding of lipid droplets. We also demonstrated that peroxisomes are not associated with the lipid droplets in tobacco pollen tubes, which reduces the risk of false interpretation of microscopic data in our system.     

A new atypical short-chain dehydrogenase is required for interfungal combat and conidiation in Trichoderma guizhouense

Hypocrealean Trichoderma are the most extensively studied facultative mycoparasites against phytopathogenic fungi. Aerial hyphae of Trichoderma guizhouense can rapidly proliferate over Fusarium oxysporum hyphae, cause sporadic cell death and arrest the growth of the host. The results of the present study demonstrated that a unique short-chain dehydrogenase/reductase (SDR), designated as TgSDR1, was expressed at a high level in T. guizhouense challenged by the hosts. Similar to other SDRs family members, the TgSDR1 protein contains a cofactor-binding motif and a catalytic site. The subcellular localization assay revealed that the TgSDR1::GFP fusion protein translocated to lipid droplets in mycelia and conidia. The data obtained using reverse genetic approach indicated that TgSDR1 is associated with antifungal ability, plays an important role in providing reducing equivalents in the form of NADPH and regulates the amino sugar and nucleotide sugar metabolism in T. guizhouense upon encountering a host. Moreover, the TgSDR1 deletion mutant was defective in conidiation. Thus, TgSDR1 functions as a key metabolic enzyme in T. guizhouense to regulate mycotrophic interactions, defence against other fungi, such as F. oxysporum, and conidiation.     

Lipid droplet hijacking by intracellular pathogens

Lipid droplets were long considered to be simple storage structures, but they have recently been shown to be dynamic organelles involved in diverse biological processes, including emerging roles in innate immunity. Various intracellular pathogens, including viruses, bacteria, and parasites, specifically target host lipid droplets during their life cycle. Viruses such as hepatitis C, dengue, and rotaviruses use lipid droplets as platforms for assembly. Bacteria, such as mycobacteria and Chlamydia, and parasites, such as trypanosomes, use host lipid droplets for nutritional purposes. The possible use of lipid droplets by intracellular pathogens, as part of an anti‐immunity strategy, is an intriguing question meriting further investigation in the near future.