Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian ‘bloodstream form’ trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.     

Trypanosoma brucei gambiense and T. b. rhodesiense: Concanavalin A Binding to the Membrane and Flagellar Pocket of Bloodstream and Procyclic Forms1

ABSTRACT We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3–16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed >95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.     

Infection and development of Nosema bombycis (Microsporida: Protozoa) in a cell line of Antheraea eucalypti

Spores of Nosema bombycis derived from diseased insects were highly purified by Urografin density gradient centrifugation. Antheraea eucalypti cells were inoculated with the purified spores primed with 0.1 n KOH solution to start a continuous propagation of N. bombycis in cell culture. The first increase in the number of infected A. eucalypti cells was observed at 48 hr postinoculation, and it was caused by the secondary infective forms of N. bombycis. The secondary infective forms were produced during the course of sporoblast differentiation. The parasites in cell cultures divided synchronously until 36 hr postinoculation. Mature spores were observed initially 6 days postinoculation at 27°C. The infected cultures were subcultured extensively for more than 1 year with the addition of healthy A. eucalypti cells.     

Meloidogyne incognita Surface Antigen Epitopes in Infected Arabidopsis Roots

Surface-coat epitopes of Meloidogyne incognita were detected in root tissues of Arabidopsis thaliana during migration and feeding site formation. A whole-mount root technique was used for immunolocalization of surface coat epitopes in A. thaliana, with the aid of a monoclonal antibody raised specifically against the outer surface of infective juveniles of M. incognita. The antibody, which was Meloidogyne-specific, recognized a fucosyl-bearing glycoprotein in the surface coat. During migration in host tissues the surface coat was shed, initially accumulating in the intercellular spaces next to the juvenile and later at cell junctions farther from the nematode. Upon induction of giant cell formation, the antibody bound to proximally located companion cells and sieve elements of the phloem.     

Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures

Dalgliesh R. J. and Stewart N. P. 1979. Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures. International Journal for Parasitology9: 115–120. The temperature of incubation of unfed Boophilus microplus larvae infected with Babesia bovis influenced the morphology and infectivity of the Babesia within the tick. Incubation at 37°C for 1–3 days stimulated the development of parasites morphologically similar to those usually observed in fed larvae harvested from cattle; similar forms appeared more slowly in larvae incubated at 31°C or 25°C. Extracts prepared from larvae after incubation at 37°C for 3–5 days or 30°C for 8 days were consistently infective for cattle. Prior storage of larvae at 14°C for up to 28 days enhanced the development of infectivity at 37°C; infectivity could still be produced after 65 days storage at 14°C but not after 76 days. Larvae released on a host transmitted B. bovis sooner if they had been incubated at 37°C for 4 days. It was concluded that the development of B. bovis to an infective stage in B. microplus is temperature dependent and does not require the stimulus of feeding by the host.     

Schistosoma mansoni: identification, characterization, and purification of the spine glycoprotein by monoclonal antibody

A tegumental surface membrane antigen of Schistosoma mansoni has been identified by use of a monoclonal antibody. The binding of ¹²⁵I-labeled monoclonal antibody showed that proteins sharing antigenic determinants recognized by this monoclonal antibody were present in cercariae and worms of both sexes, but were absent from schistosome egg extract. The protein molecules expressing these antigenic determinants differed in molecular weight: 120,000 in cercaria and 170,000 in male and female worms. The cercarial glycoprotein immunoprecipitated with the monoclonal antibody was also immunoprecipited by sera of infected humans, as shown by two-dimensional gel electrophoresis and tryptic peptide mapping. The location of the glycoprotein identified by the monoclonal antibody was restricted to the spines of the schistosomular surface, the tubercle-associated spines of the male worm, and the dorsal spines of the female worm. The spine glycoprotein was readily purified by immunoaffinity chromatography. These findings are discussed in relation to parasite development and the relevance of this antibody for serodiagnosis and immunoprophylaxis.     

Studies on the Development of Metacyclic Trypanosoma brucei sspp. Cultivated at 27°C with Insect Cell Lines1

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T. b. rhodesiense were grown at 27°C in 25-cm² flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 × 10⁵ metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

Trypanosoma cruzi: Role of δ-Amastin on Extracellular Amastigote Cell Invasion and Differentiation

Trypanosoma cruzi is a protozoan parasite that comprises different phylogenetic groups and is the causative agent of Chagas’ disease. Different T. cruzi strains present differences in infectivity in in vitro and in vivo experimental models, which are likely related to the expression of different virulence factors. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the parasite. In this study, we showed that a highly infective strain (G strain) of extracellular amastigote (EA) invasive forms expressed reduced RNA levels of amastin compared to a less infective strain (CL). The treatment of HeLa cells with recombinant δ-amastin reduced infectivity of EA forms. However, the ectopic expression of δ-amastin accelerated amastigote differentiation into trypomastigotes. Corroborating the virulence behavior in association with amastin expression, the EAs overexpressing amastin were precociously and robustly observed in the liver of susceptible mouse strains (A/JUnib), whereas parasitemia was never detected in in vivo assays. This is the first report on the regulatory role of amastin in the course of both in vitro and in vivo T. cruzi infection.     

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

Background

This study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.

Methodology

Three series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.

Principal Findings

MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.

Conclusions/Significance

These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.     

Role of lymphocyte surface determinants in lymph node homing

Thoracic duct lymphocytes briefly incubated in vitro with trypsin and then infused into syngeneic rats are unable to migrate into lymph nodes. Trypsin-treated lymphocytes incubated at 37 °C in the absence of enzyme for 12 hr recovered their lymph node homing properties. In vitro recovery did not occur if the cells were cultured at 17 °C. Evidence was obtained that trypsin cleaved sialyglycoproteins from the surface of lymphocytes and that these determinants reappeared after the cells were maintained at 37 °C for 24 hr.Puromycin added to cultures of normal lymphocytes for 3 hr before infusion markedly reduced the recovery of donor cells in lymph nodes. The results suggest that surface determinants of recirculating lymphocytes essential for homing into lymph nodes may be rapidly turned over.     

Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

The isolation of plasmids containing dna complementary to messenger rna for variant surface glycoproteins of Trypanosoma brucei

We have isolated poly(A)⁺ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)⁺ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs.The total translation products from the four poly(A)⁺ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins.We have made duplex DNA copies of these poly(A)⁺ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)⁺ RNA showed that 7–10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)⁺ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation.Poly(A)⁺ RNA from each of the variants only hybridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.     

Structure of deoxy hemoglobin C (beta six Glu replaced by Lys) in two crystal forms

Five crystal forms of the abnormal human hemoglobin Hb³ C (beta six Glu → Lys) have been grown. Two of them are grown with liganded Hb C, three with deoxy Hb C. The structures of two of the deoxy crystal forms were determined by the method of molecular replacement, using deoxy Hb A as the model structure. Fourier maps were calculated for each Hb C structure, using data to a resolution of 5 Å in one case and 4 Å in the second case. The structural differences between each deoxy Hb C structure and the deoxy Hb A model are found mostly at the molecular surface. Energetically favorable interactions involving the variant residue, beta six lysine, occur in both Hb C crystal forms, and could explain the lowered solubility and enhanced tendency of deoxy Hb C to crystallize in vivo.     

Aspects of the cross transmission to Galleria mellonella of a Baculovirus from the alfalfa looper, Autographa californica

The nuclear polyhedrosis virus originally isolated from the alfalfa looper, Autographa californica, was successfully transmitted to the greater wax moth, Galleria mellonella. Both the many polyhedra per nucleus (MP) and the few polyhedra per nucleus (FP) plaque variants of this virus were found to be infective when injected intracoelomically. When polyhedra of each plaque variant were fed to G. mellonella larvae, a difference in response was observed; the MP plaque variant was estimated to be 30 times more infective than the FP variant.     

De Novo Genome Assembly Shows Genome Wide Similarity between Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Background

Trypanosoma brucei is a eukaryotic pathogen which causes African trypanosomiasis. It is notable for its variant surface glycoprotein (VSG) coat, which undergoes antigenic variation enabled by a large suite of VSG pseudogenes, allowing for persistent evasion of host adaptive immunity. While Trypanosoma brucei rhodesiense (Tbr) and T. b gambiense (Tbg) are human infective, related T. b. brucei (Tbb) is cleared by human sera. A single gene, the Serum Resistance Associated (SRA) gene, confers Tbr its human infectivity phenotype. Potential genetic recombination of this gene between Tbr and non-human infective Tbb strains has significant epidemiological consequences for Human African Trypanosomiasis outbreaks.

Results

Using long and short read whole genome sequencing, we generated a hybrid de novo assembly of a Tbr strain, producing 4,210 scaffolds totaling approximately 38.8 megabases, which comprise a significant proportion of the Tbr genome, and thus represents a valuable tool for a comparative genomics analyses among human and non-human infective T. brucei and future complete genome assembly. We detected 5,970 putative genes, of which two, an alcohol oxidoreductase and a pentatricopeptide repeat-containing protein, were members of gene families common to all T. brucei subspecies, but variants specific to the Tbr strain sequenced in this study. Our findings confirmed the extremely high level of genomic similarity between the two parasite subspecies found in other studies.

Conclusions

We confirm at the whole genome level high similarity between the two Tbb and Tbr strains studied. The discovery of extremely minor genomic differentiation between Tbb and Tbr suggests that the transference of the SRA gene via genetic recombination could potentially result in novel human infective strains, thus all genetic backgrounds of T. brucei should be considered potentially human infective in regions where Tbr is prevalent.     

Aberrant Forms of Streptococcus cremoris HP

The cheese starter strain, Streptococcus cremoris HP, produced variant colonies when streaked on the surface of solid media and incubated at 30 or 37°C or in the presence of penicillin. Serial plating and incubation at 37°C or in the presence of penicillin resulted in the production of variants. Subculture followed by incubation at 25°C or in the absence of penicillin resulted in the reversion or partial reversion to the parent form. Colony morphology and cell morphology exhibited the characteristics of the L-phase. Evidence suggested that the aberrant forms of S. cremoris at 30°C were transitional phase variants but at 37°C and in the presence of penicillin they were L-phase variants. Electron micrographs showed that the cell walls of the variant cells were defective and that there were differences in the density and the organization of the cytoplasmic constituents compared with the parent cell.     

Determination of common genetic variants in cytidine deaminase (CDA) gene in Indian ethnic population

Cytidine deaminase (CDA) is the major enzyme involved in metabolism of gemcitabine, a pyrimidine analog widely used for chemotherapy of solid tumors. While only low amounts of administered gemcitabine undergo intracellular phosphorylation into active forms and involve in antineoplastic activities, majority of it is rapidly inactivated by CDA and excreted to avoid drug toxicity. Knowledge of the genetic polymorphisms mildly effecting cellular activity of the enzyme CDA is therefore crucial to understanding drug-induced toxicities associated with gemcitabine. Functional significance and allele frequencies for common SNPs including 79A>C (*2) and 208G>A (*3) have been reported in various ethnic populations including Caucasian, African, Korean and Japanese. However, such studies have not been reported in any Indian sub-population. In the present study, conventional polymerase chain reaction (PCR) based amplification using gene specific primers and Sanger sequencing were performed to identify CDA variants in 50 healthy individuals from Indian sub-population. Established common variant 79A>C known to reduce CDA activity was observed at a frequency of 0.14 in the study cohort. In addition to other known variants, one novel variant, c.325−209T>C was detected at a frequency of 0.06. Genetic variants in CDA gene and their frequencies established in our study hold value in pharmacogenetics.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.     

Characterization of different proteolytic activities in Trypanosoma brucei brucei

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4°C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography [1]. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoproteins by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by and alkaline pH optimum and an estimated molecular mass of 80–100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 μM. The retained material eluted by addition of 1% methyl-α-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (> 70 kDa) and the other peak of lower molecular mass (30–70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.