Chemical Inhibition of Bromodomain Proteins in Insect-Stage African Trypanosomes Perturbs Silencing of the Variant Surface Glycoprotein Repertoire and Results in Widespread Changes in the Transcriptome.

The eukaryotic protozoan parasite Trypanosoma brucei is transmitted by the tsetse fly to both humans and animals, where it causes a fatal disease called African trypanosomiasis. While the parasite lacks canonical DNA sequence-specific transcription factors, it does possess histones, histone modifications, and proteins that write, erase, and read histone marks. Chemical inhibition of chromatin-interacting bromodomain proteins has previously been shown to perturb bloodstream specific trypanosome processes, including silencing of the variant surface glycoprotein (VSG) genes and immune evasion. Transcriptomic changes that occur in bromodomain-inhibited bloodstream parasites mirror many of the changes that occur as parasites developmentally progress from the bloodstream to the insect stage. We performed transcriptome sequencing (RNA-seq) time courses to determine the effects of chemical bromodomain inhibition in insect-stage parasites using the compound I-BET151. We found that treatment with I-BET151 causes large changes in the transcriptome of insect-stage parasites and also perturbs silencing of VSG genes. The transcriptomes of bromodomain-inhibited parasites share some features with early metacyclic-stage parasites in the fly salivary gland, implicating bromodomain proteins as important for regulating transcript levels for developmentally relevant genes. However, the downregulation of surface procyclin protein that typically accompanies developmental progression is absent in bromodomain-inhibited insect-stage parasites. We conclude that chemical modulation of bromodomain proteins causes widespread transcriptomic changes in multiple trypanosome life cycle stages. Understanding the gene-regulatory processes that facilitate transcriptome remodeling in this highly diverged eukaryote may shed light on how these mechanisms evolved. IMPORTANCE The disease African trypanosomiasis imposes a severe human and economic burden for communities in sub-Saharan Africa. The parasite that causes the disease is transmitted to the bloodstream of a human or ungulate via the tsetse fly. Because the environments of the fly and the bloodstream differ, the parasite modulates the expression of its genes to accommodate two different lifestyles in these disparate niches. Perturbation of bromodomain proteins that interact with histone proteins around which DNA is wrapped (chromatin) causes profound changes in gene expression in bloodstream-stage parasites. This paper reports that gene expression is also affected by chemical bromodomain inhibition in insect-stage parasites but that the genes affected differ depending on life cycle stage. Because trypanosomes diverged early from model eukaryotes, an understanding of how trypanosomes regulate gene expression may lend insight into how gene-regulatory mechanisms evolved. This could also be leveraged to generate new therapeutic strategies.

A recombinant protein based on Trypanosoma cruzi surface molecule gp82 induces apoptotic cell death in melanoma cells.

Trypanosoma cruzi infection is known to confer resistance to tumor development in mice, and in-vitro studies have shown the toxic effects of parasite extracts on cancer cell cultures. Investigations in which T. cruzi molecules exhibit antitumor activity have just begun. Here, we used a tumorigenic cell line Tm5, derived from mouse melanocytes melan-a, to test the effect of J18, a recombinant protein based on T. cruzi surface molecule gp82 fused to glutathione-S-transferase (GST). J18 induced actin cytoskeleton disruption in Tm5 but not in melan-a cells. Several changes indicative of apoptosis were detected in Tm5 melanoma cells but not in melan-a cells treated with J18, such as the flipping of phosphatidylserine from the inner to the external side of the plasma membrane, altered nuclear morphology, DNA fragmentation, increase in mitochondria depolarization, and in caspase-3 activity. Retention of NF-kappaB in the cytoplasm was another alteration observed specifically in J18-treated Tm5 cells. No such alterations were found in Tm5 cells treated with GST. In-vivo experiments showed that C57BL/6 mice inoculated with Tm5 cells, treated at the site of tumor cell inoculation with J18, developed tumors of smaller size than mice treated with phosphate-buffered saline or GST and survived longer.

Glycosylinositolphosphate soluble variant surface glycoprotein inhibits IFN-gamma-induced nitric oxide production via reduction in STAT1 phosphorylation in African trypanosomiasis.

Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-gamma, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-gamma vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-gamma followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-alpha, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-gamma stimulation resulted in a marked reduction of IFN-gamma-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-gamma-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-gamma response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.

An accessory role for the diacylglycerol moiety of variable surface glycoprotein of African trypanosomes in the stimulation of bovine monocytes.

The membrane-associated form of the variable surface glycoprotein (mfVSG) from African trypanosomes is a potent macrophage activator capable of inducing production of tumor necrosis factor alpha (TNFalpha) in both bovine and murine models. Using a bovine model, we have re-investigated the hypothesis that the diacylglycerol moiety of the glycosylphosphatodylinositol (GPI) anchor is involved in macrophage activation and might be the actual parasite toxin. The anchor of the variable surface glycoprotein (VSG) was labeled with (3)H-myristic acid and VSG purified in its membrane-associated form. The dimyristylglycerol moiety of the anchor was released by phospholipase C cleavage. Integrity of the anchor and efficiency of cleavage was verified by autoradiography and methanol:hexane extraction. For analysis of biological function, bovine monocytes were used which had been incubated with bovine interferon gamma (primed) or with culture medium (unprimed). The VSG purified in its membrane-associated form was found to stimulate both primed and unprimed cells to secrete TNFalpha. The same preparation from which the dimyristylglycerol moiety had been cleaved was no longer able to stimulate unprimed cells but could still stimulate primed cells. Our data indicate that the presence of the dimyristylglycerol is not an absolute requirement for induction of TNFalpha production but can substitute for the interferon gamma priming. Therefore, we favor the hypothesis that stimulation of macrophages to secrete TNFalpha by the mfVSG is mediated by an as yet unknown trigger moiety and is facilitated by the dimyristylglycerol anchor.

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein.

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.

Glycosylphosphatidylinositol toxin of Trypanosoma brucei regulates IL-1 alpha and TNF-alpha expression in macrophages by protein tyrosine kinase mediated signal transduction.

A purified, structurally defined glycosylphosphatidylinositol (GPI) derived from the Variant Surface Glycoprotein (VSG) of Trypanosoma brucei, and its biosynthetic precursor P2, was able at submicromolar concentrations to regulate cytokine expression when added directly as pharmacological agonist to host macrophages, by activation of an endogenous protein tyrosine-kinase (PTK) mediated signal transduction pathway. GPI induces rapid onset tyrosine phosphorylation of multiple intracellular substrates, within minutes of addition to LPS-nonresponsive cells, followed shortly thereafter by IL-1 alpha secretion. The PTK antagonists genistein and tyrphostin inhibit both tyrosylphosphorylation and cytokine expression. A monoclonal antibody to GPI also blocks IL-1 alpha induction by total parasite extracts. Thus, as in malaria infection, GPI may induce the cytokine excess causing certain pathological states associated with trypanosomiasis.

The kinetics of gene expression and maturation of IL-1 alpha after induction with the surface coat of Trypanosoma brucei rhodesiense or lipopolysaccharide.

The purpose of this study was threefold: to determine if the variant surface coat glycoprotein (VSG) of Trypanosoma brucei rhodesiense induces IL-1 alpha; to study the kinetics of IL-1 alpha transcription, maturation and secretion; and to compare VSG to LPS in its ability to induce IL-1 alpha. VSG was added to cultures of the P388D1 murine macrophage cell line. RNA was dotted onto nitrocellulose and hybridized with a murine IL-1 alpha cDNA probe. Maximal production of IL-1 alpha mRNA occurred in a dose- and time-dependent manner, peaking at 25 micrograms/ml VSG, within 2 h. Induction of IL-1 alpha was not due to contaminants because 1) absorption of VSG with a mAb abrogated IL-1 alpha mRNA synthesis, 2) the addition of polymyxin B did not affect mRNA levels, and 3) cellular IL-1 alpha was detectable in VSG-treated splenocytes from endotoxin nonresponder C3H/HeJ mice. Murine splenic macrophages also had enhanced levels of IL-1 alpha mRNA after administration of VSG in vivo or during an acute infection. Antiserum generated against the synthetic peptide SGDDSKYPV (amino acids 177-185 from the murine IL-1 alpha sequence) was used to measure the levels of the 33-, 22-, and 14-kDa proteins in cell lysates and medium of VSG-stimulated P388D1 cells. The 22-kDa protein was the predominant cellular form until secretion started. Secretion of the 14-kDa form began abruptly 6 to 8 h after the addition of VSG. By 12 h, the 33-kDa precursor was the major cytoplasmic form. In comparative analyses, LPS-stimulated P388D1 cells produced more transcript, generated peak levels of 22-kDa protein 3 h earlier, and began to secrete the 14-kDa molecule 5 h earlier. The rate of IL-1 alpha accumulation in the medium was linear between 6 and 24 h after LPS treatment, but began to drop by 8 h in VSG-treated cells. Functional (comitogenic) IL-1 activity was also detected in media from VSG-treated splenic macrophages and P388D1 cells. Activity peaked at 50 micrograms/ml and was lost if 0.2% IL-1 antisera were added to the cultures.