Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.
- MeSH
- Cell Line MeSH
- Chagas Disease parasitology MeSH
- CRISPR-Cas Systems MeSH
- Eukaryotic Initiation Factor-2 genetics metabolism MeSH
- Phosphorylation MeSH
- Humans MeSH
- Mutation MeSH
- Cell Line, Tumor MeSH
- Parasitemia MeSH
- Proteome metabolism MeSH
- Protein Biosynthesis MeSH
- Protozoan Proteins analysis biosynthesis metabolism MeSH
- Gene Expression Regulation MeSH
- Life Cycle Stages MeSH
- Trypanosoma cruzi growth & development metabolism pathogenicity MeSH
- Virulence MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.
- MeSH
- Chagas Disease diagnosis enzymology parasitology MeSH
- Mass Spectrometry MeSH
- Kinetics MeSH
- Leucyl Aminopeptidase genetics isolation & purification MeSH
- Humans MeSH
- High-Throughput Screening Assays * MeSH
- Amino Acid Sequence genetics MeSH
- Substrate Specificity MeSH
- Trypanosoma cruzi enzymology isolation & purification pathogenicity MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, results in chronic infection that leads to cardiomyopathy with increased mortality and morbidity in endemic regions. In a companion study, our group found that a high-fat diet (HFD) protected mice from T. cruzi-induced myocardial damage and significantly reduced post-infection mortality during acute T. cruzi infection. METHODS: In the present study metabolic syndrome was induced prior to T. cruzi infection by feeding a high fat diet. Also, mice were treated with anti-diabetic drug metformin. RESULTS: In the present study, the lethality of T. cruzi (Brazil strain) infection in CD-1 mice was reduced from 55% to 20% by an 8-week pre-feeding of an HFD to induce obesity and metabolic syndrome. The addition of metformin reduced mortality to 3%. CONCLUSIONS: It is an interesting observation that both the high fat diet and the metformin, which are known to differentially attenuate host metabolism, effectively modified mortality in T. cruzi-infected mice. In humans, the metabolic syndrome, as presently construed, produces immune activation and metabolic alterations that promote complications of obesity and diseases of later life, such as myocardial infarction, stroke, diabetes, Alzheimer's disease and cancer. Using an evolutionary approach, we hypothesized that for millions of years, the channeling of host resources into immune defences starting early in life ameliorated the effects of infectious diseases, especially chronic infections, such as tuberculosis and Chagas disease. In economically developed countries in recent times, with control of the common devastating infections, epidemic obesity and lengthening of lifespan, the dwindling benefits of the immune activation in the first half of life have been overshadowed by the explosion of the syndrome's negative effects in later life.
- MeSH
- Adiposity drug effects MeSH
- Survival Analysis MeSH
- Adipose Tissue, White drug effects immunology metabolism parasitology MeSH
- Cell Line MeSH
- Chagas Disease blood immunology metabolism parasitology MeSH
- Cytokines blood metabolism MeSH
- Energy Metabolism drug effects MeSH
- Hypoglycemic Agents pharmacology therapeutic use MeSH
- Mice, Inbred Strains MeSH
- Leptin blood metabolism MeSH
- Humans MeSH
- Metabolic Syndrome drug therapy etiology immunology parasitology MeSH
- Metformin pharmacology therapeutic use MeSH
- Models, Immunological * MeSH
- Random Allocation MeSH
- Obesity blood immunology metabolism physiopathology MeSH
- Foreskin drug effects immunology metabolism parasitology MeSH
- Heart Ventricles drug effects immunology metabolism parasitology MeSH
- Trypanosoma cruzi drug effects immunology isolation & purification pathogenicity MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Comparative Study MeSH
- MeSH
- Chagas Disease * genetics parasitology transmission MeSH
- Chronic Disease MeSH
- Host-Parasite Interactions * genetics immunology MeSH
- Humans MeSH
- Retraction of Publication as Topic MeSH
- Gene Transfer, Horizontal genetics MeSH
- DNA, Protozoan genetics MeSH
- Trypanosoma cruzi * genetics immunology pathogenicity growth & development MeSH
- Check Tag
- Humans MeSH
