Trypanosoma cruzi Archives - Page 3 of 6 - Center for Tropical and Emerging Global Diseases

Trypanosoma cruzi Letm1 is involved in mitochondrial Ca 2+ transport, and is essential for replication, differentiation, and host cell invasion

Donna Huber on June 7, 2021

Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1) is a mitochondrial inner membrane protein involved in Ca²⁺ and K⁺ homeostasis in mammalian cells. Here, we demonstrate that the Letm1 orthologue of Trypanosoma cruzi, the etiologic agent of Chagas disease, is important for mitochondrial Ca²⁺ uptake and release. The results show that both mitochondrial Ca²⁺ influx and efflux are reduced in TcLetm1 knockdown (TcLetm1-KD) cells and increased in TcLetm1 overexpressing cells, without alterations in the mitochondrial membrane potential. Remarkably, TcLetm1 knockdown or overexpression increases or does not affect mitochondrial Ca²⁺ levels in epimastigotes, respectively. TcLetm1-KD epimastigotes have reduced growth, and both overexpression and knockdown of TcLetm1 cause a defect in metacyclogenesis. TcLetm1-KD also affected mitochondrial bioenergetics. Invasion of host cells by TcLetm1-KD trypomastigotes and their intracellular replication is greatly impaired. Taken together, our findings indicate that TcLetm1 is important for Ca²⁺ homeostasis and cell viability in T cruzi.

Guilherme Rodrigo Rm Dos Santos, Ana Catarina Rezende Leite, Noelia Lander, Miguel Angel Chiurillo, Aníbal Eugênio Vercesi, Roberto Docampo. FASEB J. 2021 Jul;35(7):e21685. doi: 10.1096/fj.202100120RR

Immune exhaustion in chronic Chagas disease: Pro-inflammatory and immunomodulatory action of IL-27 in vitro

Donna Huber on June 1, 2021

In chronic Chagas disease, Trypanosoma cruzi-specific T-cell function decreases over time, and alterations in the homeostatic IL-7/IL-7R axis are evident, consistent with a process of immune exhaustion. IL-27 is an important immunoregulatory cytokine that shares T-cell signaling with IL-7 and other cytokines of the IL-12 family and might be involved in the transcriptional regulation of T-cell function. Here, we evaluated the expression and function of IL-27R in antigen-experienced T cells from subjects with chronic Chagas disease and assessed whether in vitro treatment with IL-27 and IL-7 might improve T. cruzi-specific polyfunctional T-cell responses. In vitro exposure of PBMCs to T. cruzi induced a downregulation of IL-27R in CD4+ T cells and an upregulation in CD8+ T cells in subjects without heart disease, while IL-27R expression remained unaltered in subjects with more severe clinical stages. The modulation of IL-27R was associated with functional signaling through STAT3 and STAT5 and induction of the downstream genes TBX21, EOMES and CXCL9 in response to IL-27. In vitro treatment of PBMCs with IL-27 and IL-7 improved monofunctional and polyfunctional Th1 responses, accompanied by the induction of IL-10 and Bcl-2 expression in subjects without heart disease but did not improve those in subjects with cardiomyopathy. Our findings support the process of desensitization of the IL-27/IL-27R pathway along with disease severity and that the pro-inflammatory and immunomodulatory mechanisms of IL-27 might be interconnected.

María Ailén Natale, Todd Minning, María Cecilia Albareda, Melisa Daiana Castro Eiro, María Gabriela Álvarez, Bruno Lococo, Gonzalo Cesar, Graciela Bertocchi, María Josefina Elias, María Belén Caputo, Rick Lee Tarleton, Susana Adriana Laucella. PLoS Negl Trop Dis. 2021 Jun 1;15(6):e0009473. doi: 10.1371/journal.pntd.0009473.

Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi

Donna Huber on April 6, 2021

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruzi MPC1 (TcMPC1) and TcMPC2 with 3×c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O₂ consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O₂ consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca²⁺ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.

IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruzi MPC1 (TcMPC1) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.

Raquel S. Negreiros, Noelia Lander, Miguel A. Chiurillo, Anibal E. Vercesi, Roberto Docampo. mBio Apr 2021, 12 (2) e00540-21; DOI: 10.1128/mBio.00540-21

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA

Donna Huber on March 31, 2021

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU’s) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

Padilla AM, Yao PY, Landry TJ, Cooley GM, Mahaney SM, Ribeiro I, VandeBerg JL. Tarleton RL. (2021) High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA. PLoS Negl Trop Dis 15(3): e0009141. https://doi.org/10.1371/journal.pntd.0009141

Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication

Donna Huber on March 15, 2021

Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.

Ramakrishnan S, Unger LM, Baptista RP, Cruz-Bustos T, Docampo R (2021) Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn²⁺ in protein glycosylation needed for host cell invasion and intracellular replication. PLoS Pathog 17(3): e1009399. https://doi.org/10.1371/journal.ppat.1009399

Reduced Trypanosoma cruzi-specific humoral response and enhanced T cell immunity after treatment interruption with benznidazole in chronic Chagas disease

Donna Huber on March 7, 2021

Background: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease.

Methods: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months.

Results: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption.

Conclusions: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.

Melisa D Castro Eiro, María A Natale, María G Alvarez, Huifeng Shen, Rodolfo Viotti, Bruno Lococo, Jacqueline Bua, Myriam Nuñez, Graciela L Bertocchi, María C Albareda, Gonzalo Cesar, Rick L Tarleton, Susana A Laucella. J Antimicrob Chemother. 2021 Mar 7;dkab054. doi: 10.1093/jac/dkab054

Strain-specific genome evolution in Trypanosoma cruzi, the agent of Chagas disease

Donna Huber on January 28, 2021

The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite numbers. The consistent, decades-long persistence of T. cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome-largely a result of these same extensive families of genes-have prevented a full understanding of the extent of gene diversity and its maintenance in T. cruzi. In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T. cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T. cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.

Wang W, Peng D, Baptista RP, Li Y, Kissinger JC, Tarleton RL (2021) Strain-specific genome evolution in Trypanosoma cruzi, the agent of Chagas disease. PLoS Pathog 17(1): e1009254. https://doi.org/10.1371/journal.ppat.1009254

The Inositol Pyrophosphate Biosynthetic Pathway of Trypanosoma cruzi

Donna Huber on January 7, 2021

Inositol phosphates (IPs) are phosphorylated derivatives of myo-inositol involved in the regulation of several cellular processes through their interaction with specific proteins. Their synthesis relies on the activity of specific kinases that use ATP as phosphate donor. Here, we combined reverse genetics and liquid chromatography coupled to mass spectrometry (LC-MS) to dissect the inositol phosphate biosynthetic pathway and its metabolic intermediates in the main life cycle stages (epimastigotes, cell-derived trypomastigotes, and amastigotes) of Trypanosoma cruzi, the etiologic agent of Chagas disease. We found evidence of the presence of highly phosphorylated IPs, like inositol hexakisphosphate (IP₆), inositol heptakisphosphate (IP₇), and inositol octakisphosphate (IP₈), that were not detected before by HPLC analyses of the products of radiolabeled exogenous inositol. The kinases involved in their synthesis (inositol polyphosphate multikinase (TcIPMK), inositol 5-phosphate kinase (TcIP5K), and inositol 6-phosphate kinase (TcIP6K)) were also identified. TcIPMK is dispensable in epimastigotes, important for the synthesis of polyphosphate, and critical for the virulence of the infective stages. TcIP5K is essential for normal epimastigote growth, while TcIP6K mutants displayed defects in epimastigote motility and growth. Our results demonstrate the relevance of highly phosphorylated IPs in the life cycle of T. cruzi.

Brian S Mantilla, Leticia D Do Amaral, Henning J Jessen, Roberto Docampo. ACS Chem Biol. 2021 Jan 7. doi: 10.1021/acschembio.0c00759

The IP 3 receptor and Ca 2+ signaling in trypanosomes

Donna Huber on January 6, 2021

Trypanosoma cruzi, and the T. brucei group of parasites cause neglected diseases that affect millions of people around the world. These unicellular microorganisms have complex life cycles involving an insect vector and a mammalian host. Both groups of pathogens possess an inositol 1,4,5-trisphosphate (IP₃)/diacylglycerol (DAG) signaling pathway, and an IP₃ receptor, but with lineage-specific adaptations that make them different from their mammalian counterparts. The phospholipase C (PLC), which hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP₂) to IP₃ is N-terminally myristoylated and palmitoylated. Acidocalcisomes, which are lysosome-related organelles rich in polyphosphate, are the main intracellular Ca²⁺ stores. The inositol 1,4,5-trisphosphate receptor (IP₃R) localizes to acidocalcisomes instead of the endoplasmic reticulum. The trypanosome IP₃R is stimulated by luminal phosphate and pyrophosphate, which are hydrolysis products of polyphosphate (polyP), and inhibited by tripolyphosphate (polyP₃), which is the most abundant polyP in acidocalcisomes. Ca²⁺ signaling is important for host cell invasion and differentiation and to maintain cellular bioenergetics.

Roberto Docampo, Guozhong Huang. Biochim Biophys Acta Mol Cell Res. 2021 Jan 6;118947. doi: 10.1016/j.bbamcr.2021.118947

Affinity‐based proteomics reveals novel targets of inositol pyrophosphate (5‐IP7)‐dependent phosphorylation and binding in Trypanosoma cruzi replicative stages

Donna Huber on December 22, 2020

Diphosphoinositol-5-pentakisphosphate (5-PP-IP₅ ), also known as inositol heptakisphosphate (5-IP₇ ), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a post-translational modification involving a β-phosphoryl transfer. In this study, utilizing an immobilized 5-IP₇ affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP₇ -regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. We endogenously tagged four protein candidates and immunopurified them to assess whether 5-IP₇ -driven phosphorylation is conserved in T. cruzi. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP₇ -mediated phosphorylation events at a polyserine tract (Ser^578-580 ). We also identified a novel SPX domain-containing phosphoribosyl transferase [EC 2.7.6.1] herein termed as TcPRPPS4. Our data revealed new possible functional roles of 5-IP₇ in this divergent eukaryote, and provided potential new targets for chemotherapy.

Brian S Mantilla, Karunakaran Kalesh, Nathaniel W Brown Jr, Dorothea Fiedler, Roberto Docampo. Mol Microbiol. 2020 Dec 22. doi: 10.1111/mmi.14672.

Tag: Trypanosoma cruzi