Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus

Parasitology ◽  
2012 ◽  
Vol 139 (6) ◽  
pp. 735-743 ◽  
Author(s):  
F. O. R. OLIVEIRA ◽  
C. R. ALVES ◽  
F. SOUZA-SILVA ◽  
C. M. CALVET ◽  
L. M. C. CÔRTES ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rhodnius Prolixus ◽  
Insect Vector ◽  
Heparin Binding ◽  
Molecular Masses ◽  
Pre Treatment

SUMMARYHeparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

Serotonin triggers cAMP and PKA-mediated intracellular calcium waves in Malpighian tubules of Rhodnius prolixus

2014 ◽  
Vol 307 (7) ◽  
pp. R828-R836 ◽  
Author(s):  
Paula Gioino ◽  
Brendan G. Murray ◽  
Juan P. Ianowski
Keyword(s):  
Second Messenger ◽  
Fluid Secretion ◽  
Rhodnius Prolixus ◽  
Malpighian Tubules ◽  
Insect Vector ◽  
Excess Water ◽  
Intracellular Ca ◽  
Single Meal ◽  
Hematophagous Insect

Rhodnius prolixus is a hematophagous insect vector of Chagas disease capable of ingesting up to 10 times its unfed body weight in blood in a single meal. The excess water and ions ingested with the meal are expelled through a rapid postprandial diuresis driven by the Malpighian tubules. Diuresis is triggered by at least two diuretic hormones, a CRF-related peptide and serotonin, which were traditionally believed to trigger cAMP as an intracellular second messenger. Recently, calcium has been suggested to act as a second messenger in serotonin-stimulated Malpighian tubules. Thus, we tested the role of calcium in serotonin-stimulated Malpighian tubules from R. prolixus. Our results show that serotonin triggers cAMP-mediated intracellular Ca2+ waves that were blocked by incubation in Ca2+-free saline containing the cell membrane-permeant Ca2+ chelator BAPTA-AM, or the PKA blocker H-89. Treatment with 8-Br-cAMP triggered Ca2+ waves that were blocked by H-89 and BAPTA-AM. Analysis of the secreted fluid in BAPTA-AM-treated tubules showed a 75% reduction in fluid secretion rate with increased K+ concentration, reduced Na+ concentration. Taken together, the results indicate that serotonin triggers cAMP and PKA-mediated Ca2+ waves that are required for maximal ion transport rate.

scholarly journals Dual Role for Transforming Growth Factor β-Dependent Signaling in Trypanosoma cruzi Infection of Mammalian Cells

2000 ◽  
Vol 68 (4) ◽  
pp. 2077-2081 ◽  
Author(s):  
Belinda S. Hall ◽  
Miercio A. Pereira
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Growth Arrest ◽  
Transforming Growth Factor Β

ABSTRACT Expression of functional transforming growth factor β (TGF-β) receptors (TβR) is required for the invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi. However, the precise role of this host cell signaling complex in T. cruzi infection is unknown. To investigate the role of the TGF-β signaling pathway, infection levels were studied in the mink lung epithelial cell lines JD1, JM2, and JM3. These cells express inducible mutant TβR1 proteins that cannot induce growth arrest in response to TGF-β but still transmit the signal for TGF-β-dependent gene expression. In the absence of mutant receptor expression, trypomastigotes invaded the cells at a low level. Induction of the mutant receptors caused an increase in infection in all three cell lines, showing that the requirement for TGF-β signaling at invasion can be divorced from TGF-β-induced growth arrest. TGF-β pretreatment of mink lung cells expressing wild-type TβR1 caused a marked enhancement of infection, but no enhancement was seen in JD1, JM2, and JM3 cells, showing that the ability of TGF-β to stimulate infection is associated with growth arrest. Likewise, expression of SMAD7 or SMAD2SA, inhibitors of TGF-β signaling, did not block infection by T. cruzi but did block the enhancement of infection by TGF-β. Taken together, these results show that there is a dual role for TGF-β signaling in T. cruzi infection. The initial invasion of the host cell is independent of both TGF-β-dependent gene expression and growth arrest, but TGF-β stimulation of infection requires a fully functional TGF-β signaling pathway.

scholarly journals Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

2009 ◽  
Vol 83 (11) ◽  
pp. 5640-5647 ◽  
Author(s):  
Ronald L. Knight ◽  
Kimberly L. W. Schultz ◽  
Rebekah J. Kent ◽  
Meera Venkatesan ◽  
Diane E. Griffin
Keyword(s):  
Mammalian Cells ◽  
Sindbis Virus ◽  
Glycosylation Site ◽  
Surface Glycoprotein ◽  
Heparin Binding ◽  
Virus Binding ◽  
Bhk Cells ◽  
Mosquito Cells ◽  
Intrathoracic Inoculation

ABSTRACT Each Sindbis virus (SINV) surface glycoprotein has two sites for N-linked glycosylation (E1 positions 139 and 245 [E1-139 and E1-245] and E2 positions 196 and 318 [E2-196 and E2-318]). Studies of SINV strain TE12 mutants with each site eliminated identified the locations of carbohydrates by cryo-electron microscopy (S. V. Pletnev et al., Cell 105:127-136, 2001). In the current study, the effects of altered glycosylation on virion infectivity, growth in cells of vertebrates and invertebrates, heparin binding, virulence in mice, and replication in mosquitoes were assessed. Particle-to-PFU ratios for E1-139 and E2-196 mutant strains were similar to that for TE12, but this ratio for the E1-245 mutant was 100-fold lower than that for TE12. Elimination of either E2 glycosylation site increased virus binding to heparin and increased replication in BHK cells. Elimination of either E1 glycosylation site had no effect on heparin binding but resulted in an approximately 10-fold decrease in virus yield from BHK cells compared to the TE12 amount. No differences in pE2 processing were detected. E2-196 and E2-318 mutants were more virulent in mice after intracerebral inoculation, while E1-139 and E1-245 mutants were less virulent. The E1-245 mutant showed impaired replication in C7/10 mosquito cells and in Culex quinquefasciatus after intrathoracic inoculation. We conclude that the increased replication and virulence of E2-196 and E2-318 mutants are primarily due to increased efficiency of binding to heparan sulfate on mammalian cells. Lack of glycosylation at E1-139 or E1-245 impairs replication in vertebrate cells, while E1-245 also severely affects replication in invertebrate cells.

Induction of Trypanosoma cruzi Metacyclogenesis in the Gut of the Hematophagous Insect Vector, Rhodnius prolixus, by Hemoglobin and Peptides Carrying αD-Globin Sequences

1995 ◽  
Vol 81 (3) ◽  
pp. 255-261 ◽  
Author(s):  
E.S. Garcia ◽  
M.S. Gonzalez ◽  
P. Deazambuja ◽  
F.E. Baralle ◽  
D. Fraidenraich ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Rhodnius Prolixus ◽  
Insect Vector ◽  
Hematophagous Insect

Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity

Parasitology ◽  
2012 ◽  
Vol 140 (2) ◽  
pp. 171-180 ◽  
Author(s):  
F. O. R. OLIVEIRA-JR ◽  
C. R. ALVES ◽  
F. S. SILVA ◽  
L. M. C. CÔRTES ◽  
L. TOMA ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Binding Proteins ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Biochemical Properties ◽  
Heparin Binding ◽  
Flagellar Membrane ◽  
The Stability ◽  
Ph Range

SUMMARYHeparin-binding proteins (HBPs) play a key role in Trypanosoma cruzi-host cell interactions. HBPs recognize heparan sulfate (HS) at the host cell surface and are able to induce the cytoadherence and invasion of this parasite. Herein, we analysed the biochemical properties of the HBPs and also evaluated the expression and subcellular localization of HBPs in T. cruzi trypomastigotes. A flow cytometry analysis revealed that HBPs are highly expressed at the surface of trypomastigotes, and their peculiar localization mainly at the flagellar membrane, which is known as an important signalling domain, may enhance their binding to HS and elicit the parasite invasion. The plasmon surface resonance results demonstrated the stability of HBPs and their affinity to HS and heparin. Additionally, gelatinolytic activities of 70 kDa, 65·8 kDa and 59 kDa HBPs over a broad pH range (5·5–8·0) were revealed using a zymography assay. These proteolytic activities were sensitive to serine proteinase inhibitors, such as aprotinin and phenylmethylsulfonyl fluoride, suggesting that HBPs have the properties of trypsin-like proteinases.

scholarly journals Analysis of DNA Binding Proteins Associated With Hemin-Induced Transcriptional Inhibition. The Hemin Response Element Binding Protein Is a Heterogeneous Complex That Includes the Ku Protein

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1793-1801
Author(s):  
S.V. Reddy ◽  
O. Alcantara ◽  
D.H. Boldt
Keyword(s):  
Binding Proteins ◽  
Repeat Sequence ◽  
Tartrate Resistant Acid Phosphatase ◽  
Response Element ◽  
Transcriptional Inhibition ◽  
Trap Gene ◽  
Element Binding Protein ◽  
Molecular Masses ◽  
Ku Protein

Hemin inhibits transcription of the tartrate resistant acid phosphatase (TRAP) gene. Using deletion mutagenesis of the mouse TRAP 5′-flanking region, we previously identified a 27-bp DNA segment containing a central GAGGC tandem repeat sequence (the hemin response element [HRE]), which bound nuclear proteins (hemin response element binding proteins [HREBPs]) from hemin-treated cells and appeared to be responsible for mediating transcriptional inhibition in response to hemin. We now have used affinity binding to HRE-derivatized beads to identify four HREBP components with apparent molecular masses of 133-, 90-, 80-, and 37-kD, respectively. The 80- and 90-kD components correspond to the p70 and p80/86 subunits of Ku antigen (KuAg) as documented by partial amino acid microsequencing of tryptic digests and immunologic reactivity. Based on reactivity of the HREBP gel shift band with antibodies to the redox factor protein (ref1) in shift Western experiments, it is shown that the 37-kD component represents ref1. The 133-kD component appeared to be a unique protein. KuAg participation in HREBP complexes was specific as it was present in HREBPs bound to HRE microcircles. Results of depletion/reconstitution experiments suggested that KuAg does not bind alone or directly to HRE DNA, but does so only in conjunction with the 133- and/or 37-kD proteins. We conclude that HREBP is a heterogeneous complex composed of KuAg, ref1, and a unique 133-kD protein. We speculate that the role of heme may be to promote interactions among these components, thereby facilitating HRE binding and downregulation of hemin responsive genes.

scholarly journals Transcriptome-wide assessment of the m6A methylome of intestinal porcine epithelial cells treated with deoxynivalenol

2020 ◽  
Author(s):  
Zhengchang Wu ◽  
Chao Xu ◽  
Haifei Wang ◽  
Song Gao ◽  
Shenglong Wu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Tumor Necrosis ◽  
Cytotoxic Compound ◽  
Wide Range ◽  
Food And Feed ◽  
Functional Relevance ◽  
And Control ◽  
Necrosis Factor

Abstract Background: Deoxynivalenol (DON) is a cytotoxic compound found in various food and feed products. N6-Methyladenosine (m6A) is a highly abundant epitranscriptomic marker that modifies a wide range of mRNA molecules in mammalian cells. However, the role of the m6A methylome in DON-induced damage remains poorly understood.Results: In this study, we assessed the transcriptome-wide m6A profile of intestinal porcine epithelial cells (IPEC-J2) treated with 1000 ng/mL DON by m6A sequencing and RNA sequencing. Overall, 5406 new m6A peaks appeared with the disappearance of 2615 peaks in DON-treated IPEC-J2 cells. Genes that were uniquely m6A-modified following DON treatment were found to be associated with the tumor necrosis factor (TNF) signaling pathway. On comparing DON-treated and control cells, we identified 733 differentially expressed mRNAs bearing hyper- or hypomethylated m6A peaks. Further experimental data suggested that METTL3-dependent m6A methylation might also play a role in DON-induced inflammatory response, and CSF2 marker is key functional relevance in the context of DON-induced toxicity. Conclusions: This is the first study to perform a transcriptome-wide assessment of the m6A methylome of IPEC-J2 cells treated with DON. We believe that our findings should be useful for identifying mechanisms whereby m6A modifications influence the outcomes of DON exposure.

scholarly journals Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells

2019 ◽  
Vol 221 (10) ◽  
pp. 1612-1622 ◽  
Author(s):  
Evgeny A Semchenko ◽  
Tsitsi D Mubaiwa ◽  
Christopher J Day ◽  
Kate L Seib
Keyword(s):  
Epithelial Cells ◽  
Cell Aggregation ◽  
Host Cells ◽  
Heparin Binding ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
Treatment And Prevention ◽  
New Treatment ◽  
Reduced Adherence

Abstract The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.

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