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Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction.

Moraes KC, Diniz LF, Bahia MT - Mem. Inst. Oswaldo Cruz (2015)

Bottom Line: However, how the parasite interaction modulates COX-2 activity is poorly understood.Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures.Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular, Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho, Rio Claro, SP, Brasil.

ABSTRACT
Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

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molecular interaction between cyclooxygenase-2 (Cox-2) mRNA and CUGPP2protein. A: fluorescence microscopy analyses of the cross-talk between Cox-2mRNA and CUGBP2 in H9c2 cells after parasitic infection. The CUGBP2 protein(fluorescein isothiocyanate labelled) localised preferentially to the nucleus(4’,6 diamino-2-phenylindole labelled) and co-localised in the cytoplasmicregion with Cox-2 mRNA (Alexa Fluor labelled and indicated with arrowheads) andwith eukaryotic initiation factor (eIF4G) (cyanine 5 labelled). Bars = 50 μm;B: co-immunoprecipitation (Co-IP) of CUGBP2 and Cox-2 mRNA in H9c2 cellsfollowed by western blotting analyses (using total cell extract). RecombinantCUGBP2 (rCUGBP2) and total H9c2 extracts were used as control in the assay. TheCo-IP precisely captured the protein from H9c2 total cell extract and themolecules that bind to it; C: formaldehyde RNA cross-linking assay using Cox-2mRNA and H9c2 cell extract was performed followed by the Co-IP to capture themolecules that binds to CUGBP2. Quantitative polymerase chain reaction (qPCR)quantified the total amount of Cox-2 mRNA that binds to CUGBP2 and error barsrepresent the standard deviation of the mean from three independentexperiments. ANOVA testing showed significant differences between the controland cell samples and was set at p < 0.001 (***); M: marker.
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f03: molecular interaction between cyclooxygenase-2 (Cox-2) mRNA and CUGPP2protein. A: fluorescence microscopy analyses of the cross-talk between Cox-2mRNA and CUGBP2 in H9c2 cells after parasitic infection. The CUGBP2 protein(fluorescein isothiocyanate labelled) localised preferentially to the nucleus(4’,6 diamino-2-phenylindole labelled) and co-localised in the cytoplasmicregion with Cox-2 mRNA (Alexa Fluor labelled and indicated with arrowheads) andwith eukaryotic initiation factor (eIF4G) (cyanine 5 labelled). Bars = 50 μm;B: co-immunoprecipitation (Co-IP) of CUGBP2 and Cox-2 mRNA in H9c2 cellsfollowed by western blotting analyses (using total cell extract). RecombinantCUGBP2 (rCUGBP2) and total H9c2 extracts were used as control in the assay. TheCo-IP precisely captured the protein from H9c2 total cell extract and themolecules that bind to it; C: formaldehyde RNA cross-linking assay using Cox-2mRNA and H9c2 cell extract was performed followed by the Co-IP to capture themolecules that binds to CUGBP2. Quantitative polymerase chain reaction (qPCR)quantified the total amount of Cox-2 mRNA that binds to CUGBP2 and error barsrepresent the standard deviation of the mean from three independentexperiments. ANOVA testing showed significant differences between the controland cell samples and was set at p < 0.001 (***); M: marker.

Mentions: Next, the molecular connections between Cox-2 mRNA and the CUGBP2 protein inH9c2-infected cells were investigated with immunofluorescence analyses. Fig. 3A demonstrates that CUGBP2 preferentiallyco-localises to the cell nucleus (Roberts et al.1997, Savkur et al. 2001, Timchenko et al. 2001) and under a pro-inflammatorystimulus, CUGBP2 shuffling to the cytoplasm was correlated with the Cox-2 mRNAdistribution, as previously described (Timchenko et al.2001). More intense translocation of CUGBP2 from the nucleus to cytoplasmicstructures defined as stress granules (SGs) (Kedersha etal. 2005, Gareau et al. 2011) wasobserved at the 2 h time-point during T. cruzi cellular infection,which corresponded to the highest transcriptional level of Cox-2 mRNA produced by theinfected H9c2 cells. To confirm whether the subcellular structures observed undermicroscopy were real SGs, the eIF4G protein was positively labelled and visualised(Fig. 3A). In addition, CUGBP2 gene silencingwas also performed in H9c2 cells to verify the relevance of the protein in the molecularshuffling of messenger RNAs. However no single clone survived longer than 48 h (data notshown). Next, to corroborate the physical interaction of the above two molecules thatsupport the molecular trafficking, Co-IP and formaldehyde RNA cross-linking wereperformed. The results presented in Fig. 3Bdemonstrated that CUGBP2 is present in H9c2 cell extract and rCUGBP2 was used aspositive control of the assay. To prove the physical interaction between the protein andCox-2 mRNA, the messenger molecule was cross-linked to the protein before the Co-IPprocedure. The linkage of the target RNA and CUGBP2 was verified in qPCR (Fig. 3C).


Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction.

Moraes KC, Diniz LF, Bahia MT - Mem. Inst. Oswaldo Cruz (2015)

molecular interaction between cyclooxygenase-2 (Cox-2) mRNA and CUGPP2protein. A: fluorescence microscopy analyses of the cross-talk between Cox-2mRNA and CUGBP2 in H9c2 cells after parasitic infection. The CUGBP2 protein(fluorescein isothiocyanate labelled) localised preferentially to the nucleus(4’,6 diamino-2-phenylindole labelled) and co-localised in the cytoplasmicregion with Cox-2 mRNA (Alexa Fluor labelled and indicated with arrowheads) andwith eukaryotic initiation factor (eIF4G) (cyanine 5 labelled). Bars = 50 μm;B: co-immunoprecipitation (Co-IP) of CUGBP2 and Cox-2 mRNA in H9c2 cellsfollowed by western blotting analyses (using total cell extract). RecombinantCUGBP2 (rCUGBP2) and total H9c2 extracts were used as control in the assay. TheCo-IP precisely captured the protein from H9c2 total cell extract and themolecules that bind to it; C: formaldehyde RNA cross-linking assay using Cox-2mRNA and H9c2 cell extract was performed followed by the Co-IP to capture themolecules that binds to CUGBP2. Quantitative polymerase chain reaction (qPCR)quantified the total amount of Cox-2 mRNA that binds to CUGBP2 and error barsrepresent the standard deviation of the mean from three independentexperiments. ANOVA testing showed significant differences between the controland cell samples and was set at p < 0.001 (***); M: marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4489448&req=5

f03: molecular interaction between cyclooxygenase-2 (Cox-2) mRNA and CUGPP2protein. A: fluorescence microscopy analyses of the cross-talk between Cox-2mRNA and CUGBP2 in H9c2 cells after parasitic infection. The CUGBP2 protein(fluorescein isothiocyanate labelled) localised preferentially to the nucleus(4’,6 diamino-2-phenylindole labelled) and co-localised in the cytoplasmicregion with Cox-2 mRNA (Alexa Fluor labelled and indicated with arrowheads) andwith eukaryotic initiation factor (eIF4G) (cyanine 5 labelled). Bars = 50 μm;B: co-immunoprecipitation (Co-IP) of CUGBP2 and Cox-2 mRNA in H9c2 cellsfollowed by western blotting analyses (using total cell extract). RecombinantCUGBP2 (rCUGBP2) and total H9c2 extracts were used as control in the assay. TheCo-IP precisely captured the protein from H9c2 total cell extract and themolecules that bind to it; C: formaldehyde RNA cross-linking assay using Cox-2mRNA and H9c2 cell extract was performed followed by the Co-IP to capture themolecules that binds to CUGBP2. Quantitative polymerase chain reaction (qPCR)quantified the total amount of Cox-2 mRNA that binds to CUGBP2 and error barsrepresent the standard deviation of the mean from three independentexperiments. ANOVA testing showed significant differences between the controland cell samples and was set at p < 0.001 (***); M: marker.
Mentions: Next, the molecular connections between Cox-2 mRNA and the CUGBP2 protein inH9c2-infected cells were investigated with immunofluorescence analyses. Fig. 3A demonstrates that CUGBP2 preferentiallyco-localises to the cell nucleus (Roberts et al.1997, Savkur et al. 2001, Timchenko et al. 2001) and under a pro-inflammatorystimulus, CUGBP2 shuffling to the cytoplasm was correlated with the Cox-2 mRNAdistribution, as previously described (Timchenko et al.2001). More intense translocation of CUGBP2 from the nucleus to cytoplasmicstructures defined as stress granules (SGs) (Kedersha etal. 2005, Gareau et al. 2011) wasobserved at the 2 h time-point during T. cruzi cellular infection,which corresponded to the highest transcriptional level of Cox-2 mRNA produced by theinfected H9c2 cells. To confirm whether the subcellular structures observed undermicroscopy were real SGs, the eIF4G protein was positively labelled and visualised(Fig. 3A). In addition, CUGBP2 gene silencingwas also performed in H9c2 cells to verify the relevance of the protein in the molecularshuffling of messenger RNAs. However no single clone survived longer than 48 h (data notshown). Next, to corroborate the physical interaction of the above two molecules thatsupport the molecular trafficking, Co-IP and formaldehyde RNA cross-linking wereperformed. The results presented in Fig. 3Bdemonstrated that CUGBP2 is present in H9c2 cell extract and rCUGBP2 was used aspositive control of the assay. To prove the physical interaction between the protein andCox-2 mRNA, the messenger molecule was cross-linked to the protein before the Co-IPprocedure. The linkage of the target RNA and CUGBP2 was verified in qPCR (Fig. 3C).

Bottom Line: However, how the parasite interaction modulates COX-2 activity is poorly understood.Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures.Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular, Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho, Rio Claro, SP, Brasil.

ABSTRACT
Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

Show MeSH
Related in: MedlinePlus