Limits...
The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro.

Stadelmann B, Hanevik K, Andersson MK, Bruserud O, Svärd SG - BMC Microbiol. (2013)

Bottom Line: In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro.Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell- and Molecular Biology, Uppsala University, BMC, Box 596, Uppsala SE-751 24, Sweden. staffan.svard@icm.uu.se.

ABSTRACT

Background: Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections.

Results: RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.

Conclusions: Giardia affects the host's arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

Show MeSH

Related in: MedlinePlus

Giardia up-regulates flavohemoglobin upon nitric oxide (NO) stress. Human intestinal epithelial cells (HCT-8) were stimulated for NO production by addition of cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)). Giardia trophozoites of the isolate WB were added to the NO-producing host cells and to control cells after 40 h. Samples were measured for expression of the NO-detoxifying protein flavohemoglobin (FlHb) at indicated time points. A, Upon interaction with NO-producing cells FlHb was induced in trophozoites on the RNA level compared to the control gene GL50803_17364 as assessed by qPCR in technical quadruplicates. This highly significant difference is indicated by asterisks. B, Western blot detecting the expression of FlHb and the control protein Tat1 in Giardia upon interaction with HCT-8 cells with and without NO-induction. C, Quantification of the Western blot bands (B) by image J software clearly shows the induction of FlHb protein in Giardia trophozoites upon interaction with NO-induced host cells. The results are representative for similar results obtained by three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4225669&req=5

Figure 5: Giardia up-regulates flavohemoglobin upon nitric oxide (NO) stress. Human intestinal epithelial cells (HCT-8) were stimulated for NO production by addition of cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)). Giardia trophozoites of the isolate WB were added to the NO-producing host cells and to control cells after 40 h. Samples were measured for expression of the NO-detoxifying protein flavohemoglobin (FlHb) at indicated time points. A, Upon interaction with NO-producing cells FlHb was induced in trophozoites on the RNA level compared to the control gene GL50803_17364 as assessed by qPCR in technical quadruplicates. This highly significant difference is indicated by asterisks. B, Western blot detecting the expression of FlHb and the control protein Tat1 in Giardia upon interaction with HCT-8 cells with and without NO-induction. C, Quantification of the Western blot bands (B) by image J software clearly shows the induction of FlHb protein in Giardia trophozoites upon interaction with NO-induced host cells. The results are representative for similar results obtained by three independent experiments.

Mentions: Giardia trophozoites were interacted with host IECs that were previously induced to produce NO by addition of cytokines (as described above). Compared to non-stimulated IEC controls, Giardia trophozoites up-regulated FlHb expression on the RNA and protein level (Figure 5) when the IECs produced NO. This could provide another layer of NO protection for the parasite (Figure 1).


The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro.

Stadelmann B, Hanevik K, Andersson MK, Bruserud O, Svärd SG - BMC Microbiol. (2013)

Giardia up-regulates flavohemoglobin upon nitric oxide (NO) stress. Human intestinal epithelial cells (HCT-8) were stimulated for NO production by addition of cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)). Giardia trophozoites of the isolate WB were added to the NO-producing host cells and to control cells after 40 h. Samples were measured for expression of the NO-detoxifying protein flavohemoglobin (FlHb) at indicated time points. A, Upon interaction with NO-producing cells FlHb was induced in trophozoites on the RNA level compared to the control gene GL50803_17364 as assessed by qPCR in technical quadruplicates. This highly significant difference is indicated by asterisks. B, Western blot detecting the expression of FlHb and the control protein Tat1 in Giardia upon interaction with HCT-8 cells with and without NO-induction. C, Quantification of the Western blot bands (B) by image J software clearly shows the induction of FlHb protein in Giardia trophozoites upon interaction with NO-induced host cells. The results are representative for similar results obtained by three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225669&req=5

Figure 5: Giardia up-regulates flavohemoglobin upon nitric oxide (NO) stress. Human intestinal epithelial cells (HCT-8) were stimulated for NO production by addition of cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)). Giardia trophozoites of the isolate WB were added to the NO-producing host cells and to control cells after 40 h. Samples were measured for expression of the NO-detoxifying protein flavohemoglobin (FlHb) at indicated time points. A, Upon interaction with NO-producing cells FlHb was induced in trophozoites on the RNA level compared to the control gene GL50803_17364 as assessed by qPCR in technical quadruplicates. This highly significant difference is indicated by asterisks. B, Western blot detecting the expression of FlHb and the control protein Tat1 in Giardia upon interaction with HCT-8 cells with and without NO-induction. C, Quantification of the Western blot bands (B) by image J software clearly shows the induction of FlHb protein in Giardia trophozoites upon interaction with NO-induced host cells. The results are representative for similar results obtained by three independent experiments.
Mentions: Giardia trophozoites were interacted with host IECs that were previously induced to produce NO by addition of cytokines (as described above). Compared to non-stimulated IEC controls, Giardia trophozoites up-regulated FlHb expression on the RNA and protein level (Figure 5) when the IECs produced NO. This could provide another layer of NO protection for the parasite (Figure 1).

Bottom Line: In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro.Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell- and Molecular Biology, Uppsala University, BMC, Box 596, Uppsala SE-751 24, Sweden. staffan.svard@icm.uu.se.

ABSTRACT

Background: Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections.

Results: RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline.

Conclusions: Giardia affects the host's arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

Show MeSH
Related in: MedlinePlus