Limits...
Regulation of Virulence of Entamoeba histolytica by the URE3-BP Transcription Factor.

Gilchrist CA, Moore ES, Zhang Y, Bousquet CB, Lannigan JA, Mann BJ, Petri WA - MBio (2010)

Bottom Line: A comparison of in vivo to in vitro parasite gene expression demonstrated that 39% of in vivo regulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P < 0.0001).Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% +/- 6% versus 14% +/- 5%; P = 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P = 0.017), and a 3-fold increase in liver abscess size (0.1 +/- 0.1 g versus 0.036 +/- 0.1 g; P = 0.03).These results support a role for URE3-BP in virulence regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, School of Medicine, University of Virginia, Charlottesville, Virginia, USA.

ABSTRACT
It is not understood why only some infections with Entamoeba histolytica result in disease. The calcium-regulated transcription factor upstream regulatory element 3-binding protein (URE3-BP) was initially identified by virtue of its role in regulating the expression of two amebic virulence genes, the Gal/GalNac lectin and ferredoxin. Here we tested whether this transcription factor has a broader role in regulating virulence. A comparison of in vivo to in vitro parasite gene expression demonstrated that 39% of in vivo regulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P < 0.0001). Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% +/- 6% versus 14% +/- 5%; P = 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P = 0.017), and a 3-fold increase in liver abscess size (0.1 +/- 0.1 g versus 0.036 +/- 0.1 g; P = 0.03). These results support a role for URE3-BP in virulence regulation.

No MeSH data available.


Related in: MedlinePlus

Amebae expressing constitutively active URE3-BP exhibited an elongated trophozoite morphology. A representative graph is shown with the output of the circular morphology feature (y axis) and trophozoite area (x axis). (A) Trophozoites expressing dominant positive URE3-BP. (B) Induced control pSTOP transfectants. There was a statistically significant increase in the number of elongated amebae detected in trophozoites expressing dominant positive URE3-BP (χ2P value, <0.0001). (C) Graph of average data derived from experiments on 2 separate days. The y axis indicates the percentage of elongated amebae, and the x axis shows the plasmid carried (n = 5; P = 0.0014; standard errors are shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2912668&req=5

f2: Amebae expressing constitutively active URE3-BP exhibited an elongated trophozoite morphology. A representative graph is shown with the output of the circular morphology feature (y axis) and trophozoite area (x axis). (A) Trophozoites expressing dominant positive URE3-BP. (B) Induced control pSTOP transfectants. There was a statistically significant increase in the number of elongated amebae detected in trophozoites expressing dominant positive URE3-BP (χ2P value, <0.0001). (C) Graph of average data derived from experiments on 2 separate days. The y axis indicates the percentage of elongated amebae, and the x axis shows the plasmid carried (n = 5; P = 0.0014; standard errors are shown).

Mentions: One measure of alterations in surface composition is changes in cell morphology. The morphological impact of URE3-BP was measured in trophozoites expressing dominant positive URE3-BP. The Amnis Imagestream cytometer was used to compare trophozoites expressing dominant positive URE3-BP [pEF(2)URE3-BP] with those of the equivalent control strain (induced pSTOP) (Fig. 1 and 2.) A two-dimensional bright-field image of the three-dimensional ameba was analyzed (Fig. 1A). To minimize the effects of the orientation of the amebae in flow, extended-depth-of-field imaging was used. The IDEAS program was used to identify single in-focus trophozoites and to derive the aspect ratio or circularity of the image (Fig. 1B to D). To achieve the latter goal, the IDEAS software repeatedly measured cell radius; a high “circularity score” resulted from an internally consistent measurement. The automated process allowed detailed information on cell morphology to be gathered at the population level. To define the amebic shape, two different functions were used: trophozoite area and circular morphology (Fig. 2A and B). An arbitrary gate was drawn to capture the most elongated amebae. Transfectants that expressed dominant positive URE3-BP had a statistically significant 2-fold increase in the number of trophozoites with an elongated phenotype [30% ± 6% elongated in pEF(2)URE3-BP versus 14% ± 5% in pSTOP; n = 5, P = 0.0014] (Fig. 2C). The elongated phenotype could reflect surface changes related to motility and chemotaxis. Induction of URE3-BP had previously been shown to increase transwell migration, suggesting a possible role in the regulation of cellular motility (6).


Regulation of Virulence of Entamoeba histolytica by the URE3-BP Transcription Factor.

Gilchrist CA, Moore ES, Zhang Y, Bousquet CB, Lannigan JA, Mann BJ, Petri WA - MBio (2010)

Amebae expressing constitutively active URE3-BP exhibited an elongated trophozoite morphology. A representative graph is shown with the output of the circular morphology feature (y axis) and trophozoite area (x axis). (A) Trophozoites expressing dominant positive URE3-BP. (B) Induced control pSTOP transfectants. There was a statistically significant increase in the number of elongated amebae detected in trophozoites expressing dominant positive URE3-BP (χ2P value, <0.0001). (C) Graph of average data derived from experiments on 2 separate days. The y axis indicates the percentage of elongated amebae, and the x axis shows the plasmid carried (n = 5; P = 0.0014; standard errors are shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2912668&req=5

f2: Amebae expressing constitutively active URE3-BP exhibited an elongated trophozoite morphology. A representative graph is shown with the output of the circular morphology feature (y axis) and trophozoite area (x axis). (A) Trophozoites expressing dominant positive URE3-BP. (B) Induced control pSTOP transfectants. There was a statistically significant increase in the number of elongated amebae detected in trophozoites expressing dominant positive URE3-BP (χ2P value, <0.0001). (C) Graph of average data derived from experiments on 2 separate days. The y axis indicates the percentage of elongated amebae, and the x axis shows the plasmid carried (n = 5; P = 0.0014; standard errors are shown).
Mentions: One measure of alterations in surface composition is changes in cell morphology. The morphological impact of URE3-BP was measured in trophozoites expressing dominant positive URE3-BP. The Amnis Imagestream cytometer was used to compare trophozoites expressing dominant positive URE3-BP [pEF(2)URE3-BP] with those of the equivalent control strain (induced pSTOP) (Fig. 1 and 2.) A two-dimensional bright-field image of the three-dimensional ameba was analyzed (Fig. 1A). To minimize the effects of the orientation of the amebae in flow, extended-depth-of-field imaging was used. The IDEAS program was used to identify single in-focus trophozoites and to derive the aspect ratio or circularity of the image (Fig. 1B to D). To achieve the latter goal, the IDEAS software repeatedly measured cell radius; a high “circularity score” resulted from an internally consistent measurement. The automated process allowed detailed information on cell morphology to be gathered at the population level. To define the amebic shape, two different functions were used: trophozoite area and circular morphology (Fig. 2A and B). An arbitrary gate was drawn to capture the most elongated amebae. Transfectants that expressed dominant positive URE3-BP had a statistically significant 2-fold increase in the number of trophozoites with an elongated phenotype [30% ± 6% elongated in pEF(2)URE3-BP versus 14% ± 5% in pSTOP; n = 5, P = 0.0014] (Fig. 2C). The elongated phenotype could reflect surface changes related to motility and chemotaxis. Induction of URE3-BP had previously been shown to increase transwell migration, suggesting a possible role in the regulation of cellular motility (6).

Bottom Line: A comparison of in vivo to in vitro parasite gene expression demonstrated that 39% of in vivo regulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P < 0.0001).Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% +/- 6% versus 14% +/- 5%; P = 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P = 0.017), and a 3-fold increase in liver abscess size (0.1 +/- 0.1 g versus 0.036 +/- 0.1 g; P = 0.03).These results support a role for URE3-BP in virulence regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, School of Medicine, University of Virginia, Charlottesville, Virginia, USA.

ABSTRACT
It is not understood why only some infections with Entamoeba histolytica result in disease. The calcium-regulated transcription factor upstream regulatory element 3-binding protein (URE3-BP) was initially identified by virtue of its role in regulating the expression of two amebic virulence genes, the Gal/GalNac lectin and ferredoxin. Here we tested whether this transcription factor has a broader role in regulating virulence. A comparison of in vivo to in vitro parasite gene expression demonstrated that 39% of in vivo regulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P < 0.0001). Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% +/- 6% versus 14% +/- 5%; P = 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P = 0.017), and a 3-fold increase in liver abscess size (0.1 +/- 0.1 g versus 0.036 +/- 0.1 g; P = 0.03). These results support a role for URE3-BP in virulence regulation.

No MeSH data available.


Related in: MedlinePlus