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

qRT-PCR measurement of in vitro expression levels and verification of primer specificity. qRT-PCR was conducted on the URE3-BP mRNAs. The y axis indicates double-stranded DNA-dependent SYBR green 1 fluorescence at 530 nm. The x axis represents the PCR cycle number. (A) Primers specific for the dominant positive URE3-BP expression plasmid. (B) Primers specific for the control transcript of the induced pSTOP transfectant. ■, RNA isolated from dominant positive URE3-BP; ☐, induced pSTOP transfectant control; ●, background. Lower control mRNA levels were consistently observed in vitro, as shown in panel B, presumably due to the reduced stability of the untranslated transcript.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: qRT-PCR measurement of in vitro expression levels and verification of primer specificity. qRT-PCR was conducted on the URE3-BP mRNAs. The y axis indicates double-stranded DNA-dependent SYBR green 1 fluorescence at 530 nm. The x axis represents the PCR cycle number. (A) Primers specific for the dominant positive URE3-BP expression plasmid. (B) Primers specific for the control transcript of the induced pSTOP transfectant. ■, RNA isolated from dominant positive URE3-BP; ☐, induced pSTOP transfectant control; ●, background. Lower control mRNA levels were consistently observed in vitro, as shown in panel B, presumably due to the reduced stability of the untranslated transcript.

Mentions: To test whether the expression of dominant positive URE3-BP conferred a competitive advantage in establishing an invasive infection, CBA/J mice were infected intracecally with a 1:1 ratio of amebae induced to express dominant positive URE3-BP to the control pSTOP strain. We first established that we were able to distinguish dominant positive URE3-BP transcripts from the induced control pSTOP plasmid in mRNA isolated from the initial mixed inocula (Fig. 3). Lower levels of control mRNA transcribed from pSTOP were consistently observed in vitro, as shown in Fig. 3B, presumably due to the reduced stability of the untranslated transcripts, and verified in vivo the inducible expression of the URE3-BP transcript 7 days after infection (data not shown). We compared the ratios of amebic transfectants isolated from the intestinal lumen and the epithelium. Amebae were considered luminal if they were isolated from the cecal contents and tissue associated if they were isolated from the mucosa removed from the luminal surface of the cecum by scraping. Mice were sacrificed 3 weeks after infection. Primer specificity allowed quantitative PCR (qPCR) to be used as a surrogate marker for the proportion of the amebic population carrying the dominant URE3-BP expression construct. The ratio of infecting amebae determined by qPCR was used to correct the qPCR ratio of amebae isolated from three different infected mice (Fig. 3). Comparisons of uninduced amebae showed no statistically significant difference. In the induced amebae, the ratio of dominant positive URE3-BP to the induced pSTOP transfectant control doubled in the tissue-associated amebae but not in the amebae isolated from the cecal lumen (2.4 ± 0.3 versus 1.3 ± 0.4; unpaired t test P = 0.017, paired t test P = 0.0071) (Fig. 4). Amebae expressing the dominant positive URE3-BP protein were therefore more effective at associating with the host epithelium, indicating a greater potential for establishing an invasive infection within the gut.


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)

qRT-PCR measurement of in vitro expression levels and verification of primer specificity. qRT-PCR was conducted on the URE3-BP mRNAs. The y axis indicates double-stranded DNA-dependent SYBR green 1 fluorescence at 530 nm. The x axis represents the PCR cycle number. (A) Primers specific for the dominant positive URE3-BP expression plasmid. (B) Primers specific for the control transcript of the induced pSTOP transfectant. ■, RNA isolated from dominant positive URE3-BP; ☐, induced pSTOP transfectant control; ●, background. Lower control mRNA levels were consistently observed in vitro, as shown in panel B, presumably due to the reduced stability of the untranslated transcript.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: qRT-PCR measurement of in vitro expression levels and verification of primer specificity. qRT-PCR was conducted on the URE3-BP mRNAs. The y axis indicates double-stranded DNA-dependent SYBR green 1 fluorescence at 530 nm. The x axis represents the PCR cycle number. (A) Primers specific for the dominant positive URE3-BP expression plasmid. (B) Primers specific for the control transcript of the induced pSTOP transfectant. ■, RNA isolated from dominant positive URE3-BP; ☐, induced pSTOP transfectant control; ●, background. Lower control mRNA levels were consistently observed in vitro, as shown in panel B, presumably due to the reduced stability of the untranslated transcript.
Mentions: To test whether the expression of dominant positive URE3-BP conferred a competitive advantage in establishing an invasive infection, CBA/J mice were infected intracecally with a 1:1 ratio of amebae induced to express dominant positive URE3-BP to the control pSTOP strain. We first established that we were able to distinguish dominant positive URE3-BP transcripts from the induced control pSTOP plasmid in mRNA isolated from the initial mixed inocula (Fig. 3). Lower levels of control mRNA transcribed from pSTOP were consistently observed in vitro, as shown in Fig. 3B, presumably due to the reduced stability of the untranslated transcripts, and verified in vivo the inducible expression of the URE3-BP transcript 7 days after infection (data not shown). We compared the ratios of amebic transfectants isolated from the intestinal lumen and the epithelium. Amebae were considered luminal if they were isolated from the cecal contents and tissue associated if they were isolated from the mucosa removed from the luminal surface of the cecum by scraping. Mice were sacrificed 3 weeks after infection. Primer specificity allowed quantitative PCR (qPCR) to be used as a surrogate marker for the proportion of the amebic population carrying the dominant URE3-BP expression construct. The ratio of infecting amebae determined by qPCR was used to correct the qPCR ratio of amebae isolated from three different infected mice (Fig. 3). Comparisons of uninduced amebae showed no statistically significant difference. In the induced amebae, the ratio of dominant positive URE3-BP to the induced pSTOP transfectant control doubled in the tissue-associated amebae but not in the amebae isolated from the cecal lumen (2.4 ± 0.3 versus 1.3 ± 0.4; unpaired t test P = 0.017, paired t test P = 0.0071) (Fig. 4). Amebae expressing the dominant positive URE3-BP protein were therefore more effective at associating with the host epithelium, indicating a greater potential for establishing an invasive infection within the gut.

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