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

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.

FIG 1

Measurement of amebic morphology. The Amnis Imagestream imaging cytometer was used to measure the morphology of fixed amebic trophozoites. (A) Bright-field image of an elongated trophozoite. (B) The pixels which constituted the bright-field image of the trophozoites (MASK). From the mask, the computational features of area and circular morphology were derived. A high circularity score resulted from an internally consistent measurement of cell radius. Representative images show trophozoites characterized and “tagged” as either circular (C) or elongated (D).

FIG 2

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 (χ² P 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).

FIG 3

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.

FIG 4

Competitive advantage in intestinal invasion conferred by the expression of dominant positive URE3-BP in the murine model of amebic colitis. CBA mice were infected intracecally with a 1:1 ratio of amebae induced to express dominant positive URE3-BP to the control induced pSTOP strain (A) or with uninduced amebae (B). Mice were sacrificed 3 weeks after infection, and the ratios of dominant positive URE3-BP to the control pSTOP strain were determined by qPCR for both the tissue-associated and luminal amebae (n = 3). Values were corrected by the input data, and statistical significance was determined using a paired t test (P = 0.007).

FIG 5

Increase in liver abscess size following infection with amebae expressing dominant positive URE3-BP. (A) Infected liver abscess from amebae expressing the dominant positive URE3-BP protein injected into the right lobe of the liver (black arrow) and abscess from the control induced pSTOP transfectants injected in the left liver lobe (white arrow). (B) Effect of induction of dominant positive URE3-BP on abscess size (n = 16; statistical significance [P = 0.0342] was determined using the Wilcoxon matched-pair signed-rank test). The use of the unpaired Mann-Whitney test also gave statistical significance (P = 0.019).