Comparison of hemolytic activity of the intermediate subunit of Entamoeba histolytica and Entamoeba dispar lectins

Abstract

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica has roles in pathogenicity and induction of protective immunity in rodent models of amoebiasis. Recently, the intermediate subunit of the lectin, Igl1, of E. histolytica has been shown to have hemolytic activity. However, the corresponding lectin is also expressed in a non-virulent species, Entamoeba dispar, and another subunit, Igl2, is expressed in the protozoa. Therefore, in this study, we compared the activities of Igl1 and Igl2 subunits from E. histolytica and E. dispar using various regions of recombinant Igl proteins expressed in Escherichia coli. The recombinant E. dispar Igl proteins had comparable hemolytic activities with those of E. histolytica Igl proteins. Furthermore, Igl1 gene-silenced E. histolytica trophozoites showed less hemolytic activity compared with vector-transfected trophozoites, indicating that the expression level of Igl1 protein influences the activity. These results suggest that the lower hemolytic activity in E. dispar compared with E. histolytica reflects the lower expression level of Igl1 in the E. dispar parasite.

Fig 1. Recombinant Igl proteins used in the study.

Recombinant Igl proteins were constructed with a His-tag at the N-terminus. Full length (F-Igl), N-terminus and middle (NM-Igl), middle (M-Igl), and C-terminus (C-Igl) Igl1 and Igl2 of Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) were expressed in E. coli and purified using Ni columns. Estimated molecular weights of each protein including the His-tag are shown [ExPASy Compute pI/Mw tool (http://web.expasy.org/compute_pi/)].

Fig 2. Time-course of hemolytic activities of EhF-Igl1, EhF-Igl2, EdF-Igl1, EdF-Igl2 and EhNM-Igl1 proteins.

Recombinant Igl proteins (2 μM, 50 μl) were incubated with 50 μl of 2% (v/v) HoRBCs in PBS for the indicated periods. A. Protein purity and amount were confirmed by SDS-PAGE using NuPAGE Novex Bis-Tris (4–12% gradient) gels with 1 μg of each protein. B. HoRBCs were incubated in a 96-well plate after the indicated periods with Igls. Representative images of 5 independent studies are shown. C. Concentrations of hemoglobin (Hb) released in the supernatant of samples incubated for 8 h. Data are the mean ± SD from 5 independent experiments. EhF-Igls and EdF-Igls showed significantly (**p< 0.01 by ANOVA with Dunn test) higher hemolytic activities than EhNM-Igl1 and PBST. EhF1: EhF-Igl1, EhF2: EhF-Igl2, EdF1: EdF-Igl1, EdF2: EdF-Igl2, NM: EhNM-Igl1.

Fig 3. Time-course of hemolytic activities of EhC-Igl1, EdC-Igl1 and EhM-Igl1 proteins.

Recombinant Igl proteins (2 μM, 50 μl) were incubated with 50 μl of 2% (v/v) HoRBCs in PBS for the indicated periods. A. Protein purity and amount were confirmed by SDS-PAGE using NuPAGE Novex Bis-Tris (4–12% gradient) gels with 1 μg of each protein. B. HoRBCs were incubated in a 96-well plate after the indicated periods with Igls. Representative images of 5 independent studies are shown. C. Concentrations of hemoglobin (Hb) released in the supernatant of samples incubated for 8 h. Data are the mean ± SD from 5 independent experiments. *p< 0.05, **p< 0.01 by ANOVA with Dunn test. EhC1: EhC-Igl1, EdC1: EdC-Igl1, EhM1: EhM-Igl1.

Fig 4. Hemolytic activities of trophozoites of Entamoeba dispar strains.

A and B. E. histolytica (Eh), E. dispar SAW1734RclAR cultured with Pseudomonas aeruginosa (Ed) or E. dispar CYNO9:TPC (Cyno9) trophozoites (1×10⁵) were incubated with HoRBCs at 37°C for 1 h. A. Images of HoRBCs in a 96-well plate just after mixing with trophozoites (0 hr) and after incubation for 1 h with trophozoites (1 hr). B. Released hemoglobin (Hb) concentration in the supernatant of the mixture of trophozoites and HoRBCs after incubation for 1 h at 37°C. C and D. E. histolytica (Eh), E. dispar SAW1734RclAR cultured with Crithidia fasciculata (Ed) or E. dispar CYNO9:TPC (Cyno9) trophozoites (1×10⁵) were incubated with HoRBCs at 37°C for 1 h. C. Images of HoRBCs in a 96-well plate just after mixing with trophozoites (0 hr) and after incubation for 1 h with trophozoites (1 hr). D. Released hemoglobin (Hb) concentration in the supernatant of the mixture of trophozoites and HoRBCs after incubation for 1 h at 37°C. Data are the mean ± SD from 5 independent experiments. **p< 0.01, *p< 0.05 by ANOVA with Dunn test.

Fig 5. Real-time PCR analysis of Igl genes from E. histolytica and E. dispar.

Expression levels of Igl1 (open bars) and Igl2 (filled bars) in trophozoites from E. histolytica strain with an empty vector (Cont), E. histolytica gsIgl1 strains (gsIgl1A and gsIgl1B) and E. dispar SAW1734RclAR strain (Ed) were compared using actin as an internal standard. Expression levels are shown as values relative to the mean expression level of Igl2 from Cont. Vertical bars indicate the S.E. of the mean from 3 experiments. *p< 0.05, **p< 0.01 by ANOVA with Dunn test.

Fig 6. Establishment of Igl-attenuated Entamoeba histolytica trophozoites.

A. Dot blot analysis of reactivity of ED2-495 against E. histolytica and E. dispar Igls. B. Western blot of Igls in control, gene-silenced E. histolytica and E. dispar trophozoites. C. Relative quantification of Igls expressed in control, gene-silenced E. histolytica and E. dispar trophozoites. D. Suppression of Igl1 protein expression by gene-silencing confirmed by IFA. Control: vector transfected E. histolytica trophozoite, gsIgl1: Igl1 gene-silenced E. histolytica trophozoite, AS: ATP sulfurylase, DIC: differential interference contrast. Amino acid sequence alignment of ATP sulfurylase between E. histolytica and E. dispar is shown in S1 Fig.

Fig 7. Hemolytic activities of Igl1-attenuated Entamoeba histolytica trophozoites.

Vector transfected or Igl1 gene-silenced E. histolytica trophozoites (1×10⁵) were incubated with HoRBCs at 37°C for 1 h. A and C. Images of HoRBCs in a 96-well plate just after mixing with trophozoites (0 hr) and after incubation for 1 h with trophozoites (1 hr). B and D. Released hemoglobin (Hb) concentration in the supernatant of a mixture of trophozoites and HoRBCs after incubation for 1 h at 37°C. Data are the mean ± SD from 5 independent experiments. **p< 0.01 by ANOVA with Dunn test. Control: vector transfected E. histolytica trophozoite, gsIgl1A and gsIgl1B: Igl1 gene-silenced E. histolytica trophozoite.