Downregulation of an Entamoeba histolytica rhomboid protease reveals roles in regulating parasite adhesion and phagocytosis

Abstract

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

Fig. 1.

The G3-based approach downregulates expression of EhROM1. (A) Schematic of the G3 EhROM1 silencing construct used in our studies. A 538-bp 5′ region of EhROM1 was fused downstream of the EhAp-A cassette. (B) Northern blot analysis of EhROM1 expression in G3 and ROM(KD) parasites. Twenty micrograms of total RNA was resolved on an a formaldehyde gel and probed sequentially with probes for EhROM1 and Lgl1, which served as a loading control. (C) RT-PCR analysis of EhROM1 expression levels in G3 and ROM(KD) parasites. One-microgram aliquots of cDNA from G3 and ROM(KD) cell lines were subjected to 35 cycles of PCR for EhROM1; ssRNA was used as a loading control. PCR from samples without RT served as controls to exclude genomic DNA contamination. (D) Western blot analysis of EhROM1 in G3 and ROM(KD) parasites. Parasite lysates were resolved on a 10% SDS-PAGE gel and probed with anti-EhROM1 antibody (1:1,000), detected with HRP-conjugated secondary antibody (1:5,000), and developed using ECL. Blots were scanned on a Kodak Image Station 4000R.

Fig. 2.

ROM(KD) parasites form caps that are morphologically indistinguishable from G3 parasites. Capping was induced in G3 and ROM(KD) parasites in vitro by incubation of parasites with ConA on ice followed by incubation at 37°C for 15 min. Parasites were then fixed and stained with anti-EhROM1 (1:20) (A) or anti-Hgl (3F4 [1:30] plus 7F4 [1:30] antibodies) (B) followed by incubation with fluorescent secondary antibodies. Parasites were stained with Texas Red-streptavidin (1:500) in order to image caps (ConA contains a biotin tag). All imaging was performed on a Leica CTR6000 microscope, using a BD CARVII confocal unit. Image analysis and deconvolution were performed using the LAS-AF program from Leica. Deconvolution was performed in 10 iterations, with a single deconvolved slice shown for each sample.

Fig. 3.

Complement resistance and motility are not significantly altered in ROM(KD) parasites. (A) Complement resistance was measured by incubation of trophozoites with 10% NHS for 20 or 40 min at 37°C. Following incubation with complement, parasites were stained with 0.2% trypan blue to assess cell viability. The averages of two experiments, each with a single replicate, are shown with standard deviations. (B) Motility was measured by fluorescently labeling parasites with the cell tracker dye CMFDA. Parasites were added to the upper chamber of a transwell system in serum-free medium and allowed to migrate into the lower chamber containing complete medium for 4 h at 37°C. After incubation parasites in the lower chamber were quantified by reading on a fluorescence plate reader. The averages of three independent experiments, each with three replicates, are shown with standard deviations. Data are shown as the percentage of G3 motility.

Fig. 4.

Rhomboid protease inhibition results in a defect in adhesion to untreated CHO cells. (A) Adhesion was measured with a CHO cell rosette assay for G3 and ROM(KD) parasites. Parasites were mixed with CHO cells at 4°C for 2 h, after which parasites with three or more CHO cells attached were counted as positive. Data shown are an average of six experiments, each with two replicates. Standard deviations are shown. *, P < 0.001. (B) Adhesion for HM-1:IMSS parasites. The CHO cell rosette assay was modified by applying 100 μM DMSO (mock control),100 μM DCI (a serine protease inhibitor with activity for rhomboid proteases), or 100 μM PMSF (a serine protease inhibitor with no activity against rhomboid proteases) during the parasite-CHO cell 2-h incubation. Data shown are averages of two or three independent experiments, each with two replicates. Standard deviations are shown. *, P < 0.002.

Fig. 5.

No significant changes in expression, protein levels, or localization of the Gal/GalNAc lectin were noted in the ROM(KD) parasites. (A) Expression levels of Hgl and Lgl-1 transcripts were assayed by RT-PCR and matched the array data (Table 1); ssRNA was used as a control. Microarray data have been deposited at NCBI (see Materials and Methods). (B) Hgl protein expression was measured by Western blot analysis. Blots were probed with antibodies to Hgl (H85; 1:50) or actin (1:250) as a loading control. Western blot detection was performed using ECL. (C) ELISA measurement of E. histolytica lectin from the whole-cell lysates of G3 and ROM(KD) parasites. The parasites were lysed, and the final protein concentration was adjusted to 50 ng. The amount of lectin was quantified by measuring the optical density at 450 nm. (D) ELISA measurement of E. histolytica lectin from conditioned medium of G3 or ROM(KD) parasites, which was prepared by growing log-phase trophozoites in serum-free medium for 24 h. The protein concentration was adjusted to 400 ng, and lectin was quantitated using the manufacturer's instructions. Data shown are averages of three independent experiments with standard deviations. (E) Subcellular localization of Hgl was analyzed by staining both permeabilized (P) and nonpermeabilized (NP) parasites with anti-Hgl antibodies (3F4 [1:30] plus 7F4 [1:30]). All imaging was performed on a Leica CTR6000 microscope, using a BD CARVII confocal unit. Image analysis and deconvolution were performed using the LAS-AF program from Leica. Deconvolution was performed in 10 iterations, with a single deconvolved slice shown for each sample.

Fig. 6.

ROM(KD) parasites show a defect in phagocytosis of red blood cells and rice starch. (A) A total of 5 × 10⁷ HRBC were incubated with 5 × 10⁵ trophozoites in a ratio of 100:1 in PBS for 15 min at 37°C, followed by lysis of extracellular RBC and measurement of ingested erythrocytes by lysis in 90% formic acid, followed by spectrophotometric determinations at 397 nm. The results represent the means and standard deviations of four independent experiments and are expressed as a percentage of the parent G3 strain erythrophagocytosis level. *, P < 0.001. (B) A total of 1 × 10⁵ trophozoites were incubated for 1 h with 0.004% rice starch solution followed by fixation, permeabilization, and staining with 1% Lugol's solution at room temperature for 5 min. Parasites with one or more ingested starch grains were considered positive for rice starch phagocytosis. Data are averages of three independent experiments with standard deviations. *, P < 0.006.

Fig. 7.

Adherence to apoptotic treated cells was not reduced in ROM(KD) parasites, but phagocytosis of these cells was decreased. (A) Adherence to staurosporine-treated cells decreased in the parental strain but not in ROM(KD) parasites. CHO cells were treated either with 100 μM staurosporine aglycone or with 100 μM DMSO for 1 h at 4°C, and a CHO cell rosette assay was performed. Data are averages of three independent experiments. Standard deviations are shown. *, P < 0.002 for G3 (DMSO treated) versus G3 (staurosporine treated) or P < 0.003 for G3 (DMSO treated) versus ROM(KD) (staurosporine treated) cells. (B) Phagocytosis of staurosporine-treated CHO cells was decreased in ROM(KD) parasites. CHO cells were treated with 100 μM staurosporine or with 100 μM DMSO for 1 h at 4°C and subsequently incubated for 15 min at 37°C with parasites (ratio, 1:1). The cells were fixed, permeabilized, and stained with 1% (vol/vol) trichrome stain. Parasites with one or more ingested CHO cells were considered positive for phagocytosis. Data are expressed as the percentage of G3 DMSO-treated levels and are averages of three independent experiments. Standard deviations are shown. *, P < 0.003 (DMSO treated) or P < 0.007 (staurosporine treated).