A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

Amoebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. This parasite undergoes a fundamental cell differentiation process from proliferative trophozoite to dormant cyst, termed “encystation.” The cysts formed by encystation are solely responsible for the transmission of amoebiasis; therefore, Entamoeba encystation is an important subject from both biological and medical perspectives. Here, we have established a flow cytometry strategy for not only determining the percentage of formed cysts but also for monitoring changes in cell populations during encystation. This strategy together with fluorescence microscopy enables visualization of the cell differentiation process of Entamoeba encystation. We also standardized another flow cytometry protocol for counting live trophozoites. These two different flow cytometry techniques could be integrated into 96-well plate-based bioassays for monitoring the processes of cyst formation and trophozoite proliferation, which are crucial to maintain the Entamoeba life cycle. The combined two systems enabled us to screen a chemical library, the Pathogen Box of the Medicine for Malaria Venture, to obtain compounds that inhibit either the formation of cysts or the proliferation of trophozoites, or both. This is a prerequisite for the development of new drugs against amoebiasis, a global public health problem. Collectively, the two different 96-well plate-based Entamoeba bioassay and flow cytometry analysis systems (cyst formation and trophozoite proliferation) provide a methodology that can not only overcome the limitations of standard microscopic counting but also is effective in applied as well as basic Entamoeba biology

Characterization of the receptors for mycobacterial cord factor in Guinea pig.

Guinea pig is a widely used animal for research and development of tuberculosis vaccines, since its pathological disease process is similar to that present in humans. We have previously reported that two C-type lectin receptors, Mincle (macrophage inducible C-type lectin, also called Clec4e) and MCL (macrophage C-type lectin, also called Clec4d), recognize the mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM). Here, we characterized the function of the guinea pig homologue of Mincle (gpMincle) and MCL (gpMCL). gpMincle directly bound to TDM and transduced an activating signal through ITAM-bearing adaptor molecule, FcRγ. Whereas, gpMCL lacked C-terminus and failed to bind to TDM. mRNA expression of gpMincle was detected in the spleen, lymph nodes and peritoneal macrophages and it was strongly up-regulated upon stimulation of zymosan and TDM. The surface expression of gpMincle was detected on activated macrophages by a newly established monoclonal antibody that also possesses a blocking activity. This antibody potently suppressed TNF production in BCG-infected macrophages. Collectively, gpMincle is the TDM receptor in the guinea pig and TDM-Mincle axis is involved in host immune responses against mycobacteria

gpMincle is associated with FcRγ.

<p>(A) Interaction of gpMincle with gpFcRγ. HEK293T cells were transfected with HA-tagged gpMincle alone or together with Flag-tagged gpFcRγ Total lysates were immunoprecipitated with anti-HA mAb and blotted with anti-Flag and anti-HA polyclonal antibodies (pAbs). Total lysates were also blotted with anti-Flag pAb. (B) Surface expression of gpMincle. HEK293T cells were transfected with gpMincle-HA alone or together with Flag-gpFcRγ. Surface expression of gpMincle was detected by anti-HA pAb. Data are presented as representative of two separate experiments.</p

Expression of gpMincle mRNA.

<p>(A) Tissue distribution of gpMincle mRNA. mRNA expression of gpMincle in indicated tissues (cLN, cutaneous lymph node; mLN, mesenteric lymph node) and cells (BALC, bronchoalveolar lavage cell; PBC, peripheral blood cell; BMC, bone marrow cell; PMø, thioglycollate-elicited peritoneal macrophage) was detected by PCR. PCR was performed by increased cycle numbers (20, 24, 28 for β-actin and 32, 36, 40 for Mincle). (B) gpMincle mRNA is induced upon stimulation. Macrophages were stimulated with indicated concentrations of zymosan (left panel) or TDM (right panel). mRNA expression of gpMincle was analyzed by RT-PCR at 8 h after stimulation. Data are presented as mean ± s.d. (B) and representative of two separate experiments.</p

gpMincle directly binds to TDM.

<p>Ig-fusion proteins of guinea pig Mincle (gpMincle-Ig), mouse Mincle (mMincle-Ig) and human Mincle (hMincle-Ig) were incubated with plate-coated TDM. Bound proteins were detected with anti-hIgG-HRP. Data are presented as mean ± s.d. and representative of two separate experiments.</p

gpMincle functions as an activating receptor for TDM.

<p>(A) gpMincle transduces activation signal through gpFcRγ. NFAT-GFP reporter cells were transfected with Flag-tagged gpMincle alone or together with gpFcRγ. Reporter cells were stimulated with plate-coated anti-Flag mAb for 24 h. Induction of NFAT-GFP was analyzed by flow cytometry. (B) gpMincle is a TDM receptor. Indicated reporter cells were stimulated with plate-coated TDM for 24 h. Induction of NFAT-GFP was analyzed by flow cytometry. (C) gpMincle recognizes mycobacteria. Reporter cells were stimulated with <i>M. bovis</i> BCG (left panel) and heat-killed <i>M. tuberculosis</i> H37Ra (right panel) for 24 h. Induction of NFAT-GFP was analyzed by flow cytometry. Data are presented as mean ± s.d. and representative of two or three separate experiments.</p

Establishment of anti-gpMincle mAb.

<p>(A) Anti-gpMincle mAb blocks interaction of gpMincle-Ig with TDM. gpMincle-Ig (3 µg/ml) were incubated with plate-coated TDM in the presence of anti-gpMincle mAb or mouse IgG. Bound proteins were detected with anti-hIgG-HRP. (B) Surface staining by anti-gpMincle mAb. Indicated reporter cells were stained with anti-gpMincle mAb (5H4, upper panels), anti-mMincle mAb (4A9, middle panels) or anti-hMincle mAb (13D10-H11, lower panels). Open histograms show staining with isotype control IgG. (C) Anti-gpMincle mAb activates NFAT-GFP reporter cells. Indicated reporter cells were stimulated with plate-coated anti-gpMincle mAb for 24 h. Induction of NFAT-GFP was analyzed by flow cytometry. (D) Anti-gpMincle mAb blocks TDM recognition. Indicated reporter cells were treated with anti-gpMincle followed by stimulation with TDM (10 ng/well) for 24 h. Induction of NFAT-GFP was analyzed by flow cytometry. Data are presented as mean ± s.d. (A, C, D) and representative of two or three separate experiments.</p

Characterization of gpMCL.

<p>(A) Immunoblot of gpMCL. HEK293T cells were transfected with HA-tagged mMCL or gpMCL. Total lysates were blotted with anti-HA mAb (left panel) or immunoprecipitated with anti-HA pAb and blotted with anti-HA mAb (right panel). (B) gpMCL is associated with gpFcRγ. HEK293T cells were transfected with HA-tagged gpMCL alone or together with Flag-tagged gpFcRγ Total lysates were immunoprecipitated with anti-HA pAb and blotted with anti-Flag pAb and anti-HA mAb. Total lysates were also blotted with anti-Flag pAb. (C) Surface expression of gpMCL. HEK293T cells were transfected with HA-tagged gpMCL alone or together with gpFcRγ-IRES-GFP. HEK293T cells were also transfected with HA-tagged mMCL together with mFcRγ-IRES-GFP. Surface expression of gpMCL or mMCL was detected by anti-HA pAb. (D) gpMCL fails to recognize TDM. Indicated reporter cells were stimulated with plate-coated TDM for 24 h. Induction of NFAT-GFP was analyzed by flow cytometry. Data are presented as mean ± s.d. (D) and representative of two or three separate experiments.</p