Chitinase secretion by encysting Entamoeba invadens and transfected Entamoeba histolytica trophozoites: localization of secretory vesicles, endoplasmic reticulum, and Golgi apparatus

Abstract

Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.

FIG. 1

RT-PCR of arf, erd2, and chitinase (chit) mRNAs in nontransfected E. histolytica trophozoites and E. invadens encysting for 0 to 48 h. Ethidium staining of PCR products, separated on agarose gels, was electronically reversed for ease of reproduction.

FIG. 2

Confocal micrographs of chitinase-associated secretory vesicles of encysting E. invadens identified with anti-chitinase antibodies. (A) Chitinase-associated secretory vesicles were absent from trophozoites (data not shown) but were widely distributed in amebae encysting for 24 h. (B) Anti-chitinase antibodies in nonpermeabilized amebae mark secreted products on the parasite surface. Bars, 5 μm.

FIG. 3

Confocal micrographs of E. histolytica trophozoites transfected with their own chitinase gene under an actin promoter. Anti-chitinase antibodies (red) demonstrated secretory vesicles in transfected, fixed, and permeabilized E. histolytica trophozoites. These vesicles were independent of pinocytotic vacuoles marked with FITC-dextran (green in panel A) or phagocytotic vacuoles marked with GFP-labeled bacteria (green in panel B). Anti-chitinase antibodies failed to stain the surface of nonpermeabilized, transfected parasites, thus demonstrating that the enzyme is secreted and shed (data not shown). Chitinase-associated vesicles were about the same size and abundance as lysosomes, as marked by the fluorescent substrate (Arg–Arg–4-methoxy-2-naphthylamide) of cysteine proteases (yellow in panel C). Bars, 5 μm.

FIG. 4

Confocal micrographs of nontransfected E. histolytica trophozoites which were fixed, permeabilized, and stained with antibodies to Ariel, a homologue of the vaccine candidate SREHP. While most Ariel-associated secretory vesicles (red) were independent of pinocytosed FITC-dextran (green in panel A), some localized with phagocytotic vacuoles marked with GFP-labeled bacteria (green in panel B). Anti-Ariel antibodies outlined the surface of nonpermeabilized trophozoites (red in panel C). Bars, 5 μm.

FIG. 5

Confocal micrographs of amebic secretory vesicles, cytosol, and putative ER. (A) Intact chitinase was targeted in transfected E. histolytica trophozoites to secretory vesicles, which were small and numerous. (B) Similar vesicles were visualized with anti-Ariel antibodies in nontransfected parasites. (C) A truncated chitinase lacking an N-terminal signal sequence went to the cytosol, which has dark vesicles against a bright background. (D) This is the same appearance as that of nontransfected amebae stained with antibodies to ADH1. (E) A modified chitinase with a C-terminal KDEL peptide was retained in a putative ER, which was composed of many fewer vesicles than the secretory vesicles marked by unmodified chitinase or Ariel. (F) A similar pattern of staining was observed with myc-labeled BiP, which is retained by its native C-terminal KDEL in the putative ER. Bars, 5 μm.

FIG. 6

ARF alignment. Predicted ORF of a segment of the E. histolytica (Eh) arf gene aligned with ARF segments of E. invadens (Ei), G. lamblia (Gl; GenBank accession number S29008), Plasmodium falciparum (Pf, U57370), Dictyostelium discoideum (Dd; AJ000063), Saccharomyces cerevisiae (Sc; M35158), and Bos taurus (Bt; A45422). Dashes indicate identities with the E. histolytica ARF, while periods indicate gaps. Vertical boxes indicate locations of conserved pyrophosphate-binding loop (GLDAAGKT), switch region (DVGG), and guanine-recognition motif (NKQD) (3, 34).

FIG. 7

NJ tree of ARF shown in Fig. 6 as well as ARF of Xenopus laevis (Xl; GenBank accession number U31350), Drosophila melanogaster [Dm(A); S62079], Cryptococcus neoformans (Cn; L25115), and Schizosaccharomyces pombe (Sp; L09551), and ARF-like proteins of D. melanogaster [Dm(AL); A40438], Homo sapiens (Hs; L28997), P. falciparum [Pf(AL); U57370], and Leishmania tarentolae (Lt; X97072). Branch lengths are proportional to differences between adjacent sequences, while numbers at each node indicate the extent of bootstrap support (of 100 replicates). Unmarked nodes occurred in fewer than 50 replicates.

FIG. 8

Confocal micrographs of E. invadens parasites stained with heterologous anti-ARF antibodies. (A) ARF was present in large vesicles (presumed Golgi apparatus) surrounding the nucleus of trophozoites. (B) The ARF-stained vesicles of trophozoites were disrupted by Brefeldin A. (C) The Golgi apparatus of encysting parasites was similar when stained with anti-ARF. Micrographs shown in panels A and B represent a single section each, while the micrograph in panel C is a composite of multiple sections. Bars, 5 μm.

FIG. 9

Confocal micrographs of E. histolytica and E. invadens parasites stained with antibodies to Golgi-associated coatomer protein ɛ-COP. Vesicles that contain ɛ-COP were relatively few and large in trophozoites of E. histolytica (A), trophozoites of E. invadens (B), or encysting E. invadens (C). The micrograph shown in panel A represents a single section, while the micrographs in panels B and C are each composed of multiple sections. Bars, 5 μm.