Subcellular Distribution and In Situ Localization of the Acid Phosphatase of Entamoeba histolytica

Abstract

Introduction In invasive amebiasis, attempts have been made to correlatethe biochemical and functional properties observed in vitro with the virulence of trophozoites, using as a measure of vir-ulence their ability to produce amebic hepatic abscess in ani-mal models (1). Invasion of host tissues by the trophozoitesis accompanied by contact-dependent cell lysis and phagocy-tosis, as well as by the secretion of lytic components (2). Re-cently, we reported that Entamoeba histolytica contains andsecretes an acid phosphatase (AP), in contrast with Enta-moeba dispar , which possesses the activity but is unable to se-crete it (3). Additionally, we have described the purificationand properties of the E. histolytica acid phosphatase (4). Inthis work, we present data concerning the subcellular local-ization by immunofluorescence of the AP in fixed trophozo-ites, as well as its in situ localization in infected tissue by usingpolyclonal antibodies prepared against purified AP. Materials and Methods Cell culture. E. histolytica HM-1:IMSS strain was grownas described (4). Purification of AP. The enzyme was purified as reportedpreviously (4). Polyclonal anti-AP antibody production. Balb/c mice wereimmunized subcutaneously at least four times with DEAE-enriched fractions. Samples were emulsified with Titer Maxthe first and second times, and with Melox at subsequenttimes. Immunofluorescence analysis. Trophozoites were fixedwith 4% paraformaldehyde for 1 h at 37 8 C and permeabi-lized with PBS-T (0.2% Triton X-100) for 20 min at roomtemperature. Cells were incubated with anti-AP antibodies(1:200), then incubated with a FITC-conjugated secondarygoat-antimouse antibody (1:50), and visualized by confocalmicroscopy. Immunohistochemistry for light and electron microscopydetection of AP. For abscess development, the reportedtechnique was followed (5). After different times postinocu-lation, animals were anesthetized with sodium pentobarbital(94.5 mg/kg of body weight) and the portal vein uncoveredto perfuse the liver with 4% paraformaldehyde, 1% glutaral-dehyde, and 15% picric acid in 0.1 M sodium cacodylatebuffer for 30 min. The liver was dissected and small frag-ments 0.1–0.2 cm thick were transferred to the same fixa-tive and incubated for at least 2 h. For light microscopy,paraffin sections were treated as reported previously (5), us-ing the anti-AP antibody at a 1:100 dilution. For electronmicroscopy, liver samples were included in LR-White, theanti-AP antibody used at a 1:50 dilution, and the secondaryantibody (a 20-nm coloidal gold-labeled goat-antimouseIgG) at a 1:20 dilution (5). Results and Discussion Subcellular localization of E. histolytica AP . To learn moreabout the subcellular localization of AP in trophozoites, weprepared polyclonal monospecific antibodies against AP.DEAE-eluted material was used directly to immunize mice.We found that the antibodies recognized a high-molecular-weight component of approximately 120 kDa present in sol-ubilized fraction (data not shown). Immunofluorescence localization of E. histolytica AP. Thestaining pattern of the anti-AP antibody showed a peripheralstaining in nonpermeabilized cells, whereas in permeabi-