Entamoeba dispar Contains but Does Not Secrete Acid Phosphatase as Does Entamoeba histolytica*1

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Experimental Parasitology 92, 219–222 (1999) Article ID expr.1999.4416, available online at http://www.idealibrary.com on

RESEARCH BRIEF Entamoeba dispar Contains but Does Not Secrete Acid Phosphatase as Does Entamoeba histolytica

P. Talama´s-Rohana,1 M. M. Aguirre-Garcı´a, M. Anaya-Ruiz, and J. L. Rosales-Encina Experimental Pathology Department, CINVESTAV-IPN, Avenida IPN 2508, Colonia San Pedro Zacatenco, 07000 Me´xico, D.F., Me´xico

Talama´s-Rohana, P., Aguirre-Garcı´a, M. M., Anaya-Ruiz, M., and Rosales-Encina, J. L. 1999. Entamoeba dispar contains but does not secrete acid phosphatase as does Entamoeba histolytica. Experimental Parasitology 92, 219–222. q 1999 Academic Press Index Descriptors and Abbreviations: acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2); Entamoeba histolytica; parasitic protozoan; pathogenicity; virulence; AP, acid phosphatase; MAP, membrane-bound acid phosphatase; p-NPP, pnitrophenylphosphate; SAP, secreted acid phosphatase.

invadens IP and PZ; E. histolytica-like Laredo (Lo´pez-Revilla and Go´mez-Domı´nguez 1988); and E. dispar (Espinosa-Cantellano et al. 1997). They were harvested during logarithmic growth by centrifugation at 200g (Diamond et al. 1978). Conditioned medium to assay SAP content was prepared by collecting E. histolytica HM-1 trophozoites at different time points during growth and then subculturing them in serum-free medium for 5 h and for 8 h in the case of SAP analysis from different Entamoeba strains. AP activity was determined using p-nitrophenylphosphate ( p-NPP) as substrate (Dissing et al. 1979). “In situ” activity of AP was detected in 10% SDS–PAGE, mixing samples with b-mercapthoethanol-free sample buffer (15% sucrose and methylene blue in 0.125 M Tris–HCl, pH 6.8) and loaded onto the gel without boiling. The gel was washed with 50 mM sodium acetate, 10 mM MgCl2, pH 5.5, and incubated overnight at 378C in a buffer containing 400 ml of 2.5 mol/L sodium acetate, pH 5.2, 400 ml of 12.5 mg/ml of Naphtol AS-BI in N,N8-dimethylformamide, and 9.2 ml of H2O in the presence of Fast Garnet GBC. Subcellular fractionation of trophozoites was carried out as described (Aley et al. 1980) and solubilized fraction was prepared by lysing cells in buffer A (10 mM Imidazole–HCl, pH 7.2, 5 mM EDTA, 1 mM EGTA, 0.1% b-mercaptoethanol) in a nitrogen pump (300 psi, 10 min); cell lysate was centrifuged at 16,000g for 15 min and the pellet was resuspended in buffer B (buffer A plus 2% Triton X-100 and 2 mM instead of 5 mM EDTA) and centrifuged as before. Since most of the strains used in this study had been maintained in axenic condition for several years, it was necessary to recover them from liver before testing its virulence. This was done as reported (Shibayama et al. 1997). After recovering and adapting the strains in axenic culture for 6 weeks, they were evaluated in their capacity for abscess production (Tsutsumi et al. 1992). As shown in Fig. 1A, we confirmed that AP is actively secreted by trophozoites to the culture medium. We also found that the end of the log phase (between 45 and 68 h of cultivation) was the optimal secretion time of the enzyme. Maximum secretion in serum-free medium occurs

Amoebiasis is the human infection caused by the protozoan parasite Entamoeba histolytica. Its pathogenic process is a multifactorial phenomenon that occurs in three steps: adhesion, cytotoxic and cytolytic effect, and phagocytosis (Martı´nez-Palomo et al. 1993). Several authors have proposed that lytic enzymes may participate during the second part of this process. Among these enzymes, acid phosphatase (AP) has been suggested as a virulence factor (Fastag-de-Shor 1973) and to date it hasn’t been possible to detect whether or not AP really participates in damaging the host. Amoebic AP was originally described as a membrane-associated (MAP) (Serrano et al. 1977; Aley et al. 1980) ´ as well as a secreted (SAP) (Aguirre-Garcıa et al. 1997) enzyme. In this work, we present data comparing the secreted and membranebound AP activity present in E. histolytica HM-1 and the levels of SAP and MAP in different pathogenic strains (with different degrees of virulence) and E. dispar, a nonpathogenic Entamoeba, and make a correlation with their ability to produce amoebic liver abscess. Strains used were E. histolytica HM-1, HM-2, HM-38, HK-9; E.

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0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. SAP and MAP determination in Entamoeba histolytica HM-1. (A) Trophozoites were grown and followed for different periods (18 to 91 h), collected, washed, and subcultured in serum-free medium for 5 h. SAP activity was measured as optical densities (405 nm) of p-nitrophenol produced by 1 3 106 cells. (B) Trophozoites were fractionated according to Aley et al. (1980) to obtain soluble components (SC), plasma membranes (PM), internal membranes (IM), and nonvesiculated membranes (NVM). Data are presented as percentage of activity associated with each of the subcellular fractions, considering as 100% the activity present in a total homogenate. “In situ” detection of activity in 10% SDS–PAGE (insets) with a chromogenic substrate. The mobility of molecular mass standards is indicated on the left. Data are representative of three separate experiments.

between 10 and 12 h (Aguirre-Garcı´a et al. 1997). Acid phosphatases are considered as markers for lysosomal compartments and are found in the lumen of these organelles (Lemansky et al. 1985); in the case of E. histolytica, this parasite does not have typical lysosomes. Instead, ´ the cell is full of vacuoles (Martınez-Palomo et al. 1993). Eaton et al. (1969) postulated that AP was associated with surface-active lysosomes. Fastag de Shor et al. (1973) presented evidence of a positive

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signal for AP present in the filiform processes at the periphery of the trophozoites, as well as in the pseudopoda. Serrano et al. (1977) suggested that AP is present in fraction V, a fraction containing plasma membranes as well as digestive vacuoles, whereas Aley et al. (1980) showed that AP is associated mainly with internal membranes. Therefore, it was important to determine how the nonsecreted AP activity was subcellularly distributed in the trophozoites. We found that 83.68% of AP activity was associated with internal membranes (Fig. 1B), confirming the results reported by Aley (1980). When fixed trophozoites were incubated with the chromogenic substrate, the enzymatic activity was heterogeneously distributed among the trophozoite population. Some of the trophozoites were heavily labeled, whereas others gave a very weak signal; in some cells, the enzymatic label was observed on the trophozoite surface (data not shown). One possible explanation for this heterogeneous distribution could be that trophozoites are in different stages of the cell cycle. On the other hand, the enzyme activity may be induced during the active process of phagocytosis in mature cells. However, in Acanthamoeba, phagocytosis does not lead to an increase in AP activity in the cells or in the medium (Ryter and Bowers 1976). Heterogeneity of AP activity on electrophoretic behavior has been reported previously (Agrawal et al. 1987) . By “in situ” detection of AP on SDS–gel, we found for SAP two bands: a weak band with a molecular weight of 200 kDa and a stronger band of 97 kDa (Fig. 1A, inset). For MAP, we found only the 97-kDa band (Fig. 1B, inset). Differences in molecular weight isoforms of SAP and MAP could be due to different levels of glycosylation and/or aggregation. In some microorganisms, AP activity has been associated with pathogenic mechanisms. In the pathogenesis of primary amoebic meningoencephalitis caused by Naegleria fowleri, the specific activity of AP is higher than in other Naegleria spp (De Jonckheere and Dierickx 1982). In Giardia, the presence of AP in the peripheral vacuoles suggests a possible role in giardiosis (Feely and Dyer 1987). Some authors (Fastag-de-Shor et al. 1973; Palacios-Beristain et al. 1986) have proposed that amoebic AP could participate in the pathogenic mechanism of this parasitic protozoa. To test whether or not AP could be related with pathogenicity and/or virulence, we analyzed the content of both MAP and SAP in different strains. To be sure about the pathogenicity and/or virulence of these strains, trophozoites (HM1, HM-38, HM-2, and HK-9) were recovered after 3 days post-infection from intraperitoneally inoculated hamsters. E. dispar has been recently tested for its ability to infect but not to develop liver abscess (EspinosaCantellano et al. 1997). The more virulent of the pathogenic strains, HM-1 and HM-38, were able to produce amoebic liver abscess with a percentage of damage around 40%; however, HM-2 and HK-9, the low-virulence E. histolytica strains, as well as the nonpathogenic E. dispar were unable to develop amoebic abscess (data not shown). Considering the AP activity in HM1 as 100%, the enzyme activity ranged from 84% for MAP and 57% for SAP in HK-9 to 116% for MAP and 118% for SAP in HM2. When we measured MAP content in E. dispar, we found a rather high content of the enzyme (87%), quite similar to the content in E. histolytica. However, this strain was unable to secrete the activity to the culture medium. E. histolytica-like Laredo had the lower content of MAP (6%) (Fig. 2). SAP activity in this strain was not determined due to its intrinsic low content of AP activity. There is considerable confusion in the literature regarding the use of the terms “pathogenicity” and “virulence”, at least with respect to E. histolytica. The best definition of both terms was given by Gladstone (1970): “Pathogenicity is distinct from virulence. Pathogenicity is the

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E. dispar CONTAINS BUT DOES NOT SECRETE ACID PHOSPHATASE

FIG. 2. Comparison of SAP and MAP content between different E. histolytica strains, E. dispar and E. histolytica-like Laredo. All E. histolytica strains were recovered from liver and after five to six subcultures were tested for AP content. Due to variability in lot activity during sample preparation, results were expressed as percentage of AP activity present in E. histolytica strain HM-1. Data are representative of three different experiments. E. histolytica strains (HM1, HM38, HM2, HK9); E. dispar (DIS); E. histolytica-like Laredo (LAR). SAP (M); MAP ( ).

ability to produce disease without specifying the conditions. Virulence is the capacity to produce disease in a given set of circumstances. Various degrees of virulence may be found depending on conditions . . . but the organisms are either pathogenic or not”. Strains isolated from dysenteric patients (HM1, HM-2, HK-9, HM-38), are pathogenic, although they show different degrees of virulence (Clark and Diamond 1994). Since continuous culture in axenic conditions largely affects the virulence of strains (Tsutsumi et al. 1992), we used liver-passaged strains for the assessment of virulence. In the present study, we found that all the pathogenic strains had a high content of AP activities. Strikingly, the infective but nonpathogenic strain E. dispar, although with a high content of MAP, was unable to secrete the AP activity (only 12%). Strains that are not infective for humans and/or hamster had less AP activity, 41% for SAP and 39% for MAP in IP strain of E. invadens (data not shown) and as low as 6% for MAP in E. histolytica-like Laredo. In conclusion, we have shown that this enzyme has at least two isoforms, 200 and 97 kDa. Given the differences between E. histolytica and E. dispar regarding the ability to secrete AP, we believe that this enzyme can be an important element of the pathogenic mechanisms in these parasites. However, the fact that AP content did not correlate with virulence suggests that other existing differences between virulent and avirulent strains may participate in the production of the disease. We cannot ignore the possibility that this enzyme may participate in digestive processes as has been reported previously (Mora-Galindo 1988). We have reported a partial purification and characterization of MAP (Anaya-Ruiz et al. 1997), but additional studies are in progress to further purify and characterize this enzymatic activity.

donated by Dr. Rube´n Lo´pez-Revilla, from the Cell Biology Department, and E. dispar by Dr. Martha Espinosa-Cantellano, from the Experimental Pathology Department, both at CINVESTAV. We thank ´ ˜´ Adrian Trejo-Carmona and Ma. Guadalupe Castanon for the culture ´ of the amoeba strains, Amelia Angel and Leticia Perez for liver abscess ´ production in the hamster model, and Amelia Rıos for her technical assistance. We also thank Dr. Krishanu Sengupta for critically reading the manuscript.)

(This work was partially supported by Grant 3687-M9607 from ´ CONACyT, Mexico. M.M.A.G. and M.A.R. are recipients of CONACyT fellowships. HM-2, HK-9, HM-38, IP, and PZ strains were kindly

Clark, C. G., and Diamond, L. S. 1994. Pathogenicity, virulence and Entamoeba histolytica. Parasitology Today 10, 46–47.

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Received 30 November 1998; accepted with revision 25 March 1999

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