Parasite Immunology, 2011, 33, 255–264
DOI: 10.1111/j.1365-3024.2010.01275.x
Myeloperoxidase binds to and kills Entamoeba histolytica trophozoites J. PACHECO-YPEZ,1 V. RIVERA-AGUILAR,2 E. BARBOSA-CABRERA,3 S. ROJAS HERNNDEZ,3 R. A. JARILLO-LUNA4 & R. CAMPOS-RODRGUEZ5 1 Laboratorio de Microscopia Electrnica, Facultad Mexicana de Medicina, Universidad La Salle Fuentes 17, Tlalpan, Mxico, 2Departamento de Microbiologa, Unidad de Biologa Tecnologa y Prototipos (UBIPRO), Facultad de Estudios Superiores (FES)-Iztacala, Universidad Nacional Autnoma de Mxico, Tlalnepantla Estado. de Mxico, Mxico, 3Seccin de Estudios de Posgrado e Investigacin, Escuela Superior de Medicina, Instituto Politcnico Nacional, Plan de San Lus y Daz Mirn, Mxico, 4Departamento de Morfologa, Escuela Superior de Medicina, Instituto Politcnico Nacional, Plan de San Lus y Daz Mirn, Mxico, 5Departamento Bioqumica, Escuela Superior de Medicina, Instituto Politcnico Nacional, Plan de San Lus y Daz Mirn, Mxico
SUMMARY During amebic invasion, neutrophils are a key component in either protecting against invading trophozoites or contributing to tissue damage. Upon degranulating or being lysed, neutrophils release toxic substances that can kill amebas as well as damage host tissue. In a previous study we identified a protein from nonspecifically stimulated peritoneal exudates of hamster that has peroxidase and marked amebicidal activity. In the current study we analyzed the in vitro amebicidal effect of purified hamster myeloperoxidase (MPO). The results demonstrate that MPO must bind directly to the surface of Entamoeba histolytica trophozoites in order to carry out amebicidal activity by using the H2O2 produced by the amebas themselves. Myeloperoxidase-incubated amebas showed important morphological and ultrastructural alterations that increased with incubation time. Changes included an increase of vacuoles in the cytoplasm, a decrease of glycogen, alterations of nuclear morphology and disturbances in the plasma membrane culminating in complete ameba destruction. Keywords amebicidal activity, E. histolytica, peroxide, myeloperoxidase, neutrophil
hydrogen
INTRODUCTION Entamoeba histolytica is an enteric human protozoan parasite that causes amoebic colitis and liver abscess. Several Correspondence: R. Campos-Rodrguez, Departamento de Bioqumica. Escuela Superior de Medicina, Instituto Politcnico Nacional, Plan de San Lus y Daz Mirn, C.P. 11340, Mxico (e-mail:
[email protected]). Disclosures: None. Received: 9 July 2010 Accepted for publication: 22 November 2010 2011 Blackwell Publishing Ltd
studies have established that innate immunity is of importance in the host defense against amebiasis (1–3). Neutrophils, a key component of the innate immune response, are the first cell to interact with an invading ameba in both humans and animals (4–6). In resistant individuals and naturally resistant species (e.g. mice and rats), neutrophils are competent effector cells that control amebic infection (7–9). On the other hand, in susceptible individuals and naturally susceptible species (e.g. hamsters and gerbils), the lysis of neutrophils by amebas causes the release of toxic mediators that contribute to the extensive tissue damage seen in invasive diseases (2,5,10,11). The factor determining which of these contrary roles of neutrophils dominate seems to be whether or not the parasite is able to overcome host defenses, a question that is related to the pathogenicity of the parasite, the activation state of neutrophils, and the genetic background of the host (2). It has been proposed that reactive oxygen species (ROS) and nitric oxide produced by activated neutrophils are important innate defense mechanisms against E. histolytica (8,12–16). However, neutrophils either massively degranulate once contacting virulent amebas or are lysed by the amebas, and in both cases release toxic substances such as defensins, serine proteases, superoxide anion, collagenases, lactoferrin, and myeloperoxidase (MPO), which potentially are able to kill trophozoites (17). Myeloperoxidase (MPO; EC 1.11.1.7) is a cationic enzyme found in primary azurophilic granules of neutrophils and primary lysosomes of monocytes. In activated neutrophils, MPO uses hydrogen peroxide (H2O2) generated during the oxidative burst to oxidize chloride ions (Cl), thus producing highly cytotoxic hypochlorous acid (HOCl) (18–20). MPO–H2O2–Cl is the most efficient microbicidal system in neutrophils (19–21). Because MPO
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can also be expressed by cells, the MPO-mediated microbicidal system can operate in the interstitial fluid (20) where it may kill amebas (14). We identified a protein with peroxidase and marked amebicidal activity in the nonspecifically stimulated peritoneal exudates of hamster (22). Upon analysis it was found that this enzyme is a MPO that mediates the killing of trophozoites, but only after directly binding to the surface of E. histolytica. Since the addition of H2O2 was not necessary for MPO amebicidal activity, it seems that the amebas are the source of this molecule.
MATERIALS AND METHODS Amebic cultures Trophozoites of the E. histolytica strain HMI:IMSS were maintained axenically in BI-S33 medium, as previously described (23). The strain was subsequently cloned and passed three times through hamster liver to increase its virulence. The inoculum was prepared from amoebic cultures at 72 h, when they were in the logarithmic growth phase.
Crude preparation of MPO from peritoneal supernatant Male adult golden hamsters (Mesocricetus auratus) were used; handling and experimentation to which they were subjected were carried out in accordance with the institutional animal care committee. Groups of 10–12 hamsters were inoculated intraperitoneally with 8 mL of mineral oil (Sigma, St. Louis, MO, USA) or saline. Seven days after inoculation, the animals were euthanized and the peritoneal cavities of control and mineral oil-treated hamsters were washed with 10 mL of cold Hanks balanced saline solution containing 5 U ⁄ mL heparin. The supernatants were collected by centrifugation at 400 · g for 10 min at room temperature (RT), and stored at )70C.
Purification of MPO The supernatant of the peritoneal lavage was concentrated 10 times by lyophilization, dialyzed in loading buffer (10 mM phosphate, pH 8Æ5), and applied to a DEAESepharose column (2Æ5 · 48 cm). Fraction II, eluted from the DEAE-Sepharose column, was concentrated by lyophilization, and then was dissolved in 2 mL of 10 mM phosphate, pH 8Æ5, and applied to a Sepharose 4B-CL column as previously reported (22). The amebicidal activity in the different fractions was assayed using a trypan blue exclusion assay. Purified MPO was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), and analyzed by Western blotting with a rabbit
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polyclonal anti human IgG MPO antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
SDS-PAGE and Western blot analysis The purified MPO peritoneal fractions were mixed with buffer (2% of SDS), 10% 500 mM Tris, pH 6Æ8, and glycerol, and boiled for 3 min. Twenty microliters of MPO sample were electrophoresed using 10% SDS-PAGE. The gel was washed and stained with Coomassie Brilliant Blue (Bio-Rad, Mexico City, Mexico) for detection of proteins. Additional gel was transferred to a nitrocellulose membrane. The membrane was incubated, blocked by incubation with PBS buffer (pH 7Æ4, containing 10% nonfat dry milk) for 1 h, and subsequently incubated with rabbit polyclonal anti-MPO antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 250. The membrane was washed with 0Æ05% Tween-20 in PBS for 10 min with agitation, and incubated for 1 h with radish peroxidase labeled goat antirabbit IgG antibody (Santa Cruz Biotechnology) diluted 1 : 1000. Finally, the recognized proteins were revealed with the substrate solution (H2O2, 3Æ6 mM 4-chloro-1napthol; Pierce, Rockford, IL, USA). Densitometric analysis of digitalized images was done with MULTI GAUGE 3.0 software (Fuji Photo Film Co., Burbank, CA, USA).
Spectrophotometric assay for quantification of viable amebas A modification of the procedure of Gilbert et al. (24) was carried out using 96-well microtiter plates (Costar, New York, NY, USA). Trophozoites were harvested by centrifuging at 500 · g for 5 min at 4C, then resuspended and adjusted to 1 · 106 trophozoites per mL in PBS. Amebas (1 · 105 amebas in 100 lL) were added to each well, and incubated in the presence of the MPO protein fraction II-1 (1 and 10 lg ⁄ mL) for 2 h. Additional wells with the trophozoite-MPO fraction II-1 were incubated with H2O2 (1 mM), catalase 1 mg ⁄ mL, anti MPO antibody or several MPO inhibitors (see below). Plates were incubated at 37C for 2 h, and then washed with PBS. The remaining attached amebas were incubated with 100 lL of 99% methanol per well at RT, and after 2 min the plates were emptied. Next, wells were stained with 100 lL of 1% violet crystal solution (Difco, Kansas City, KS, USA) for 10 min at RT. The dye was discarded and stained plates were washed with distilled water and allowed to dry. The dye bound to each ameba was solubilized with 100 lL per well of 1% SDS in 50% ethanol. Finally, the optical density of the resulting solution was recorded at 570 ⁄ 620 nm using a Bio-Rad Instruments microplate reader. Each experiment was performed in triplicate. The percentage of viable 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 255–264
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trophozoites was determined by using a standard curve elaborated with 5 · 103 to 5 · 104 amebas per well, since the optical density was proportional to the number of viable amebas.
Amebicidal effect of MPO in presence of anti-MPO antibodies or MPO inhibitors Myeloperoxidase at 1 and 10 lg ⁄ mL was incubated in microcentrifuge tubes during 10 min at RT with anti MPO antibody (Sigma-Aldrich) diluted 1 : 100 or with different MPO inhibitors: (a) 25 lM of salicylhydroxamic acid, (b) 25 lM of benzohydroxamic acid, (c) 25 lM of potassium cyanide, (d) 0Æ1% bovine albumin, and (e) 1000 lM of sodium azide. Entamoeba histolytica trophozoites (2 · 105) in PBS were incubated with MPO at 1 and 10 lg ⁄ mL in a 96-well plate and the supernatant was eliminated and washed with PBS. Some ameba samples in the plate were incubated with the MPO + anti MPO antibody or MPO + MPO inhibitor mixture. In other wells, MPO inhibitors were incubated with amebas, but without MPO. Control amebas were incubated in PBS. Each plate was incubated for 2 h at 37C, after which time the supernatant of the wells was discarded and the plate gently washed with PBS. The amebas were fixed as mentioned above. Then quantification of viable amebas was conducted with violet crystal as previously mentioned. Some incubations of amebas and MPO were made in ameba culture medium containing 15% albumin, and the trophozoite viability was assayed using a trypan blue exclusion analysis.
Binding of MPO to fixed Entamoeba histolytica trophozoites To determine the binding of MPO to amebas, we previously fixed parasites (1 · 106) with paraformaldehyde and then incubated them with 18Æ0 lg ⁄ mL of MPO. Control fixed parasites were incubated in PBS. Then, the parasites were analyzed immunocytochemically.
Amebic alterations induced by MPO To determine the binding and effect of MPO on live E. histolytica trophozoites, amebas (1 · 106) were initially incubated with 18Æ0 lg ⁄ mL of MPO during 5, 10 and 30 min at RT or without treatment (control) and centrifuged at 500 · g for 5 min. The supernatant was discarded and the protozoa were washed with PBS. Then 2% paraformaldehyde in PBS (v ⁄ v) was added, and the amebas were incubated for 30 min at RT with agitation. The paraformaldehyde was eliminated and PBS was added to the 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 255–264
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amebas, which were left until use for immunocytochemical analysis. For transmission electron microscopy (TEM) analysis, samples of live trophozoites were incubated in the presence or absence of MPO for 5, 10 and 30 min, fixed with 2% glutaraldehyde in 100 mM sodium cacodylate buffer for 60 min, and post-fixed with 2Æ0% osmium tetroxide in the same buffer for 60 min. The tetroxide was removed and the samples were dehydrated with increasing concentrations of ethanol and treated with propylene oxide. The parasites were embedded in epoxy resins (Electron Microscopy Science, Hatfiel, PA, USA) and polymerized at 60C for 24 h. Thin sections were stained with uranyl acetate followed by lead citrate. Sections were examined in a Philips EM 208 S transmission electron microscope (Royal Philips Electronics, Amsterdam, The Netherlands).
Immunocytochemical MPO analysis Samples were incubated with 50 mM NH4Cl during 30 min. Then all parasites were washed with PBS and incubated for 1 h at RT with rabbit polyclonal anti-MPO antibody (Sigma-Aldrich) diluted 1 : 100. To samples with MPO, an irrelevant antibody was also added to test antibody specificity. Amebas were incubated for 1 h at RT with peroxidase goat anti-rabbit IgG diluted 1 : 100 (Santa Cruz Biotechnology). The reaction was carried out with a DAB substrate kit (Pierce). Finally, the samples were counterstained with hematoxylin and analyzed by light microscopy (ECLIPSE E600, Nikon Inc., Melville, NY, USA).
Data analysis The data obtained from each experiment were expressed as the mean € SD and the statistical difference between experimental groups was calculated using a one-way analysis of variance (ANOVA). If a significant difference was identified (P < 0Æ05), the means of the respective groups were compared using the Bonferroni t-test. Differences between two means were analyzed by the non-paired Student’s t-test. All analyses were performed using the statistical program SIGMASTAT for Windows version 2.03 (Systat Software Inc., San Jose, CA, USA).
RESULTS Purification of myeloperoxidase The MPO enzyme is a glycoprotein that presents three major isoforms (I, II and III), each having a dimeric structure composed of one heavy and one light subunit (25).
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(a)
(b)
150 K
150 K
100 K
100
100 K
50 K
75 K 60–57
MPO heavy chain
37 K 39
37 K
MPO light chain
13
1
2
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80
MPO
95 60 57
2
Figure 1 SDS-PAGE and Western blot analysis of myeloperoxidase (MPO). The chromatographically purified MPO from peritoneal exudates was subjected to SDS-polyacrylamide gel electrophoresis (panel a) and Western blotting (panel b). Lane 1, standard molecular weight, and lane 2, hamster MPO (2 U ⁄ mg). Proteins were transferred to a nitrocellulose membrane as described in ‘Materials and methods’. The strip was first incubated with the anti-MPO antibody, then with peroxidase labeled goat anti-rabbit IgG. Western blot detected bands at 95 kDa (MPO), 60–57 kDa (MPO a or heavy chain), and 13 kDa (MPO b or light chain); the band of 39 kDa could be an inmature form of MPO.
The purified forms show distinct molecular weights of their heavy and light polypeptide chains (26). In the Western blot analysis the anti-MPO antibody recognized a wide 57–60 kDa band that corresponds to the MPO heavy subunit, a 13 kDa band that corresponds to the MPO light subunit, and two bands of 95 and 39 kDa (Figure 1, panel b) that frequently are found in different MPO preparations (25–32). In the gel (Figure 1, panel a, lane 2) two bands were observed, but four bands were detected by Western blotting using the anti-MPO antibody, probably due to the higher sensitivity of the latter method.
Amebic viability (%)
75 K
60
40
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0
C
MPO MPO H2O2 MPO 1 μg 10 μg H2O2
CAT
H2O2 CAT
H2O2 CAT MPO
Figure 2 Killing of Entamoeba histolytica trophozoites by myeloperoxidase (MPO). Entamoeba histolytica trophozoites were incubated with MPO (1 and 10 lg ⁄ mL), catalase (1 mg ⁄ mL) or H2O2 (1 mm), and amebic viability was assessed by a spectrophotometric assay. Data represent the mean € SD of at least three independent determinations. In a dose dependent manner, MPO, as well as H2O2 alone or in combination with MPO (1 lg ⁄ mL), reduced the number of viable amebas (P < 0Æ001, Student’s t-test). Catalase alone increased the amebic viability, and partially reverted the amebicidal effect of H2O2 (P < 0Æ001, Student’s t-test). Myeloperoxidase exerted an important amebicidal effect in spite of the presence of catalase (P < 0Æ01 compared to H2O2 + CAT).
to a solution with amebas, resulting in a significant increase (10%) in amebic viability compared with the control (P < 0Æ001, Student’s t-test), and a reduction (approximately 50%) in the toxic activity of H2O2. However, catalase had less effect when MPO was present, evidenced by the fact that 75% of amebas were killed in the mixture containing MPO, H2O2 and catalase (Figure 2).
Killing of Entamoeba histolytica trophozoites by MPO
Effect of myeloperoxidase antibody and myeloperoxidase inhibitors
Myeloperoxidase (specific activity: 2Æ5 U ⁄ mg) significantly reduced the number of viable amebas (Figure 2, P < 0Æ001, Student t-test) without any addition of H2O2, killing approximately 90% of the amebas present after 2 h of incubation. More trophozoites were lysed when using the higher concentration (10 lg) of MPO, indicating that the effect was dose dependent. Since the 50% maximum effective concentration (EC50) of MPO was 1Æ5 nM, or approximately 0Æ2 lg ⁄ mL, we used 5 and 50 times this concentration. In addition, the lysis of amebas occurred even when the unbound MPO was previously removed by washing. Although H2O2 was not added, we supposed that it was involved in the amebicidal activity of MPO. To confirm this hypothesis, catalase (a scavenger of H2O2) was added
To confirm that the observed amebicidal effect was dependent on MPO enzymes and on the MPO enzymatic reaction product, an anti-MPO antibody and several MPO inhibitors were tested. Amebas incubated with MPO (2Æ5 U ⁄ mg) plus anti-MPO antibodies showed a significant reduction (75%) of MPO amebicidal activity (P < 0Æ001) compared with amebas incubated with MPO alone (Figure 3). Albumin (0Æ1%), salicylhydroxamic acid and benzohydroxamic acid (25 lM) significantly reduced the amebicidal effect of MPO (Figure 3). Albumin had the highest inhibitory effect, reducing the toxic effect of MPO by approximately 80%, while salicylhydroxamic acid reduced this toxic effect by 15% and benzohydroxamic acid by 40%. The 50% maximum effective concentrations (EC50) of albumin [0Æ2 mg ⁄ mL = 0Æ02%], salicylhydroxamic
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Table 1 Effect of culture medium on amebic cytotoxicity
MPO 1 μg MPO 10 μg
Amebic viability (%)
Amebic viability (%)
80
Control MPO + PBS MPO + medium
60
40
20
0 C MPO
Ab α MPO
Alb
Sal
Ben
Figure 3 Effect of myeloperoxidase (MPO) inhibitors. Amebas were incubated in a solution containing 1 or 10 lg ⁄ mL of MPO, or MPO plus anti myeloperoxidase antibodies (Ab a MPO) in the presence or absence of inhibitors: albumin (Alb, 0Æ1 %), salicylhydroxamic acid (Sal, 25 lm) or benzohydroxamic acid (Ben, 25 lm). After incubation during 2 h, amebic viability was assessed by a spectrophotometric assay. Data represent the mean € SD of at least three independent determinations. The amebicidal activity of MPO was significantly inhibited by the anti MPO antibody (P £ 0Æ001, compared to MPO alone, Bonferroni t-test) and all inhibitors (P < 0Æ01, compared to MPO alone). Albumin had the highest inhibitory effect (P < 0Æ01 compared to Sal and Ben, Bonferroni t-test).
and benzohydroxamic acids were 3, 5, and 50 lM, respectively. The effect of the inhibitors was less evident when a higher concentration (10 lg) of MPO was used (Figure 3). Other MPO inhibitors, such as sodium azide (1000 lM) and potassium cyanide (25 lM) significantly reduced parasite viability (P < 0Æ01) when used alone, and therefore were not useful in this study. The fact that the amebicidal effect was reduced by anti-MPO antibodies and MPO inhibitors seems to indicate that MPO mediates such an effect. To clarify the inhibitory role of albumin in the binding of MPO to E. histolytica trophozoites, we did experiments in which culture medium (containing 15% albumin) was used instead PBS. The amebicidal effect of MPO (7 U ⁄ mg) was significantly reduced in the presence of this culture medium (P < 0Æ001) compared to assays done with PBS (Table 1).
MPO binds to and damages Entamoeba histolytica trophozoites The binding of MPO to microbial surfaces is believed to be the first step in its microbicidal activity (33–36). In the current study MPO (7 U ⁄ mg) was incubated with 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 255–264
82 € 8 10 € 5a 18 € 4
P
10, it avidly binds to a negatively charged surface (20), such as that of the surface membrane (glycocalyx) of E. histolytica and Entamoeba dispar (55,56). The higher negative surface charge of E. dispar than E. histolytica is probably responsible for a greater production of H2O2 (12,15) and therefore a greater binding by MPO to the former species (55), leading to its reduced virulence. Once MPO binds to the surface of the ameba, it has been demonstrated that proteins such as serum albumin, as well as low molecular weight reducing agents (e.g. ascorbic acid), react rapidly with the highly reactive products of the MPO system and prevent them from reaching the trophozoites (20). A peroxiredoxin (Prxs) on the outer surface of the E. histolytica trophozoites reduces H2O2 and yields water, thus protecting trophozoites from the H2O2 produced by activated phagocytes and facilitating the amebic invasion of the host. On surface areas of trophozoites expressing high levels of Prxs, this enzyme may be able to counteract the H2O2 generated by trophozoites or produced by host cells, thus reducing the amebicidal effect of the MPO system. However, the binding of MPO to surface areas expressing low levels of Prxs probably avoids the removal of the H2O2 generated by trophozoites, thus preventing or delaying the antioxidant action of the Prxs, and allowing for the amebicidal effect of bound MPO [–H2O2– halide system]. We propose a model of the amebicidal effect of MPO (Figure 7). In the first step of the amebic invasion when E. histolytica trophozoites spread through the blood vessels to the liver, they are exposed to a high oxygen partial pressure and to circulating neutrophils (2,52,54,57). Neutrophils rapidly accumulate around the trophozoites (5,8), but the amebas are too large to be ingested by them. Therefore, these bound neutrophils are stimulated, and release MPO into a walled-off pocket of space between the neutrophils and the ameba. H2O2 formed by the stimulated neutrophils is released into the pocket, and toxicity is induced in a manner similar to that occurring in an intracellular phagosome containing an ingested microorganism (20). In addition, MPO may be released to the extracellular fluid by leakage during phagocytosis, by cell lysis, or when the neutrophils are exposed to a variety of soluble stimuli (20). However, free MPO in the extracellular environment is probably inhibited by the high concentration of albumin. Previous studies on other parasites have reported that whereas albumin does not significantly 2011 Blackwell Publishing Ltd, Parasite Immunology, 33, 255–264
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inhibit the toxicity of the eosinophil peroxidase bound to schistosomula, it completely inhibits the peroxidase free in solution (58). Thus, MPO probably is more efficient in the area of contact with the amebic membrane. Myeloperoxidase uses the H2O2 produced by the trophozoites (SOD and NADPH : flavin oxidoreductase) to oxidize chloride ions and produce the highly cytotoxic HOCl that damages and kills trophozoites. A membranal peroxiredoxin is capable of reducing H2O2 and thus decreasing the amebicidal effect of MPO. In conclusion, the killing of E. histolytica trophozoites mediated by the activity of the MPO–H2O2–Cl-system on the surface of trophozoites may be an important factor by which the innate immune response prevents the invasion of E. histolytica.
ACKNOWLEDGEMENTS The authors wish to thank Bruce Allan Larsen for reviewing the use of English in this manuscript. This work was supported by grant 20060359 from SIP-IPN, Mexico. Barbosa-Cabrera E, Rojas Hernndez S, Jarillo-Luna RA, and Campos-Rodrguez R are fellows of COFAA and DEPI-IPN.
CONFLICT OF INTEREST The authors declare that there is no conflict of interest that would interfere with the impartiality of this research work.
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