Parasitol Res (2001) 87: 651±656 DOI 10.1007/s004360100412
O R I GI N A L P A P E R
J. Walochnik á A. Obwaller á E.-M. Haller-Schober H. AspoÈck
Anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities in correlation to strain pathogenicity Received: 14 February 2001 / Accepted: 9 March 2001 / Published online: 12 May 2001 Ó Springer-Verlag 2001
Abstract Several representatives of the genus Acanthamoeba are known as causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. These occur predominantly in the immunocompromised host, but it is still unclear what primes the amoebae for pathogenicity. The aim of this study was to assess possible immunological dierences between a highly pathogenic and a nonpathogenic Acanthamoeba strain. A total of 20 sera, including two sera of Acanthamoeba keratitis patients, were tested for anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities using immunoblotting. All sera were positive for Acanthamoeba, revealing two predominant bands at 29 kDa and at 47 kDa, respectively. Interestingly, IgG and particularly IgA immunoreactivity enabled a clear discrimination between the pathogenic and nonpathogenic strains. Moreover, compared to the control sera, the two sera of Acanthamoeba keratitis patients showed rather weak immunoreactivities and they lacked the 29 kDa and the 47 kDa band in the IgA immunoblot against the pathogenic strain. The results of our study support the assumption that immunological predisposition might also be of importance in Acanthamoeba keratitis.
Introduction Free-living amoebae of the genus Acanthamoeba are known as causative agents of two well de®ned disease entities. They provoke the so-called Acanthamoeba keratitis, which is a chronic and very often seriously progressive disease occurring predominantly in contact J. Walochnik á A. Obwaller á E.-M. Haller-Schober H. AspoÈck (&) Department of Medical Parasitology, Clinical Institute of Hygiene, University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria E-mail:
[email protected] Tel.: +43-1-427779430 Fax: +43-1-42779794
lens wearers. Also, they are responsible for chronic granulomatous amoebic encephalitis (GAE) and several disseminating infections in the immunocompromised host, including dermatitis and pneumonitis (Martinez and Visvesvara 1997). GAE is fatal in almost all cases, due to the unavailability of a suciently eective treatment. Acanthamoebae represent microorganisms which occur ubiquitously world-wide. Their double-walled cysts are extremely resistant against desiccation and are spread through the air easily, making frequent contact with this microorganism almost inevitable. In fact, contact lens cases of asymptomatic individuals have also been reported to be contaminated with acanthamoebae (Donzis et al. 1987; Walochnik et al. 2000a). However, the vast majority of humans never generates an Acanthamoeba keratitis, even when supplementary predisposing factors are prevalent, such as contact lens wear and corneal trauma. It has been suggested that either additional, unidenti®ed risk factors contribute to infection or protective immunity is commonly acquired through environmental exposure to nonpathogenic strains (Alizadeh et al. 1995; Cursons et al. 1980b). Some strains of Acanthamoeba may cause transitory infection, thereby stimulating the host defence mechanisms, which subsequently control the infection and eliminate the causative agent (Visvesvara and Stehr-Green 1990). Ferrante (1991) proposed that the presence of Acanthamoeba-speci®c antibodies, together with the activation of the alternative pathway, serves as a ®rst barrier against infection in humans. In contrast to the primary amoebic meningoencephalitis caused by another free-living amoeba, Naegleria fowleri, GAE occurs almost exclusively in immunocompromised persons. This discrepancy is believed to depend on dierences in the immunity mechanisms between these two amoebae (Ferrante 1991). Anti-Acanthamoeba antibodies apparently block the cytopathic eect of acanthamoebae (Cursons et al. 1980a) and highly eective protection against Acanthamoeba meningoencephalitis after immunisation with Acanthamoeba antigen has repeatedly been demonstrated in animal models
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(Culbertson 1971; Rowan-Kelly and Ferrante 1984). It is unknown, however, whether the immunological status of the patient is also somehow important for the establishment of an Acanthamoeba keratitis. The human eye certainly is an immunologically privileged site. However, two of the most severe diseases of the cornea, namely onchocerciasis and Herpes simplex keratitis, are believed to be immunity-mediated (Pearlman 1997; Streilein et al. 1997) and, moreover, successful antibody-mediated immunisation against Acanthamoeba keratitis has already been demonstrated in an animal model (Alizadeh et al. 1995). In a recent study we demonstrated that 18S rDNA sequence-based identi®cation correlates to immunological dierentiation (Walochnik et al. 2001). The aim of the present study is to assess possible immunological dierences between a highly pathogenic and a nonpathogenic Acanthamoeba strain. We investigated the anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities of several sera, including two Acanthamoeba keratitis patients, against a highly pathogenic strain isolated from one of the Acanthamoeba keratitis patients and against a nonpathogenic contact lens case isolate. To our knowledge, this is the ®rst study on anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities of Acanthamoeba keratitis patients' sera and the ®rst study demonstrating anti-Acanthamoeba IgA in human serum.
Materials and methods Amoeba strains Two strains of Acanthamoeba, one highly pathogenic (11DS) and one nonpathogenic (5SU), were used for antigen extraction. The 11DS strain was isolated from the contact lens case of an Acanthamoeba keratitis patient (patient 1). The 5SU strain was isolated from the contact lens case of a non-Acanthamoeba keratitis patient (patient 3), whose contact lens case had been investigated routinely and had not caused any disease, although coming into contact with an aected cornea. Eye-swab samples and ulcus material from this patient were negative for Acanthamoeba. Moreover, the patient did not show any typical clinical signs of Acanthamoeba keratitis and the disease was treated successfully with antibacterial drugs. The 11DS strain is highly cytopathic in human tissue culture and shows pathogenicity-related physiologic characters, such as high temperature tolerance (42 °C), high growth rate, and migration under agarose (Walochnik et al. 2000a, b). The 5SU strain shows no growth at 42 °C, no migration under agarose, and no ability to eectively lyse a human cell monolayer (Walochnik et al. 2000b). The amoebae were cultured in sterile, ®ltered proteose peptone± yeast extract±glucose medium (Visvesvara and Balamuth 1975) in 150-cm2 tissue culture ¯asks (Corning Costar, Bodenheim, Germany) at 30 °C. Preparation of antigens Trophozoites (106 cells) were harvested from extensively growing cultures by centrifugation at 500 g for 7 min. They were then washed in sterile phosphate-buered saline (PBS) and resuspended in 300 ll sterile distilled water. Cells were disrupted by multiple freeze-thawing, before adding 100 ll 4´sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) loading buer
(63 mM TrisHCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 30 M bromophenol blue). The suspension was then boiled for 5 min, vortexed, and centrifuged at 12,000 g for 5 min. The supernatant was transferred to a new tube and stored at 20 °C until use. Sera A total of 20 sera were tested, including two sera of Acanthamoeba keratitis patients, one serum of a non-Acanthamoeba keratitis patient, four sera from patients with a diagnosed Entamoeba histolytica infection, ten control sera from healthy individuals, two sera from AIDS patients, and one newborn serum. Sera 1 and 2 derived from patients presenting a fulminate Acanthamoeba keratitis, serum 3 derived from a non-Acanthamoeba keratitis patient. The Acanthamoeba keratitis patients differed from the non-Acanthamoeba keratitis patient by showing typical clinical signs for Acanthamoeba keratitis, such as the presence of keratitis with severe pain and photophobia, stromal in®ltrates, radial keratoneuritis, no response to antibacterial or antiviral treatment, and detection of acanthamoebae in the corneal epithelium. Sera of patients with a diagnosed Entamoeba histolytica infection (sera 4±7) were included in this study because Entamoeba, being the most closely related pathogen of Acanthamoeba, is of considerable interest with respect to crossreactivity. Controls (sera 8±17) were randomly collected from healthy adult individuals, who were proven to be negative in any other parasitological test. Two sera from AIDS patients (sera 18, 19) were included in this study with respect to a possible immunode®ciency-dependent diverging immunoreactivity. A newborn serum (serum 20) was added as a potential negative control. Altogether, the age range was 0±60 years and the male:female ratio was 11:9. SDS-PAGE and immunoblot SDS-PAGE was performed according to the protocol of Laemmli (1970). Brie¯y, 100 ll of cell lysates of each strain were separated in 12.5% (w/v) PAG at 135 V for 90 min at room temperature (RT). A rainbow molecular weight marker (Amersham Pharmacia Biotech, Freiburg, Germany) was included. The SDS-PAGE were run at 135 V for 90 min at RT and separated proteins were stained with Coomassie brilliant blue R-250 (Sigma, St. Louis, Mo.). For immunoblotting, 100 ll of cell lysates were separated and transferred to nitrocellulose (Schleicher and Schuell, Dassel, Germany) by semi-dry blotting for 45 min at 10 V. Non-speci®c binding sites were blocked with PBS (pH 7.4), 3% bovine serum albumin (BSA), 0.1% Tween 20 for 1 h at RT. Strips (3 mm in width) were cut and incubated overnight at 4 °C with a range of dilutions of sera, using blocking buer. After three washing steps, strips were incubated with horseradish peroxidase-conjugated rabbit anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, USA) and horseradish peroxidase-conjugated mouse anti-human IgM and IgA antibodies (Zymed Laboratories, San Francisco, USA) at various dilutions for 3 h at RT on a rocking platform. Bands were developed using 4-chloro-1-naphthole (Sigma, St. Louis, Mo.) and H2O2. Optimal serum dilutions were assessed as 1:400 for IgG, 1:100 for IgM, and 1:20 for IgA immunoblots. Optimal conjugate dilutions were 1:1,000 for IgG and 1:100 for IgM and IgA.
Results A total of 20 sera, including two sera of Acanthamoeba keratitis patients, were tested for anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities in order to show up possible immunological dierences between a highly
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pathogenic and a nonpathogenic Acanthamoeba strain. Coomassie brilliant blue staining revealed rather complex protein pro®les for the two strains, 11DS and 5SU, as shown in Fig. 1. Multiple major bands could be observed from 16 kDa to 100 kDa. The pro®les of the two strains were rather similar, yet both strains exhibited several unique protein bands. The most prominent of these were two extra bands for the 5SU strain at 160 kDa and 250 kDa, respectively. However, these bands resulted in no signi®cant immunoreactivity with any of the sera in the following immunoblots. Generally, all sera exhibited anti-Acanthamoeba immunoreactivity. The most prominent bands were at 29 kDa and 47 kDa, being present in both strains and in all antibody isotypes. Interestingly, in the pathogenic 11DS strain, the 29-kDa band was more pronounced and, in the nonpathogenic 5SU strain, the 47-kDa band was more prominent. As shown in Fig. 2A, B, all sera revealed antibodies of the IgG isotype against both the highly pathogenic and the nonpathogenic strain of Acanthamoeba. The 11DS strain resulted in rather complex antibody pro®les, with a prominent band at 29 kDa (Fig. 2A). This band was present in all sera, except control serum 8, the two AIDS patients' sera (sera 18, 19) and the newborn serum (serum 20). The two Acanthamoeba keratitis patients (sera 1, 2) showed another rather prominent band at 44 kDa, which was also visible in all Entamoeba histolytica patients, but not in most of the control sera and also not in patient 3, who had developed no Acanthamoeba keratitis, despite coming into contact with acanthamoebae. In the Acanthamoeba keratitis patients (sera 1, 2), the non-Acanthamoeba keratitis patient (serum 3), and the E. histolytica patients (sera 4±7), a supplementary 47-kDa band was visible, which was non-existent in most of the control sera. This band was very weak in the Acanthamoeba keratitis patients and the non-Acanthamoeba keratitis patient, but was rather prominent in the E. histolytica patients, particularly in patient 7. The weakest immunoreaction was observed in serum 8, a control serum, and in the newborn serum (serum 20), in which only a single, very weak band at 40 kDa was present. In the anti-5SU IgG assay, the most prominent band was at 47 kDa and this was pre-
sent in all sera (Fig. 2B). The 29-kDa band was visible in most sera, although it was noticeably weaker than in the 11DS strain. Interestingly, the strongest immunoreactions against the pathogenic strain were seen in the E. histolytica sera (sera 4±7), while all sera of asymptomatic individuals (sera 8±17) except serum 8 showed higher reactivity against the nonpathogenic strain than any of the patients' sera. Against both Acanthamoeba strains, the weakest reactions were observed in control serum 8, the AIDS patients' sera (sera 18, 19), and in the newborn serum (serum 20). The strongest immunoreactivity was seen with IgM (Fig. 2C, D). Moreover, here all sera reacted in a rather similar way. Again, the 29-kDa and 47-kDa bands were the most prominent bands. A supplementary 33-kDa band was visible in all sera except the newborn serum (serum 20). There was no dierence between the two amoeba strains, with respect to immunoreactivity. Anti-IgA testing revealed only weak reaction to the pathogenic strain 11DS (Fig. 2E, F). Five sera (sera 8, 11, 17, 19, 20) showed no immunoreactivity at all. Both the 29-kDa band and the 47-kDa band were visible in all E. histolytica sera (sera 4±7), several of the control sera (sera 9, 10, 12, 15), and also in the non-Acanthamoeba keratitis serum (serum 3), but they were not visible in either of the Acanthamoeba keratitis sera (sera 1, 2). The Acanthamoeba keratitis sera showed a rather weak band at 44 kDa and serum 2 showed supplementary bands at 75, 38, and 12 kDa. Testing with the nonpathogenic strain 5SU resulted in an altogether stronger immunoreactivity (Fig. 2F). The prominent 47-kDa band was present in all sera, except the AIDS patients' sera (sera 18, 19) and the newborn serum (serum 20). The two Acanthamoeba keratitis sera (sera 1, 2) showed rather low immunoreactivity, while the non-Acanthamoeba serum (serum 3) revealed a very strong 47-kDa band, compared with its homologous Acanthamoeba strain. The lowest immunoreactivity was seen in serum 19 and in the newborn serum (serum 20). Moreover, it is remarkable that in IgA testing, both Acanthamoeba keratitis sera lacked both the 29 kDa and the 47 kDa band against the pathogenic strain 11DS, while both bands were present in the four E. histolytica patients and in the non-Acanthamoeba keratitis patient.
Discussion
Fig. 1 Protein pro®les of the highly pathogenic Acanthamoeba strain 11DS and the non-pathogenic Acanthamoeba strain 5SU in Coomassie brilliant blue staining. M Molecular weight marker
Immunological dierences between a highly pathogenic and a nonpathogenic Acanthamoeba strain were evaluated by testing 20 sera, including two sera from Acanthamoeba keratitis patients, for anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities, using immunoblotting. The protein pro®les of the two strains were rather similar, except for the 5SU strain possessing two extra bands at 160 kDa and 250 kDa, respectively, which were, however, not immunoreactive. Generally, anti-Acanthamoeba antibodies were detected
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in all investigated sera with two predominant bands, at 29 kDa and 47 kDa, being present in all antibody isotypes and in both strains. However, the immunoreactions diered markedly with respect to the pathogenicity of the Acanthamoeba strain used for antigen preparation. Moreover, in IgG and particularly in IgA immunoblots marked dierences were seen between patients' sera and the sera of asymptomatic individuals.
Immunoreactivities of Acanthamoeba strains In IgG and in IgA testing, the 29-kDa band was more pronounced in the pathogenic strain while, in the nonpathogenic strain, the 47-kDa band was more prominent. Moreover, in IgG and particularly in IgA testing, immunoreactivities were signi®cantly higher against the nonpathogenic strain than against the pathogenic strain; and this was most obvious in the control sera.
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Fig. 2 IgG (A, B), IgM (C, D) and IgA (E, F) immunoreactivities of two sera from Acanthamoeba keratitis patients (lanes 1, 2), one serum from a non-Acanthamoeba keratitis patient (lane 3), four sera from patients with a diagnosed Entamoeba histolytica infection (lanes 4±7), ten control sera from healthy individuals (lanes 8±17), two sera from AIDS patients (lanes 18, 19) and one newborn serum (lane 20) against blotted antigens of the highly pathogenic Acanthamoeba strain 11DS (A, C, E) and the nonpathogenic Acanthamoeba strain 5SU (B, D, F). Note the remarkable dierence in immunoreactivity between the pathogenic strain 11DS and the nonpathogenic strain 5SU, particularly in the IgA immunoblot. Acanthamoeba keratitis patients (lanes 1, 2) show almost no IgA immunoreactivity against the pathogenic strain 11DS. Both lack the 29-kDa and 47-kDa bands, particularly serum 1, which is the homologous serum to this strain (E). M Molecular weight marker
This might be due to avirulent strains being more abundant in the environment. However, it has been demonstrated that highly pathogenic acanthamoebae can, in contrast to weakly pathogenic ones, evade the antibody-dependent amoebicidal activity of macrophages and are more resistant to the lytic activity of complement (Marciano-Cabral and Toney 1998; Toney and Marciano-Cabral 1998). Moreover, it was demonstrated that acanthamoebae can degrade human IgG and IgA antibodies, utilising a 33-kDa serine protease and that this capability is restricted to pathogenic strains (Kong et al. 2000). Pathogenic strains of Acanthamoeba also seem to be more resistant to complement lysis, which is supposed to be a mechanism of host immunity evasion (Toney and Marciano-Cabral 1998). In all sera, including the newborn serum, antiAcanthamoeba antibodies were detectable, which suggests a 100% seropositivity. Other authors report 50±100% seropositivity for Acanthamoeba spp. (Cerva 1989; Cursons et al. 1980a; Powell et al. 1994). Seropositivity of newborn sera, as also demonstrated in Balamuthia by other authors (Huang et al. 1999), is most probably due to transplacental antibody transfer. Cursons et al. (1980a) could detect anti-Acanthamoeba IgG in cord blood. The very high percentage of healthy individuals carrying antibodies against Acanthamoeba can be explained by the ubiquity of Acanthamoeba spp. Acanthamoebae have been isolated from typical man-made habitats, such as swimming pools, tap water, bottled mineral water, and air-conditioning, as well as from several clinical specimens of healthy individuals (Cerva et al. 1973; De Jonckheere 1991; Michel et al. 1982). Acanthamoeba cysts are spread through the air and can easily persist and survive on mucosal sites and skin surfaces. The high abundance of acanthamoebae in the environment and their extremely resistant cysts are parameters of considerable importance for the establishment of an infection (Ferrante 1991). However, while Balamuthia GAE has infrequently been recognised in immunocompetent individuals, Acanthamoeba GAE seems to occur exclusively in immunode®cient patients. This might partially be due to a greater virulence of Balamuthia (Huang et al. 1999), but may also be attributed to variations in the host immune response.
Immunoreactivities of sera It is most remarkable that the Acanthamoeba keratitis patients generally showed lower reactivities than the Entamoeba histolytica patients, despite having a fulminate Acanthamoeba keratitis, with deep corneal ulcers and amoebae detectable in necrotic tissue. The sera of the E. histolytica patients were the only sera showing high IgG and IgA reactivity against the pathogenic strain, which might re¯ect crossreactivities, since E. histolytica is closely related to Acanthamoeba. Moreover, both Acanthamoeba keratitis sera lacked both the 29-kDa and the 47-kDa bands in IgA testing against the pathogenic strain, while both bands were present in the four E. histolytica patients and in the nonAcanthamoeba keratitis patient. Compared to the control sera, they both exhibited markedly lower IgG and IgA immunoreactivities against the nonpathogenic strain, which might refer either to not having had previous contact with these amoebae or to a possible immunode®ciency. Generally, an Acanthamoeba infection probably results from a high dose infection, together with poor antibody levels. Anti-Acanthamoeba antibodies were shown to block the action of cytopathogenic substances secreted by the amoebae, thus neutralising their cytopathogenic eects (Cursons et al. 1980a). It was also shown in several studies that antibodies increase the amoebilytic capability of the immune system (Ferrante and Abell 1986; Marciano-Cabral and Toney 1998; Stewart et al. 1992, 1994), which was shown to dier signi®cantly between serum donors (Toney and Marciano-Cabral 1998). Moreover, amoebae treated with antibodies were found to round up, losing motility and the capacity to bind to human cells (Ferrante 1991; Moore et al. 1991). Particularly, IgA seems to inhibit the binding of the amoeba to corneal epithelial surfaces (Leher et al. 1998a). The ability to adhere to surfaces, however, is a virulence factor of prime importance for many pathogens and seems to be the critical step in the Acanthamoeba cytopathic eect (Niederkorn et al. 1999). To our knowledge, this is the ®rst study demonstrating anti-Acanthamoeba IgA in human serum. IgA provides an immunological barrier for microorganisms, blocking their adherence to epithelial cells; and it is IgA which is synthesised by the mammalian immune system in larger quantities than all other isotypes combined (Mazanec et al. 1993). Being the most abundant immunoglobulin in mammalian tears (Fullard and Snyder 1990), IgA is of major importance for eye infections. Moreover, IgA has a weak capacity to activate the complement cascade, so Leher et al. (1980a) deduce that it can prevent infection without inducing an in¯ammation. It would thus be at least possible that the absence of anti-Acanthamoeba antibodies (particularly of the IgA isotype) ± either due to no previous contact to acanthamoebae or due to immunode®ciency ± is a supplementary risk factor for generating an Acanthamoeba keratitis. Monoclonal IgA antibodies have been
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demonstrated to protect against Acanthamoeba keratitis (Leher et al. 1999). Obviously, immunity cannot be acquired by ocular infection alone (Alizadeh et al. 1995), but oral immunisation with Acanthamoeba antigen protects against subsequent corneal infection by inducing the production of parasite-speci®c IgA (Leher et al. 1998b). Thus, low serum anti-Acanthamoeba IgA antibody titres might re¯ect reduced local immunity against Acanthamoeba in the eye. Taken together, the results of our study indicate the dierentiation between pathogenic and nonpathogenic acanthamoebae, provoking distinct IgG and particularly IgA antibody pro®les. Moreover, the fact that the two sera of Acanthamoeba keratitis patients lacked both the 29-kDa and the 47-kDa band in IgA testing against the pathogenic strain, while both bands were present in all E. histolytica patients and in the non-Acanthamoeba keratitis patient, makes it at least possible that there is an immunological predisposition for Acanthamoeba keratitis. It is suggested that the lack of any ability to produce antibodies indicating immunity to Acanthamoeba infections may be an important precondition for clinical manifestations, including keratitis.
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