STRONGYLOIDES STERCORALJS: ANTIGENIC ANALYSIS OF INFECTIVE LARVAE AND ADULT WORMS CAROLYN NORTHERN*
and DAVID I. GROVE~$
* Department of Medicine, University of Western Australia, Nedlands, Western Australia 6009, Australia t Department of Postgraduate Medical Education, Sir Charles Gairdner Hospital, Nedlands, Western Australia 6009, Australia (Received 21 August 1989; accepted
I January 1990)
Abstract-NoarHaaN C. and GROVE D. I. 1990. Strongyloides stercorah: antigenic analysis of infective larvae and adult worms. International Journal for Parasilology 20: 381-387. The protein composition of Strongyloides stercorulis infective larvae and adult worms solubilized sequentially in water, sodium deoxycholate and sodium dodecyl sulphate (SDS) and their excretory/s~retory products were analysed by one- and two-dimensional SDS-~lyacrylamide electrophoresis. These extracts were demonstrated to be complex mixtures containing many proteins, some of which were common and others which were stagespecific. Western blot analysis of these antigens with infected human sera showed most sero-reactivity against larval antigens, whilst normal human sera were unreactive. These data identify immunogenic antigens which may be available for detection in an antigen assay. INDEX KEY WORDS: Strong.yfoides stercoruiis; infective larvae; adult worms; antigens; stage-specificity; Western blots; human sera.
INTRODUCTION Strongyloides
stercoralis
humans
continues
is an intestinal parasite of to elude complete understanding and accurate diagnosis. Infection often persists undetected with the worm reinfecting the host over a period of many years by a process of autoinfection. The parasite cycles between a systemic phase where infective larvae penetrate the tissues in their migration throughout the body and an intestinal phase in which the adult worm resides in the intestinal wall. An understanding of any antigenic differences between these stages may shed light on the parasites and on protective host responses to them. People infected with S. stercoralis produce specific antilarval IgA, IgE, IgG and IgM antibodies. These antibodies have been measured in various diagnostic assays including the enzyme-linked immunosorbent assay (ELISA) (Carroll, Karthigasu & Grove, 1981; Genta, Frei & Linke, 1987), the immunofluorescent antibody assay (IFA) (Grove & Blair, 1981), the radioallergosorbent test (RAST) (Genta, Deuce & Walzer, 1986) and with skin tests (Neva, 1986). The problem for accurate diagnosis, however, lies in the fact that the level of antibody measured does not necessarily differentiate between past and present that
$ Present address and address for correspondence: Department of Clinical Microbiology and Infectious Diseases, The Queen Elizabeth Hospital, Woodville South, South Australia 5011, Australia.
infections (Grove, 1982). This difficulty would be solved by the development of an assay for parasite antigens in host serum or urine. In order to approach both objectives it is first necessary to characterize the antigenic profiles of tissue-penetrating infective larvae and adult worms within the intestinal mucosa. In this study, we have compared the protein composition of infective larvae and adult worms. We employed a sequential solubilization technique in the preparation of parasite material for immunochemical analysis in order to maximize the number of antigens for study. The antibodies produced against these antigens by normal and infected individuals were then identified with Western blots. MATERIALS AND METHODS Animats and parasite. Parasites were recovered originally
from a man who had been infected while a prisoner-of-war in Southeast Asia 35 years previously (Grove, 1980). The parasite has since been maintained in mongrel dogs obtained from the local pound. The animals were washed, treated with anthelmintics bunamidine hydrochloride and pyrantel pamoate, and their faeces examined to ensure they were free of helminth eggs. The methods for preparation of larvae, infection of animals, and quantitation of larvae in the faeces have been described previously (Grove & Northern, 1982). Worm burdens were increased by immunosuppressing dogs with prednisolone plus or minus azathioprine (Grove, Heenan & Northern, 1983). Preparation of antigen. Infective larvae were obtained from cultures of dog faeces, then washed three times by 381
382
C. NORTHERNand D. 1. GROVE
centrifugation in sterile water containing penicillin (Glaxo, Boronia, Vi,., Australia) and gentamicin (David Bull Laboratories, Mulgrave, Vie., Australia). The larvae were spun to a pellet and then frozen in antigen preparation buffer (APB) [20 mM-Tris-HC! pH 8.0, 5 mM-EDTA acid, 2 mt+ phenylmethylsulfonyl fluoride (Sigma, St Louis, U.S.A.) and 100 units ml ’ Aprotinin @(Bayer Pharmaceuticals, Botany, N.S.W., Austra!id)]. Adult worms were obtained from infected dogs at least 6 weeks after infection The small intestine was removed, slit longitudinally, scraped free of mucus and rinsed, then it wasincubated in PBS pH 7.4 for 2 h at 37°C to allow parasites to crawl out of the tissues. The adult worms were separated from the residual fluid by gravity sedimentation and washed several times in PBS containing penicillin and gentamicin. Somatic proteins were prepared from worms that were thawed, washed and resuspended in fresh APB. They were then homogenized in a Braun@ homogenizer; equal volumes of worm suspension and glass beads. 0.45-0.50 pm in diameter, were placed in a bottle which was placed in a rotating cylinder cooled with rapidly flowing CO, for 2 min. The homogenate was allowed to stand overnight at 4°C. The supernatant fluid was centrifuged at 5000 g for 30 min at 4°C then the supernatant fluid of this step was respun at 100,000 g for 60 min at 4°C. The final supernatant fluid was filtered through a 0.23~pm Millex GV low protein-binding filter (Millipore Corp.. Bedford, U.S.A.); this is designated as ‘water-soluble fraction’. The two pellet preparations were combined and resuspended in APB containing 1% sodium deoxychoiate (DOC) (BDH Chemicals Ltd. Poole. U.K.), and allowed to stand overnight at 4°C. The material was centrifuged and filtered as before. This supernatant f!uid is referred to as ‘DOC-soluble fraction’. The two pellets were again combined and the process was repeated in APB containing 1% SDS (BDH Chemicals). The final supernatant fluid is designated ‘SDSsoluble fraction’. The remaining pellet was discarded. Each fraction was diatysed against water at 4°C over 24 h. Protein concentrations were measured by the method of Hal-tree (1972). In the instance of adult worms. the first two steps of antigen solubilization were omitted due to the difficulty ol sufficient two-dimensional obtaining material for eiectrophoresis (2DE) and the worms were solubilired directly in
& Neuhoff (1981). Relative molecular weights (M,) of sample polypeptides were calculated from the mobility of molecular weight standards co-electrophoresed in a separate well of each gel. Molecular weight estimates of 94 or above are extrapolations and only serve as approximate guides. Two-dimensional electrophoresis (2DEl. 2DE was performed using a modification of the method of O’Farrell, Goodman & O’Farrell(1977) as described earlier (Northern &Grove, 1987). Samples were prepared in lysis buffer, 9.5 Murea (Biorad), 2% Nonidet P40 (Sigma), 2% Pharmalyte ?-10 (Pharmacia Fine Chemicals) and 5% 2-mercaptoethanol. The first dimension was separated by non-equilibrium pH gradient gel electrophoresis (NEPHGE) on a 0.7-mm thick slab gel as described by Ferreira & Eichinger (1981) The second dimension was separated by SDS-PAGE as before and the gels were stained with silver. W’extern blot anulysis. A pool of sera from patients with parasito!o~~ca!!y proven strongyloidiasis was obtained. These sera were confirmed to have high antibody titres by ELISA against S. .stercornlis antigen (Carroll et cd., 1981). Western blot analysis was undertaken as described earlier (Northern 6z Grove, 1988). In brief, proteins and prestained molecular weight standards (Bethesda Research Lahoratories, MD, U.S.A.) separated by SDS-PAGE were transferred to nitrocellulose paper (Biorad) by the method 01 Towbin, Staehlin & Gordon (1979). lmmunoreactivc bands were detected using alkaline phosphate-conjugated antibodies (My Lien Dao, 1985). The nitrocellulose was blocked with 0.5% Tween 20 in 20 met-Tris, 500 mu-NaCl. pH 7.5 (TTBS) for 30 nun at room temperature in order to saturate unused protein binding sites. It was then incubated overnight in normal or pooled immune human serum diluted I in 250 with TTBS. The nitrocel!u!osc was washed twice in Tris-buffered saline (TBS) (20 mM-Tris, 500 mM-NaC!. pH 7.5) and placed in IO mr+EDTA to inactivate any endogenous alkaline phosphatase activity. It was then washed three times in TBS and incubated with alkaline anti-hlllllan in~n~u~l~~&lohul~n phosphat;~se-conjugale~i (Silenus Laboratories, Hawthorn. Australia) diiuted I:500 in TTBS for I h. The nitrocellulose was washed three tlmcs 111 TTBS and then rinsed in substrate buffer (0.06 \I-sodium borate pH 9.7 containing 1.2 mg ml ’ magnesium sulphatci. immune complexes were visualized after staining with (Idianisidine (0.25 mg ml ‘) and B-naphthyi acid phosphate (0.25 mg ml ‘) in substrate buffer. Following enlyme staining. the nitrocellulose was washed in a solution 01’ methanol, acetic acid and water (5: 1.5:. v!\ :\ I foilowccih\ ;t final rinse in water. RESULTS One-ilimensional el~~c,tropllorc,.si.r The SDS-PAGE profiles of infective larval water-. DOC- and SDS-soluble preparations and ES proteins
SDS pol~ucr$_mid~~ ~cl electrophoresi.~ (SDS PAGE). All preparations were analysed initially by one-dimensional SDS-PAGE (Laemmli & Favre. 1973). In brief. samples containing 5 pg protein and molecular weight standards (IMW kit, Pharmacia Fine Chemicals, Uppsala. Sweden) were prepared in sample buffer. 62.5 mr+Tris--HCI pH 6.8. 4% w/v SDS, 5% v/v 2-mercaptoethanol (BDH Chemicals), 10% glycerol (BDH Chemicals) and 0.001% bromophenol blue (Biorad. Richmond. California, U.S.A.). These were then heated in a water bath at 100°C for 5 min. Samples were centrifuged at 11,000 g for 5 min and were loaded onto the gels. These were stained with silver as described by Poehling
are illustrated in Fig. 1. A large number of bands were resolved for each preparation with differences noted according to the method of solubihzation. In the water-soluble larval extract major bands were resolved at 16, 17 ,18, 28 and 59 mol. wt. Major DOC-soluble bands had mol. wts of 3 I, 36.59 and 97, while the SDSsoluble bands were resolved at 18.3 I, 36.50.55 and 62 mol. wt. The ES preparation yielded a dominant band of mol. wt 52 with additional minor components. The SDSPAGE profiles of adult worms are illustrated in Fig. 2. Five preparations were analysed: water-. DOC-.
Antigenic analysis of S. sfercoralis
383
revealed major components at 33, 44, 60 and two bands greater than 94 mol. wt. The sequential SDSsoluble extract had major bands at 30, 33, 37, 66, 78 and 83 while the SDS-total-soluble extract contained dominant bands at 21,24, 26 and 66 mol. wt. The ES extract had major bands at 16, 33 and 71 mol. wt.
-20 -14.4 12
3
4
FIG. I. SDS-PAGE of S. stercoralis infective larvae preparation, stained with silver. Water-soluble fraction (lane I), DOC-soluble fraction (lane 2) SDS-soluble fraction (lane 3) ES products (lane 4). Molecular weight standards are shown on the right.
Mr
-94 -67 -43 -30
-20 -14.4 12
3
4
5
FIG. 2. SDS-PAGE of S. stercoralis adult worm preparation,
stained with silver. Water-soluble fraction (lane l), DOCsoluble fraction (lane 2), SDS-soluble fraction (lane 3), SDStotal-soluble fraction (lane 4) ES products (lane 5). Molecular weight standards are shown on the right. SDS-, SDS-total-soluble proteins and ES products. Again, a complex array of bands were resolved with differences observed among the methods of preparation. Major water-soluble bands were resolved at 19, 5 1 and 64 mol. wt, while the DOC-soluble preparation
Two-dimensional electrophoresis Greater discrimination of these differences was provided by 2DE. Many proteins were visualized in the water-soluble fraction of infective larvae (Fig. 3a). A number of additional spots were seen in the sequential DOC-solubilized preparation (Fig. 3b). In the third preparation in which the pellet was finally solubihzed in SDS a further number of proteins were demonstrated (Fig. 3~). It was not possible to make direct comparisons between the infective larval and adult worm somatic components as the paucity of adult worms did not permit sequential solubilization of this stage of the parasite. Nevertheless increased discrimination of proteins was obtained when 2DE was performed on adult worm material extracted directly with SDS (Fig. 4a). A smaller set of proteins was isolated from larval excretory/secretory products (Fig. 3d). The pattern of distribution of proteins in the ES products derived from adult worms (Fig. 4b) was quite different from that seen with larvae thus indicating differences in the stage-specificities of the parasite. Western blots The soluble extracts of infective larvae and adult worms were analysed by Western blots with a pool of normal, uninfected human sera and a pool of immune sera from individuals with parasitologically proven strongyloidiasis. Figure 5 shows the profile of reactivity of normal sera to the antigenic mixtures and Fig. 6 illustrates the same profile of antigens reacted with immune sera. The normal serum profile is notable for its lack of reactivity against virtually all proteins. A single band in the larval DOC-soluble preparation with mol. wt 38 is faintly detected. In contrast, the immune serum profile identifies a number of antigenic bands in both the larval and adult worm preparations. The larval water-soluble preparation has major reactive bands with mol. wt 38, 48 and 58 as do the DOC-soluble preparation and the SDS-soluble preparation, the latter also having an additional band at 27 mol. wt. The ES products comprise many but fainter reactive bands; the major ones have mol wts 11, 14, 38,48 and 58 and six bands with a mol. wt greater than 75. In contrast to the larval preparations the adult worm preparations are characterized by a paucity of strongly reactive components. The adult SDS-total-soluble preparation has two major reactive bands, one with mol. wt 38 and the other a band at greater than 130. The adult ES preparation shows only a number of faintly staining bands, the strongest being with mol. wt 58.
384
C. NORTHERN andD.1. GROVE
Antigenie analysis of S. sterc~rui~s
385
Mr
-94 -67 -43 -30
ACIDIC
BASIC
FIG. 4. Two-dimensional electrophoretic profiles of S. stercoralisadult worms. Samples were analysed by NEPHGE in the first dimension and SDS-PAGE in the second dimension. SDStotal-soluble fraction (panel A) and ES products (panel B). Acidic and basic regions of the gels are indicated. Molecular weight standards are shown on the right.
DISCUSSION
In this study we have shown that the antigenic profiles of the tissue penetrating infective larvae and mucosal-dwelling adult worms differ. In addition, we have demonstrated that both the somatic extracts and excretory/secretory products are complex mixtures containing many proteins which can be separated in high resolution electrophoresis systems. The method of sequential solubilization of the worms yielded different sets of proteins reflecting differing solubitities in the detergents used. Although
exposed to strong anionic detergents such as sodium deoxycholate and sodium dodecyl sulphate the proteins retained their antigenicity when utilized in a Western blot assay. We have made similar observations in the comparison of Strongyloides ratti infective larvae and adult worms (Northern & Grove, 1987) as have others with different nematodes such as Trichinella spiralis, Nematospiroides dubius and Dipetalonema viteae (Phillip, Parkhouse & Ogilvie, 1980; Pritchard, MaizeIs, Behnke & Appleby, 1984; Baschong, 1985).
386
C. NORTHERN and D. 1. GROVE
Mr
-130 -75 -50 -39 -27
-17
12
3
4
5
6
FIG. 5. Western blot of S. srercoralk larval and adult worm antigen preparations with normal human sera. Larval watersoluble (lane I), larval DOC-soluble (lane 2). larval SDSsoluble (lane 3), larval ES products (lane 4). adult SDS-totalsoluble (lane 5) and adult ES products (lane 6). Prestained molecular weight standards are shown on the right.
Mr
-130 -75 -50 -39 -27
123456 FIG. 6. Western blot of S. stercoralis larval and adult worm antigen preparations with immune human sera. Larval water-soluble (lane I), larval DOC-soluble (lane 2), larval SDS-soluble (lane 3), larval ES products (lane 4), adult SDStotal-soluble (lane 5) and adult ES products (lane 6). Prestained molecular weight standards are shown on the right.
Others have also begun to analyse larval surface, metabolic and excretory/secretory antigens of S. stercorafis (Brindley, Gam, Pearce, Poindexter & Neva, 1988). They identified antigenic molecules from
infective larvae on the basis of immunoprecipitation reactions with human sera and purified larval ES proteins bound to a column of human IgG from immune human sera. They demonstrated a number of bands with mol. wts 12,25,30,33,35,40,50,54,60,90 and 240. We also demonstrated antigenic bands at some of these weights although direct comparison is difficult. Our interest has been to study both infective larvae and adult worms in order to compare the antigenic profiles of the two stages of the parasite. We have shown that both the somatic extracts and the excretory/secretory products of two developmental stages of S. stercoralis are highly complex but that common and stage-specific proteins could be demonstrated. The use of 2DE permitted greater discrimination of the various protein bands found by SDS-PAGE. Furthermore we have shown with Western blots utilizing sera obtained from infected humans that many of these compounds are immunogenic. The patients were suffering from strongyloidiasis and had no other concomitant parasitic infections. Most seroreactivity was directed against larval antigens. Similar major bands were seen in the water-, DOC- and SDSsoluble extracts reflecting their increasing solubility in stronger detergents. When adult worm somatic antigens were extracted using the strongest detergent. SDS, very few antigenic bands were identified. A plethora of antigenic bands was shown in the metabolic products produced by infective larvae. In contrast, only a small number of bands were seen in the metabolic products derived from adult worms. The difference in sero-reactivity between infective larvae and adult worms is not surprising when the physical locations of these stages of the parasite are considered. Infective larvae migrate through the tissues and are in close proximity to the cells of the immune system. On the other hand, adult worms live in tunnels in the small intestine epithelium and do not penetrate the basement lamina (Grove, Warton, Yu, Northern & Papadimitriou, 1987). They are therefore relatively isolated from immunoreactive cells. These studies provide a framework for the identification and separation of those antigens which are most relevant to the generation of host-protective immune responses and which may be made amenable to detection in an antigen assay. Acknowledgemenrs-This study was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES BASCHONGW. 1985. Changes in the surface of Dipefalonema vifeae (Filarioidea) during its development as shown by comparative peptide mapping. Parasikg?, 90: 35 l-356.. BRINDLEYP. J., GAM A. A.. PEARCEE. J.. POINDEX~ERR. W. & NEVA F. A. 1988. Antigens from the surface and excretions/ secretions of the filariform larva of Srrongylaides
Antigenic
analysis
stercoralis. Molecular and Biochemical Parasitology 28: 171180. CARROLL S. M., KAR~H~GASUK. T. & GROVE D. I. 1981. Serodiagnosis of human strongyloidiasis by an enzymelinked immunosorbent assay. Transactions of the Royal Society of Tropical Medicine and Hygiene 15: 706-709. FERREIRA A. & EICHINGER D. 1981. A simplified twodimensional technique. Journal of Immunological Methods 43: 291-299. GENTA R. M., DOUCE R. W. & WALTZER P. D. 1986. Diagnostic implications of parasite specific immune responses in immunocompromised patients with strongyloidiasis. Journal of Clinical Microbiology 23: 1099-I 103. GENTAR. M., FREI D. F. & LINKE M. J. 1987. Demonstration and partial characterization of parasite specific immunoglobulin A responses in human strongyloidiasis. Journal of Clinical Microbiology 25: 1505-I 510. GROVE D. I. 1980. Strongyloidiasis in Allied ex-prisoners of war in south-east Asia. British Medical Journal 280: 598% 601. GROVE D. I. & BLAIR A. J. 1981. Diagnosis of human strongyloidiasis by immunofluorescence using Strongyloides ratti and S. stercoralis larvae. American Journal of Tropical Medicine and Hygiene 30: 344-349. GROVE D. I. 1982. Treatment of strongyloidiasis with thiabendazole: an analysis of toxicity and effectiveness. Transactions of the Royal Society of Tropical Medicine and Hygiene 76: 114-l 18. GROVE D. I. &NORTHERN C. 1982. Infection and immunity in dogs infected with a human strain of Strongyloides stercoralis. Transactions of the Royal Society of Tropical Medicine and Hygiene 76: 833-837. GROVE D. I., HEENAN P. J. & NORTHERN C. 1983. Persistent and disseminated infections with Strongyloides stercoralis in immunosuppressed dogs. International Journal for Parasitology 13: 483490. GROVE D. I., WARTON A., Yu L. L., NORTHERN C. & PAPADIMITRIOUJ. M. 1987. Light and electron microscopical studies of the location of Strongyloides stercoralis
of S. stercoralis
387
in the jejunum of the immunosuppressed dog. International Journalfor Parasitology 11: 1257-1265. HARTREE E. F. 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analytical Biochemistry 48: 422427. LAEMMLIU. K. & FAVRE M. 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. Journal of Molecular Biology 80: 575-599. MY LIEN DAO 1985. An improved method of antigen detection on nitrocellulose: in situ staining of alkaline phosphatase-conjugated antibody. Journal of Immunological Methods 82: 225-23 1. NEVA F. A. 1986. Biology and immunology of human strongyloidiasis. Journal of Irzfectious Diseases 153: 397406. NORTHERN C. & GROVE D. I. 1987. Antigenic analysis of Strongyloides ratti infective larvae and adult worms. Immunology and Cell Biology 65: 231-239. NORTHERNC. & GROVE D. I. 1988. Western blot analysis of reactivity to larval and adult Strongyloides ratti antigens in mice. Parasite Immunology 10: 681-691. O’FARRELL P. Z., GOODMAN H. M. & O’FARRELL P. H. 1977. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 2: 1133-l 142. PHILLIP M., PARKHOUSE R. M. E. & OGILVIE B. M. 1980. Changing proteins on the surface of a parasitic nematode. Nature (London) 287: 538-540. POEHLING H. M. & NEUHOFF V. 1981. Visualisation of proteins with a silver ‘stain’: a critical analysis. Electrophoresis 2: 141-147. PRITCHARDD. I., MAIZELS R. M., BEHNKEJ. M. & APPLEBYP. 1984. Stage-specific antigens of Nematospiroides dubius. Immunology 53: 325-335. TOWBINH., STAEHLINT. & GORDON J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proceedings of the National Academy of Science of the United States of America 16: 435@4354.
and DAVID I. GROVE~$
* Department of Medicine, University of Western Australia, Nedlands, Western Australia 6009, Australia t Department of Postgraduate Medical Education, Sir Charles Gairdner Hospital, Nedlands, Western Australia 6009, Australia (Received 21 August 1989; accepted
I January 1990)
Abstract-NoarHaaN C. and GROVE D. I. 1990. Strongyloides stercorah: antigenic analysis of infective larvae and adult worms. International Journal for Parasilology 20: 381-387. The protein composition of Strongyloides stercorulis infective larvae and adult worms solubilized sequentially in water, sodium deoxycholate and sodium dodecyl sulphate (SDS) and their excretory/s~retory products were analysed by one- and two-dimensional SDS-~lyacrylamide electrophoresis. These extracts were demonstrated to be complex mixtures containing many proteins, some of which were common and others which were stagespecific. Western blot analysis of these antigens with infected human sera showed most sero-reactivity against larval antigens, whilst normal human sera were unreactive. These data identify immunogenic antigens which may be available for detection in an antigen assay. INDEX KEY WORDS: Strong.yfoides stercoruiis; infective larvae; adult worms; antigens; stage-specificity; Western blots; human sera.
INTRODUCTION Strongyloides
stercoralis
humans
continues
is an intestinal parasite of to elude complete understanding and accurate diagnosis. Infection often persists undetected with the worm reinfecting the host over a period of many years by a process of autoinfection. The parasite cycles between a systemic phase where infective larvae penetrate the tissues in their migration throughout the body and an intestinal phase in which the adult worm resides in the intestinal wall. An understanding of any antigenic differences between these stages may shed light on the parasites and on protective host responses to them. People infected with S. stercoralis produce specific antilarval IgA, IgE, IgG and IgM antibodies. These antibodies have been measured in various diagnostic assays including the enzyme-linked immunosorbent assay (ELISA) (Carroll, Karthigasu & Grove, 1981; Genta, Frei & Linke, 1987), the immunofluorescent antibody assay (IFA) (Grove & Blair, 1981), the radioallergosorbent test (RAST) (Genta, Deuce & Walzer, 1986) and with skin tests (Neva, 1986). The problem for accurate diagnosis, however, lies in the fact that the level of antibody measured does not necessarily differentiate between past and present that
$ Present address and address for correspondence: Department of Clinical Microbiology and Infectious Diseases, The Queen Elizabeth Hospital, Woodville South, South Australia 5011, Australia.
infections (Grove, 1982). This difficulty would be solved by the development of an assay for parasite antigens in host serum or urine. In order to approach both objectives it is first necessary to characterize the antigenic profiles of tissue-penetrating infective larvae and adult worms within the intestinal mucosa. In this study, we have compared the protein composition of infective larvae and adult worms. We employed a sequential solubilization technique in the preparation of parasite material for immunochemical analysis in order to maximize the number of antigens for study. The antibodies produced against these antigens by normal and infected individuals were then identified with Western blots. MATERIALS AND METHODS Animats and parasite. Parasites were recovered originally
from a man who had been infected while a prisoner-of-war in Southeast Asia 35 years previously (Grove, 1980). The parasite has since been maintained in mongrel dogs obtained from the local pound. The animals were washed, treated with anthelmintics bunamidine hydrochloride and pyrantel pamoate, and their faeces examined to ensure they were free of helminth eggs. The methods for preparation of larvae, infection of animals, and quantitation of larvae in the faeces have been described previously (Grove & Northern, 1982). Worm burdens were increased by immunosuppressing dogs with prednisolone plus or minus azathioprine (Grove, Heenan & Northern, 1983). Preparation of antigen. Infective larvae were obtained from cultures of dog faeces, then washed three times by 381
382
C. NORTHERNand D. 1. GROVE
centrifugation in sterile water containing penicillin (Glaxo, Boronia, Vi,., Australia) and gentamicin (David Bull Laboratories, Mulgrave, Vie., Australia). The larvae were spun to a pellet and then frozen in antigen preparation buffer (APB) [20 mM-Tris-HC! pH 8.0, 5 mM-EDTA acid, 2 mt+ phenylmethylsulfonyl fluoride (Sigma, St Louis, U.S.A.) and 100 units ml ’ Aprotinin @(Bayer Pharmaceuticals, Botany, N.S.W., Austra!id)]. Adult worms were obtained from infected dogs at least 6 weeks after infection The small intestine was removed, slit longitudinally, scraped free of mucus and rinsed, then it wasincubated in PBS pH 7.4 for 2 h at 37°C to allow parasites to crawl out of the tissues. The adult worms were separated from the residual fluid by gravity sedimentation and washed several times in PBS containing penicillin and gentamicin. Somatic proteins were prepared from worms that were thawed, washed and resuspended in fresh APB. They were then homogenized in a Braun@ homogenizer; equal volumes of worm suspension and glass beads. 0.45-0.50 pm in diameter, were placed in a bottle which was placed in a rotating cylinder cooled with rapidly flowing CO, for 2 min. The homogenate was allowed to stand overnight at 4°C. The supernatant fluid was centrifuged at 5000 g for 30 min at 4°C then the supernatant fluid of this step was respun at 100,000 g for 60 min at 4°C. The final supernatant fluid was filtered through a 0.23~pm Millex GV low protein-binding filter (Millipore Corp.. Bedford, U.S.A.); this is designated as ‘water-soluble fraction’. The two pellet preparations were combined and resuspended in APB containing 1% sodium deoxychoiate (DOC) (BDH Chemicals Ltd. Poole. U.K.), and allowed to stand overnight at 4°C. The material was centrifuged and filtered as before. This supernatant f!uid is referred to as ‘DOC-soluble fraction’. The two pellets were again combined and the process was repeated in APB containing 1% SDS (BDH Chemicals). The final supernatant fluid is designated ‘SDSsoluble fraction’. The remaining pellet was discarded. Each fraction was diatysed against water at 4°C over 24 h. Protein concentrations were measured by the method of Hal-tree (1972). In the instance of adult worms. the first two steps of antigen solubilization were omitted due to the difficulty ol sufficient two-dimensional obtaining material for eiectrophoresis (2DE) and the worms were solubilired directly in
& Neuhoff (1981). Relative molecular weights (M,) of sample polypeptides were calculated from the mobility of molecular weight standards co-electrophoresed in a separate well of each gel. Molecular weight estimates of 94 or above are extrapolations and only serve as approximate guides. Two-dimensional electrophoresis (2DEl. 2DE was performed using a modification of the method of O’Farrell, Goodman & O’Farrell(1977) as described earlier (Northern &Grove, 1987). Samples were prepared in lysis buffer, 9.5 Murea (Biorad), 2% Nonidet P40 (Sigma), 2% Pharmalyte ?-10 (Pharmacia Fine Chemicals) and 5% 2-mercaptoethanol. The first dimension was separated by non-equilibrium pH gradient gel electrophoresis (NEPHGE) on a 0.7-mm thick slab gel as described by Ferreira & Eichinger (1981) The second dimension was separated by SDS-PAGE as before and the gels were stained with silver. W’extern blot anulysis. A pool of sera from patients with parasito!o~~ca!!y proven strongyloidiasis was obtained. These sera were confirmed to have high antibody titres by ELISA against S. .stercornlis antigen (Carroll et cd., 1981). Western blot analysis was undertaken as described earlier (Northern 6z Grove, 1988). In brief, proteins and prestained molecular weight standards (Bethesda Research Lahoratories, MD, U.S.A.) separated by SDS-PAGE were transferred to nitrocellulose paper (Biorad) by the method 01 Towbin, Staehlin & Gordon (1979). lmmunoreactivc bands were detected using alkaline phosphate-conjugated antibodies (My Lien Dao, 1985). The nitrocellulose was blocked with 0.5% Tween 20 in 20 met-Tris, 500 mu-NaCl. pH 7.5 (TTBS) for 30 nun at room temperature in order to saturate unused protein binding sites. It was then incubated overnight in normal or pooled immune human serum diluted I in 250 with TTBS. The nitrocel!u!osc was washed twice in Tris-buffered saline (TBS) (20 mM-Tris, 500 mM-NaC!. pH 7.5) and placed in IO mr+EDTA to inactivate any endogenous alkaline phosphatase activity. It was then washed three times in TBS and incubated with alkaline anti-hlllllan in~n~u~l~~&lohul~n phosphat;~se-conjugale~i (Silenus Laboratories, Hawthorn. Australia) diiuted I:500 in TTBS for I h. The nitrocellulose was washed three tlmcs 111 TTBS and then rinsed in substrate buffer (0.06 \I-sodium borate pH 9.7 containing 1.2 mg ml ’ magnesium sulphatci. immune complexes were visualized after staining with (Idianisidine (0.25 mg ml ‘) and B-naphthyi acid phosphate (0.25 mg ml ‘) in substrate buffer. Following enlyme staining. the nitrocellulose was washed in a solution 01’ methanol, acetic acid and water (5: 1.5:. v!\ :\ I foilowccih\ ;t final rinse in water. RESULTS One-ilimensional el~~c,tropllorc,.si.r The SDS-PAGE profiles of infective larval water-. DOC- and SDS-soluble preparations and ES proteins
SDS pol~ucr$_mid~~ ~cl electrophoresi.~ (SDS PAGE). All preparations were analysed initially by one-dimensional SDS-PAGE (Laemmli & Favre. 1973). In brief. samples containing 5 pg protein and molecular weight standards (IMW kit, Pharmacia Fine Chemicals, Uppsala. Sweden) were prepared in sample buffer. 62.5 mr+Tris--HCI pH 6.8. 4% w/v SDS, 5% v/v 2-mercaptoethanol (BDH Chemicals), 10% glycerol (BDH Chemicals) and 0.001% bromophenol blue (Biorad. Richmond. California, U.S.A.). These were then heated in a water bath at 100°C for 5 min. Samples were centrifuged at 11,000 g for 5 min and were loaded onto the gels. These were stained with silver as described by Poehling
are illustrated in Fig. 1. A large number of bands were resolved for each preparation with differences noted according to the method of solubihzation. In the water-soluble larval extract major bands were resolved at 16, 17 ,18, 28 and 59 mol. wt. Major DOC-soluble bands had mol. wts of 3 I, 36.59 and 97, while the SDSsoluble bands were resolved at 18.3 I, 36.50.55 and 62 mol. wt. The ES preparation yielded a dominant band of mol. wt 52 with additional minor components. The SDSPAGE profiles of adult worms are illustrated in Fig. 2. Five preparations were analysed: water-. DOC-.
Antigenic analysis of S. sfercoralis
383
revealed major components at 33, 44, 60 and two bands greater than 94 mol. wt. The sequential SDSsoluble extract had major bands at 30, 33, 37, 66, 78 and 83 while the SDS-total-soluble extract contained dominant bands at 21,24, 26 and 66 mol. wt. The ES extract had major bands at 16, 33 and 71 mol. wt.
-20 -14.4 12
3
4
FIG. I. SDS-PAGE of S. stercoralis infective larvae preparation, stained with silver. Water-soluble fraction (lane I), DOC-soluble fraction (lane 2) SDS-soluble fraction (lane 3) ES products (lane 4). Molecular weight standards are shown on the right.
Mr
-94 -67 -43 -30
-20 -14.4 12
3
4
5
FIG. 2. SDS-PAGE of S. stercoralis adult worm preparation,
stained with silver. Water-soluble fraction (lane l), DOCsoluble fraction (lane 2), SDS-soluble fraction (lane 3), SDStotal-soluble fraction (lane 4) ES products (lane 5). Molecular weight standards are shown on the right. SDS-, SDS-total-soluble proteins and ES products. Again, a complex array of bands were resolved with differences observed among the methods of preparation. Major water-soluble bands were resolved at 19, 5 1 and 64 mol. wt, while the DOC-soluble preparation
Two-dimensional electrophoresis Greater discrimination of these differences was provided by 2DE. Many proteins were visualized in the water-soluble fraction of infective larvae (Fig. 3a). A number of additional spots were seen in the sequential DOC-solubilized preparation (Fig. 3b). In the third preparation in which the pellet was finally solubihzed in SDS a further number of proteins were demonstrated (Fig. 3~). It was not possible to make direct comparisons between the infective larval and adult worm somatic components as the paucity of adult worms did not permit sequential solubilization of this stage of the parasite. Nevertheless increased discrimination of proteins was obtained when 2DE was performed on adult worm material extracted directly with SDS (Fig. 4a). A smaller set of proteins was isolated from larval excretory/secretory products (Fig. 3d). The pattern of distribution of proteins in the ES products derived from adult worms (Fig. 4b) was quite different from that seen with larvae thus indicating differences in the stage-specificities of the parasite. Western blots The soluble extracts of infective larvae and adult worms were analysed by Western blots with a pool of normal, uninfected human sera and a pool of immune sera from individuals with parasitologically proven strongyloidiasis. Figure 5 shows the profile of reactivity of normal sera to the antigenic mixtures and Fig. 6 illustrates the same profile of antigens reacted with immune sera. The normal serum profile is notable for its lack of reactivity against virtually all proteins. A single band in the larval DOC-soluble preparation with mol. wt 38 is faintly detected. In contrast, the immune serum profile identifies a number of antigenic bands in both the larval and adult worm preparations. The larval water-soluble preparation has major reactive bands with mol. wt 38, 48 and 58 as do the DOC-soluble preparation and the SDS-soluble preparation, the latter also having an additional band at 27 mol. wt. The ES products comprise many but fainter reactive bands; the major ones have mol wts 11, 14, 38,48 and 58 and six bands with a mol. wt greater than 75. In contrast to the larval preparations the adult worm preparations are characterized by a paucity of strongly reactive components. The adult SDS-total-soluble preparation has two major reactive bands, one with mol. wt 38 and the other a band at greater than 130. The adult ES preparation shows only a number of faintly staining bands, the strongest being with mol. wt 58.
384
C. NORTHERN andD.1. GROVE
Antigenie analysis of S. sterc~rui~s
385
Mr
-94 -67 -43 -30
ACIDIC
BASIC
FIG. 4. Two-dimensional electrophoretic profiles of S. stercoralisadult worms. Samples were analysed by NEPHGE in the first dimension and SDS-PAGE in the second dimension. SDStotal-soluble fraction (panel A) and ES products (panel B). Acidic and basic regions of the gels are indicated. Molecular weight standards are shown on the right.
DISCUSSION
In this study we have shown that the antigenic profiles of the tissue penetrating infective larvae and mucosal-dwelling adult worms differ. In addition, we have demonstrated that both the somatic extracts and excretory/secretory products are complex mixtures containing many proteins which can be separated in high resolution electrophoresis systems. The method of sequential solubilization of the worms yielded different sets of proteins reflecting differing solubitities in the detergents used. Although
exposed to strong anionic detergents such as sodium deoxycholate and sodium dodecyl sulphate the proteins retained their antigenicity when utilized in a Western blot assay. We have made similar observations in the comparison of Strongyloides ratti infective larvae and adult worms (Northern & Grove, 1987) as have others with different nematodes such as Trichinella spiralis, Nematospiroides dubius and Dipetalonema viteae (Phillip, Parkhouse & Ogilvie, 1980; Pritchard, MaizeIs, Behnke & Appleby, 1984; Baschong, 1985).
386
C. NORTHERN and D. 1. GROVE
Mr
-130 -75 -50 -39 -27
-17
12
3
4
5
6
FIG. 5. Western blot of S. srercoralk larval and adult worm antigen preparations with normal human sera. Larval watersoluble (lane I), larval DOC-soluble (lane 2). larval SDSsoluble (lane 3), larval ES products (lane 4). adult SDS-totalsoluble (lane 5) and adult ES products (lane 6). Prestained molecular weight standards are shown on the right.
Mr
-130 -75 -50 -39 -27
123456 FIG. 6. Western blot of S. stercoralis larval and adult worm antigen preparations with immune human sera. Larval water-soluble (lane I), larval DOC-soluble (lane 2), larval SDS-soluble (lane 3), larval ES products (lane 4), adult SDStotal-soluble (lane 5) and adult ES products (lane 6). Prestained molecular weight standards are shown on the right.
Others have also begun to analyse larval surface, metabolic and excretory/secretory antigens of S. stercorafis (Brindley, Gam, Pearce, Poindexter & Neva, 1988). They identified antigenic molecules from
infective larvae on the basis of immunoprecipitation reactions with human sera and purified larval ES proteins bound to a column of human IgG from immune human sera. They demonstrated a number of bands with mol. wts 12,25,30,33,35,40,50,54,60,90 and 240. We also demonstrated antigenic bands at some of these weights although direct comparison is difficult. Our interest has been to study both infective larvae and adult worms in order to compare the antigenic profiles of the two stages of the parasite. We have shown that both the somatic extracts and the excretory/secretory products of two developmental stages of S. stercoralis are highly complex but that common and stage-specific proteins could be demonstrated. The use of 2DE permitted greater discrimination of the various protein bands found by SDS-PAGE. Furthermore we have shown with Western blots utilizing sera obtained from infected humans that many of these compounds are immunogenic. The patients were suffering from strongyloidiasis and had no other concomitant parasitic infections. Most seroreactivity was directed against larval antigens. Similar major bands were seen in the water-, DOC- and SDSsoluble extracts reflecting their increasing solubility in stronger detergents. When adult worm somatic antigens were extracted using the strongest detergent. SDS, very few antigenic bands were identified. A plethora of antigenic bands was shown in the metabolic products produced by infective larvae. In contrast, only a small number of bands were seen in the metabolic products derived from adult worms. The difference in sero-reactivity between infective larvae and adult worms is not surprising when the physical locations of these stages of the parasite are considered. Infective larvae migrate through the tissues and are in close proximity to the cells of the immune system. On the other hand, adult worms live in tunnels in the small intestine epithelium and do not penetrate the basement lamina (Grove, Warton, Yu, Northern & Papadimitriou, 1987). They are therefore relatively isolated from immunoreactive cells. These studies provide a framework for the identification and separation of those antigens which are most relevant to the generation of host-protective immune responses and which may be made amenable to detection in an antigen assay. Acknowledgemenrs-This study was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES BASCHONGW. 1985. Changes in the surface of Dipefalonema vifeae (Filarioidea) during its development as shown by comparative peptide mapping. Parasikg?, 90: 35 l-356.. BRINDLEYP. J., GAM A. A.. PEARCEE. J.. POINDEX~ERR. W. & NEVA F. A. 1988. Antigens from the surface and excretions/ secretions of the filariform larva of Srrongylaides
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analysis
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