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Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen
Molecular and Biochemical Parasitology, 31 (1988) 241-2511
241
Elsevier MBP 011)46
Construction of Onchocerca volvulus c D N A libraries and partial characterization of the c D N A for a major antigen John E. D o n e l s o n 1, B . O . L . D u k e 2, David Moser 1, Wenlin Zeng ~, Ngozi E. Erondu l, Richard Lucius 3, Alphons Renz 4, Marc Karam 5 and Guillermo Zea Flores 6 IDepartment of Biochernistrv, University of Iowa, lowa City, 1A, U.S.A. : :Dept. oJ" Infectious and Parasitic Diseases Pathology, Armed Forces Institute of Pathology, Washington, DC, U.S.A. : 3b~stitut fl~r Tropenhygiene, Universitiit Heidelberg, Heidelberg, F. R. G. ; aTropenmedizinisches lnstitut. Universitiit Tiibingen, Ti~bingen, F.R. G. ; SOnchocerciasis Control Programme, Ouagadougou, Burkina Faso: 6Ministerio de Salud Publica Asistencia Social, Dept. de EnCermaedad de Robles (Oncocercosis), Guatemala City, Guatemala (Received 9 May 1988; accepted 27 June 1988)
Adult Onchocerca volvulus were isolated from nodules removed from onchocerciasis patients at four locations - two in the West African Sudan-savanna region (near B a m a k o , Mali, and Touboro, Cameroon), one in a West African forest region ( K u m b a , Cameroon) and one near G u a t e m a l a City, Guatemala. Four different c D N A expression libraries were constructed in bacteriophage ~gt 11 using poly(A) + R N A from the adult female worms. Individual c D N A clones of single copy genes were used to compare the g e n o m e s of parasites from the different locales and to show that the haploid genome of O. volvulus is 1.5 × l/I ~ base pairs. A b o u t 1 in 700 recombinant clones in each of the four amplified c D N A libraries produces a fusion protein recognized by pooled h u m a n anti-O, volvulus antisera. Partial sequence determination of a 2.0 kb c D N A clone for an O. volvulus protein that induces an imm u n o d o m i n a n t response in rabbits revealed that this antigen has sequence similarities with Caenorhabditis elegans myosin and with schistosome paramyosin (which confers partial protection against schistosome infection). The four c D N A libraries have been deposited with American Type Culture Collection ( A T C C ) , 12301 Parklawn Drive, Rockville, MD 20852, U . S . A . , for general distribution u n d e r A T C C N u m b e r 37509. Key words: Onchocerca volvulus; c D N A library; G e n o m e size: Immunoscreening: I m m u n o d o m i n a n t antigen
Introduction An estimated 17.8 million people in sub-Saharan Africa and Latin America suffer from onchocerciasis, or river blindness [1]. The disease is caused by Onchocerca volvulus, a filarial nematode transmitted by blackflies of the genus Simulium which live near rapidly flowing rivers and streams. The infection is associated with clinical manifestations ranging from pruritis and various skin changes to severe lymphatic involvement and
Correspondence address: J.E. Donelson, Dept. of Biochemistry, University of Iowa, Iowa City, IA 52242, U.S.A. Note: Nucleotide sequence data reported in this paper have been submitted to the G e n B a n k T M Data Bank with accession n u m b e r J03995.
ocular disease. The most extreme cases occur in people of the Sudano-Guinean savanna zone of Africa, where microfilariae frequently enter the eyes and cause lesions that can lead to impaired vision and blindness [2]. In some highly endemic savanna areas children that survive beyond the age of 15 have a 40-50% chance of eventually becoming blind [3]. In contrast, the parasite burden of individuals living in the forest regions of West Africa can be as high as in the savanna but the incidence of blindness is much lower. The reason for this difference is not known but a commonly expressed possibility is that different genetic forms of the parasite may exist in the forest and savanna regions [4-9]. A major problem in the study of O. volvulus and onchocerciasis is the lack of a good laboratory model for the disease. The parasite infects
11166-6851/88/$03.51/© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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only humans, gorillas and chimpanzees [10]. None of the traditional laboratory m a m m a l s has been shown to harbor a productive infection of O. volvulus. Therefore, the main source of worm material comes from nodules removed from human patients, most of whom live in remote areas. This greatly limits the n u m b e r of investigators that can study the parasite and has led to large gaps in our understanding of the genetics of the worm and the immunology of the infection. For example, very little is known about the w o r m ' s metabolism or how the microfilariae evade the immune response of their host. To provide w o r m material for a larger n u m b e r of investigators and to examine the basic molecular biology of the parasite, we isolated adult worms from onchocerciasis patients in four widely spaced geographical areas and p r e p a r e d c D N A libraries of their poly(A) + R N A . In vitro translations and Southern blots show that the parasites from the four areas are very similar in their genetic make-up. When the c D N A libraries were immunoscreened with pooled patient antisera or with a rabbit antisera against an adult worm homogenate, m a n y positive clones in all four libraries were detected. Several c D N A clones identified during these immunoscreenings were found to cross-hybridize and one was characterized further. Materials and Methods
Collection of parasites. As summarized in Table I, nodulectomies were p e r f o r m e d under local anesthesia on 20-41 onchocerciasis patients at each of the four locales. I m m e d i a t e l y after their surgical removal, the nodules were submerged in a sterile solution of R P M I 1654 medium containing 20 m M H e p e s buffer, p H 7.5, 0.2 mg gentamicin ml 1 and 3 mg collagenase ml -~ (isolated from Clostridiurn histolyticum and purchased from Boehringer-Mannheim Biochemicals). The nodules were digested in this solution as described [11] for 12 - 48 h at t e m p e r a t u r e s between 30 and 37°C (equipment for maintaining a constant temperature was not available). Periodically, the nodules were placed in a Petri dish under a dissecting microscope and partially digested human tissue r e m o v e d with a small forceps. The diges-
tion solution was replaced with fresh solution if the incubation continued for more than 24 h. As soon as intact adult worms were sufficiently free of nodular tissue, they were removed with forceps and extensively rinsed with R P M I medium dispensed from a Pasteur pipette. Special care was taken during this step to remove all of the visible human tissue from the worms. Female worms and the separated smaller male worms were sorted into groups of 1 to 15, placed in cryopreservation tubes and immediately frozen in liquid nitrogen. As Table I shows, 1-3 nodules were obtained from each patient and 50-209 female worms were isolated at each collection site. Similar numbers of male worms were also obtained. Often the larger nodules contained calcified and dead worms that were discarded. One intermediate-sized nodule, from a patient in the forest area near K u m b a in southwestern C a m e r o o n , contained 31 live female worms, all of which were about the same size and could be separated intact from each other. These worms from the same nodule were used to make the K u m b a c D N A library.
Isolation of RNA and DNA. A porcelain mortar. was placed in a solid CO2-metfianol bath and filled with liquid nitrogen. Frozen female worms (23-33 in number) were placed in the mortar and ground for about 10 rain to a fine powder with a pestle. Liquid nitrogen in the mortar was periodically replaced during the grinding. The powder was transferred to 11 ml of a solution containing 8 M guanidinium thiocyanate, 100 mM Tris-HC1, pH 7.6, 20 mM E D T A , 2% Sarkosyl and 5 mM [3mercaptoethanol [12] and subjected to two 10 s
TABLE I Collection of adult Onchocerca volvulus Location
No. of patients
No. of nodules
-20 -20 41
21 -25 62
No. of live female worms
Guatemala Mali Touboro, C a me roon Kumba, Cameroon
(forest) (savanna)
50 54 156
(savanna) 28
71 ~'
(forest)
:' One nodule contained 31 live female worms.
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243 T A B L E I1 D N A and R N A isolation from adult female worms
Kumba Touboro Mall Guatemala
No. of female worms
DNA (p.g)
Total RNA (~g)
% Poly(A) ÷ RNA
31 33 23 30
200 135 156 198
270 191 178 172
6.2 7.3 7.9 9.0
Yield/female worm
5.1
7.(l
bursts in a tissue mincer (Tekmar Co., Cincinnati, OH). This solution was centrifuged at 4000 rpm for 10 rain. The pellet was resuspended in 1 ml of the guanidinium thiocyanate solution and the centrifugation repeated. The 1 ml of supernatant from the second centrifugation was added to the original 11 ml of supernatant. The 12 ml were layered on top of step gradients of 5.7 M CsC1 (2 ml) and 3.0 M CsC1 (2 ml) containing 0.1 M E D T A in Beckman SW41 tubes and centrifuged at 29000 rpm for 20 h. The R N A was recovered in a pellet at the bottom of the tube and the D N A from the interface between the 5.7 M and 3.0 M CsC1 layers. The R N A and D N A were then treated as described [12]. Table II summarizes the recoveries. An average of 5.1 p,g D N A and 7.0 p,g total R N A was obtained from each female worm.
Construction of cDNA libraries. Poly(A) + R N A was purified from total female R N A by two passages through oligo(dT) cellulose (Collaborative Research, Type 3) [13]. Between 6% and 9% of the total R N A was recovered as poly(A) + R N A (Table II). First strand c D N A was prepared from
5 ~xg of poly(A) + R N A using AMV reverse transcriptase and oligo(dT)12_18 as primer [14]. Following alkaline hydrolysis of R N A in the RNAD N A hybrid, second strand cDNA was synthesized by hairpin priming using the Klenow fragment of D N A polymerase I (Boehringer Mannheim Biochemicals). The hairpin was cleaved with S1 nuclease. The c D N A was then treated with T4 D N A polymerase and all four dNTPs to ensure the presence of blunt ends. After methylation of the cDNA with EcoRI methylase, EcoRI linkers were ligated to the ends of the molecules and digested with EcoRI. The c D N A was purified from linker fragments and size fractionated on a Sepharose CL-4B column. After ligating the cDNA to phosphatase-treated, EcoRI-cleaved ~.gtll D N A (Strategene) and incubating with ;~ packaging extracts (GigaPack Gold, Strategene), libraries containing 27-61 × lff' phage were obtained as summarized in Table III. ?
Amplification and distribution of the libraries. Between 4 and 10 million phage in each of the four unamplified cDNA libraries were incubated with Escherichia coli strain Y1088 and plated onto 100 Petri dishes (150 cm 2) for amplification as described [15]. A summary of these amplified libraries is given in Table IV. Note that the background of phage without c D N A inserts (yielding blue plaques) increased during the amplification step, probably because phage without inserts have a larger burst size. These amplified libraries are available from the American Type Culture Collection (ATCC). lmmunoscreening of the cDNA libraries. The libraries were introduced into E. coli strain Y1090 and immunoscreened [14,16] at a density of about
T A B L E III Construction of primary c D N A libraries Poly(A)- R N A (Ixg)
No. of )~gtll recombinant clones
% without cDNA inserts (blue plaques)
No. of positive clones with human patient antisera
< l 2.5 7 8
1 out 1 out 1 out 1 out
(x 106) Kumba Touboro Mali Guatemala
5 5 5 5
61 27 30 38
of of of of
50(1 500 500 700
244
10 000 phage per Petri dish (150 cm 2) with a 1:100 dilution of pooled h u m a n anti-O, volvulus antisera or with a 1:1000 dilution of rabbit antiserum against an O. volvulus homogenate. The human antiserum was obtained from chronically infected patients living in a forest region of Liberia, West Africa. The h o m o g e n a t e used to generate the rabbit antiserum was prepared by sonicating sliced adult female worms for 2 rain at 400 W and was injected into the rabbit subcutaneously, with Freund's complete adjuvant. Subsequent boosts were made with the h o m o g e n a t e plus Freund's incomplete adjuvant. Other methods and materials. In vitro translations using rabbit reticulocyte lysates (Promega Biotec, Madison, WI) [17] and Southern blots of genomic D N A [18] were conducted as described. D N A fragments were radiolabeled in vitro for hybridizations according to the random priming procedure [19], D N A sequences were determined according to the M a x a m and Gilbert procedure [20] on fragments that were 3' end-labeled with [o~-32p]dNTPs and Klenow fragment of D N A polymerase I. Subcloning of c D N A fragments from ,~gtll to p U C plasmids and other general procedures were as described [15]. Unless otherwise noted, enzymes and other recombinant materials were from New England Biolabs, Beverly, M A , or Bethesda Research Labs, Gaithersburg, M D , and were used under conditions r e c o m m e n d e d by the supplier.
Fig. 1. Autoradiogram of two dimensional gels [29] containing the [35S]methionine-labeled in vitro translation products of total RNA isolated from adult female O. volvulus collected in Guatemala (forest region of Central America) and near Touboro. East Cameroon (savanna region of West Africa). Arrows on the right side of each panel indicate the positions of molecular weight markers of (top to bottom) 97, 75, 50, 42 and 20 kDa. respectively.
Results
In vitro translations o f O. volvulus R N A . Since the parasite-containing nodules were incubated with collagenase for 12-48 h prior to isolating the adult worms, it was necessary to determine if degradation of the parasite R N A occurred during this digestion period. In vitro translations of the isolated R N A s with rabbit reticulocyte lysates and [35S]methionine were used for this evaluation. The translation products were displayed on two-dimensional acrylamide gels, one example of which is shown in Fig. 1. The larger proteins synthesized during these in vitro translations are greater than 80 k D a , suggesting that little, if any, degradation of parasite m R N A occurred during the
collection of the parasites and isolation of the RNA. In addition, no differences, other than some changes in relative intensity, were detected among the 200-300 major translation products from the RNAs of the parasites from G u a t e m a l a (Central American forest) and from T o u b o r o (African savanna). Identical patterns of in vitro translation products were also obtained when R N A s from Mall (African savanna) and K u m b a (African forest) parasites were used (not shown). Thus, at this level of gene expression, there are no detectable differences between parasites from the forest and the savanna regions.
245
Construction and preliminary characterizations of cDNA libraries. To provide reagents to investigate parasite antigens that elicit an immune response, c D N A expression libraries in bacteriophage )~gtll were constructed from each of the four parasite R N A s as described in Materials and Methods. These R N A s , isolated from adult females, were derived from both adult tissue and the developing stages of microfilariae in the fertilized female. In addition a few adult male worms may have contaminated the preparations of the much larger females. As summarized in Table III, between 27 and 61 million primary c D N A clones were obtained from 5 Ixg of starting poly(A) ~ R N A . Examination of over 40 randomly, or specifically, selected c D N A clones has revealed that the average insert size of the libraries is 1200 bp with a range of 3(X) to 35(X) bp. Some phage in the K u m b a library may have multiple c D N A inserts since a higher percentage of the inserts in this library have internal E c o R I sites than would be Hind III GMT
K4"4
expected for their insert sizes (on average, an E c o R I site would be expected about every 4000 bp). The other three libraries appear to have fewer of these multiple cloning events although this has not been rigorously examined. Since the parasites were isolated from human tissue, it was important to determine whether human R N A and D N A contaminated the preparations of parasite R N A and D N A . The middle panel of Fig. 2 shows a test of this possibility. A Southern blot of restricted D N A from humans and from each of the four parasite preparations was probed with radiolabeled total human D N A . As expected, a strong signal occurred in the lanes containing the restricted human D N A . In the lanes that contain parasite D N A , hybridization could only be observed to the D N A from the G u a t e m a l a parasites (lanes G). Densitometer tracings of the autoradiogram revealed that this contamination is about 6% for the G u a t e m a l a n parasite D N A and less than 1% for the others.
Eco RI
HUMAN
3 2 1GMTKS GM
T K
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K GMT
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Fig. 2. Southern blots to determine the contamination of parasite D N A w i t h h u m a n D N A and to determine the g e n o m c s i z e of O. voh, ulus. (Left panel) Ethidium bromide stain of an 0.Sff'~ agarose gel containing 5 p,g of genomic D N A from parasites from G u a t e m a l a (G), Mall (M), T o u b o r o (T) and K u m b a (K) digested with either H i n d l l l or E c o R | . Lanes 1-4 contain E e o R | digests of 5 p,g of h u m a n D N A and. respectively, 11, 22, 33, and 44 pg of the purified 840 bp c D N A insert in a randomly selected recombinant phage of the K u m b a c D N A library. Lane 4' is the same as lane 4 except that the D N A is digested with HindIll. Lane S contains trace a m o u n t s of ~2p-labeled H i n d l l l fragments of X D N A . (Middle panel) Autoradiogram of a Southern blot of the D N A s shown in the left panel probed with labeled total h u m a n D N A . (Right panel) Autoradiogram of the D N A s in the left panel probed with the same labeled 840 bp c D N A fragment that was mixed with the h u m a n D N A in lanes 1-4'.
246
Dot blot hybridizations using human D N A as the probe seemed to be less sensitive than the Southern analysis (data not shown). By analogy, the extent of contamination of the R N A preparations is probably about the same and may be reflected in the c D N A libraries as well. The higher level of contamination in the G u a t e m a l a n parasites probably occurred because the collagenase treatment of these nodules, which were collected first, was conducted before the optimal digestion conditions had been determined.
Determination o f the genome size of O. volvulus. Since the genome size of O. volvulus has not been reported, several randomly selected c D N A clones from the K u m b a c D N A library were used as probes to determine this haploid size. The right panel of Fig. 2 shows one such experiment. The single 840 bp E c o R I c D N A insert of a randomly chosen recombinant ;~gtll phage was used as a probe for this Southern blot. It contains two internal H i n d I I I sites separated by about 200 bp. In all four genomic D N A s this c D N A hybridizes to a single E c o R I D N A fragment of 3.8 kb and to three H i n d I I I fragments of 1.0 kb, 2.0 kb and the 200 bp of internal sequence (which migrated too far to be seen in Fig. 2), as would be expected for a single copy gene in the genome. This gene is not tandemly repeated in the genome because additional H i n d I I I fragments containing sequences flanking the potential terminal gene repeats were not observed. Lanes 1-4 show the hybridization of this probe to increasing amounts of its own 840 bp sequence, which via densitometer tracings were used to correlate the signal intensity to a known amount of the sequence. From this analysis, and
by averaging the signals in the four lanes containing EcoRI-digested parasite D N A , it was determined that about 29 pg of the 840 bp sequence are present in 5 ~g of genomic D N A , which is equivalent to a haploid genome size of about 1.5 × 10~ bp. An analogous calculation was made by adding the signals of the two bands in the HindIII lanes and a similar value for the genome size was obtained. In addition, a similar result was obtained when a second unrelated e D N A insert from another recombinant phage was used as the probe (not shown). A third c D N A probe, however, yielded a hybridization pattern that was consistent with the presence of three distinct hybridizing regions in the genome (not shown). Still other e D N A probes yielded more complex hybridization patterns, suggesting that at least portions of their genomic sequences are repetitive. The value of 1.5 x 1(1~ bp for the O. volvulus genome is within a factor of two of those determined for other nematodes and trematodes such as Caenorhabditis elegans (0.8 x 1()~ bp) [21], Brugia malayi (0.8 x 1()~ bp) [22] and Schistosoma mansoni (2.7 x 10a bp) [23]. It should also be noted that all of the c D N A s that have been used to date as hybridization probes yield the same Southern blot pattern of fragments in all four O. volvulus genomes; a result that, although admittedly a small sample size, is consistent with the occurrence of relatively little coding sequence variation among parasites at different geographic locations.
Characterization of a cDNA for an O. volvulus antigen that induces an immunodominant response. A b o u t 30000 clones of each of the four c D N A libraries were immunoscreened with a
T A B L E IV Amplification of the c D N A libraries
Kumba Touboro Mall Guatemala
No. of clones that were amplified ( x lff')
No. of phage ~xl ~ after amplification (x 10'~)
?i without e D N A insert (blue plaques)
No. of positive chines with h u m a n patient antisera
4 10 10 l0
0.8 4.0 4.8 3.0
4 ~* 9 12 14
1 out 1 out 1 out 1 out
~' Note that c>~.b a c k g r o u n d increased during the amplification step.
of of of of
700 700 700 7tl0
247
rabbit antiserum against an adult O. volvulus homogenate and with pooled antisera from onchocerciasis patients in a forest area of Liberia. About 1 in 500 clones in each of the libraries produced a fusion protein recognized by the rabbit antisera and about 1 in 700 clones were detected by antibodies in the h u m a n antisera (Tables III and IV). Pre-immune sera from a rabbit or from a non-infected h u m a n did not give a positive signal with recombinant clones in any of the libraries. The fact that both the rabbit and h u m a n antisera recognize the same proportion of clones in each of the libraries supports the conclusion that most antigens and their coding sequences are very similar in parasites from all four collection sites. Small-scale D N A preparations were p e r f o r m e d on 16 plaque-purified phage from the unamplified K u m b a and T o u b o r o libraries that were positive with the rabbit antiserum. Hybridization of
Southern blots containing E c o R I digests of these phage D N A s using different phage D N A s as the labeled probe revealed that 7 out of the 16 cDNAs (44%) cross-hybridized. Each of these 7 independent recombinant phage contained a c D N A insert of 2.0-2.3 kb. The 2.0 kb c D N A insert of one of the cross-hybridizing phage was chosen for further characterization and subcloned into the E c o R I site of pUC19. The nucleotide sequences at the ends of this insert were determined by M a x a m and Gilbert sequencing of end-labeled fragments generated by cleavage with E c o R I or HindlII (in the polylinker of p U C I 9 ) . The 5' sequence at one end of the insert was found to have termination codons in all three translation reading frames, suggesting that the other strand contains the sequence appearing in the antigen's mRNA. The 400 nucleotides determined at the 5' end
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Fig. 3. (A) The 5' nucleotide, and deduced amino acid, sequence of a 2.0 kb c D N A clone encoding an i m m u n o d o m i n a n t protein of O. volvulus recognized by a rabbit antiserum. (B) The deduced amino acid sequence shown in panel A arranged as heptad repeats (a-b-c-d-e-f-g), in which the boxed positions 'a' and 'd" (asterisks) are predominantly hydrophobic residues while the other positions are often hydrophilic residues. In this sequence organization the hydrophobic residues are packed between the two strands of an s-helical coiled-coil while most of the polar groups point out [30]. (C) Comparison of amino acid positions 79-106 in the O. voh'ulus sequence shown in panel A with amino acids 292-318 in the partial paramyosin sequence of Schistosoma mansoni [25] and amino acids 853-879 in the unc-54 myosin of Caenorhabditis elegans [26]. Solid boxes surround identical amino acids and dashed boxes enclose functionally similar residues.
248 of the other strand contain a single open translation reading frame shown in Fig. 3A. This nucleotide sequence likely begins at an internal EcoRI site in the original c D N A since it does not have the EcoRI linker sequence of 5' GAAT-FCC 3'. It is not known whether this is the result of subcloning from Xgtll to the plasmid or lack of methylation during the c D N A library construction. The deduced amino acid sequence has a pattern of distinct periodic heptad repeats (a-b-cd-e-f-g)n in which hydrophobic residues predominate at positions a and d (Fig. 3B). This sequence organization is characteristic of c~-helical coiled-coils found in some structural proteins such as myosin [24]. In addition, a computer search of all of the nucleotide sequences in the GenBank database revealed that the sequence in Fig. 3A has the greatest similarity to the partial sequence of paramyosin of Schistosoma mansoni [25]. Slightly less sequence similarity was detected with segments of the o~-helical coiled-coil rod regions of other myofibrillar proteins, such as myosin from Caenorhabditis elegans [26]. Fig. 3C shows a comparison of a 26-27 amino acid region from each of these three proteins at which about 37% positional identity exists. Other regions within the partial sequence of O. volvulus antigen also show similar amino acid identity to segments of paramyosin and myosin. However, within the sequences currently available for all three antigens, these similarities are not uniformly distributed throughout the sequence shown in Fig. 3A but are confined to pockets such as the one shown in Fig. 3C. A more extensive sequence comparison awaits the complete nucleotide determination of the 2.0 kb cDNA but the common properties shown in Fig. 3B and C suggest that this immunodominant antigen of O. volvulus may be a myofibrillar protein. Discussion
The availability of c D N A libraries of adult O. volvulus m R N A should help overcome some of
the research problems associated with the inability to maintain this parasite in the laboratory. For example, it has not been possible to obtain a sufficient amount of any protein or antigen from this parasite for biochemical characterization. Now it
should be possible to synthesize large quantities of the peptide backbones of most 0. volvulus proteins in an appropriate recombinant vector/host system once their cDNAs have been identified in the libraries. In addition, to date only three reports on the D N A of O. volvulus have appeared [8,9~27]. All three describe the cloning and characterization of repetitive genomic D N A sequences from the organism for potential diagnostic uses. The expression cDNA libraries described here should contribute to this diagnostic objective and also facilitate the identification of genes for parasite antigens useful in both diagnostic and immunoprophylactic studies. A first step toward the latter goal is the detection of cDNA clones for the immunodominant antigen described here. The fact that this antigen has amino acid similarity to schistosome paramyosin is of both biochemical and immunological interest. It suggests that this antigen might be an O. volvulus analog to schistosome paramyosin and that eventual comparison of their complete sequences will provide information about the structural conservation and function of these invertebrate muscle proteins. In addition, vaccination with S. mansoni paramyosin has been shown to confer partial protection against challenge by infective larvae of S. rnansoni [28]. Indeed, when mice were immunized intradermally with extracts of S. rnansoni, the predominant schistosome antigen against which antibodies were raised was paramyosin [25]. Thus, it is perhaps not surprising that injection of extracts of another helminth parasite would induce an immune response to a similar protein. Paramyosin has recently been shown by immunoelectron microscopy to be present in the external tegument structure of schistosomes [31], which may account for its immunogenicity during a schistosome infection and may also suggest a similar external location for the O. volvulus antigen described here. In contrast to schistosomes, where sufficient paramyosin for vaccination studies was obtained from laboratory-reared parasites, further characterization of this O. volvulus antigen will require its production by recombinant techniques. Finally, no evidence for polymorphisms was observed in the in vitro translations or with the
249
few c D N A s used to probe Southern blots. The parasites from forest and savanna regions of Africa, and from Guatemala in Central America, all yielded the same patterns, providing no molecular insights into the clinical differences of their infections. Nevertheless, a 153 bp probe of O. volvulus repetitive DNA has been reported to be specific for forest parasites [8]. This suggests that differences a m o n g the parasites do exist at the D N A level and that it may be possible to differentially screen the forest and savanna c D N A li-
braries for transcripts or antigens that form the basis for these clinical differences. Acknowledgements We thank Kwang S. Kim, Virginia Strand and Charles Keller of the University of Iowa for technical support on the recombinant D N A portion of this project. The work was supported by a grant from The Edna McConnell Clark Foundation.
References 1 World Health Organization (1987) Third report of the Expert C o m m i t t e e on Onchocerciasis. Technical Report Series No. 752, World Health Organization, Geneva. 2 Connor, D . H . , Gibson, D . W . , Taylor, H . R . , Mackenzie, C . D . , Meyers, W.M. and Neafie, R.C. (1984) Onchocerciasis. In: H u n t e r ' s Tropical Medicine, 6th edn. (Strickland, G . T . , ed.), pp. 667-680, W.B. Saunders Co., Philadelphia. 3 Prost, A. (1986) The burden of blindness in adult males in the savanna villages of West Africa exposed to onchocerciasis. Trans. R. Soc. Trop. Med. Hyg. 80, 525-527. 4 Duke, B . O . L . , Lewis, D.J. and Moore, P.J. (1966) Onchocerca-Sirnulium complexes 1. - Transmission of forest and Sudan-savanna strains of Onchocerca voh, ulus from C a m e r o o n by Simulium damnosum from various West African bioclimatic zones. Anal. Trop. Med. Parasitol. 611, 318-336. 5 D u k e . B.O.L. and A n d e r s o n , J. (1972) A comparison of the lesions produced in the cornea of the rabbit eye by microfilariae of the forest and Sudan-savanna strains of Onchocerca voh,ulus. T r o p e n m e d . Parasitol. 22,354-368. 6 A n d e r s o n , J., Fuglsang, H., Hamilton, P.J.S. and de-C. Marshall, T.F. (1974) Studies on onchocerciasis in the United C a m e r o o n Republic, II. Comparison of onchocerciasis in rain-forest and Sudan-savanna. Trans. R. Soc. Trop. Med. Hyg. 68, 2/19-222. 7 Lobos, E. and Weiss, N. (1985) I m m u n o c h e m i c a l comparison between worm extracts of Onchocerca voh'ulus from savanna and rain forest. Parasitol, Immunol. 7, 333-347. 8 E r n m a n n , K . D . , U n n a s c h , T . R . , G r e e n e , B.M., Albiez, E.J., Boateng, J., D e n k e , A . M . , Ferraroni, J.J., Karam, M.. Schulz-Key, H. and Williams, P.N. (1987) A D N A sequence specific for forest form Onchocerca volwdus. Nature 327,415-417. 9 Shah, J.S., Karam, B., Piessens, W. and Wirth, D.F. ( 19871 Characterization of an Onchocerca-specific D N A clone from Onchocerca voh'ulus. A m . J. Trop. Med. Hyg. 37, 376-384. 10 D u k e , B.O.L. (1980) Observations on Onchocerca volvulus in experimentally infected chimpanzees. Tropenmed. Parasitol. 31, 41-54.
11 Schulz-Key, H., Albiez, E.J. and Buttner, D.W. (19771
12
13
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16
17
18
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21 22
23
Isolation of living adult Onchocerca voh'ulus from nodules. T r o p e n m e d . Parasitol. 28, 428-4311. Chirgwin, J.M.. Przbyla, A . E . , MacDonald, R.J. and Rutter. W.J. (1979) Isolation of biologically active R N A from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Aviv, H. and Leder, P. (1972) Purilication of biologically active globin m R N A by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 14118-1412. Huynh, T.V., Young, R.A. and Davis, R.W. 11985) Constructing and screening cDNA libraries in Xgtl(I and kgll 1. In: D N A Cloning: A Practical Approach, Vol. I (D.M. Glover, ed.), pp, 4%78, 1RL Press Ltd., Oxford. Maniatis, T., Fritsch, E.F. and Sambrook, J. (19821 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY. Young, R.A. and Davis, R.W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. U S A 80, 1194-1198. Pelham, H . R . B . and Jackson, R.J. 11976) An efficient m R N A - d e p e n d e n t translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256. Southern, E.M. (1975) Detection of specitic sequences among D N A fragments separated by gel electrnphoresis. J. Mol. Biol, 98, 503-517. Feinberg, A.P. and Vogelstein, B. 11983) A technique for radiolabeling D N A restriction cndonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13. Maxam. A.M. and Gilbert, W. (1977) Sequencing end-labeled D N A with base-specific chemical cleavages. Methods Enzymol. 65,499-56t). Sulston, J.E. and Brenner. S. (1974) The D N A of Caenorhabditis elegans. Genetics 77.95-1114. McReynolds, L.A., DeSimone, S.M. and Williams, S.A. (1986) Cloning and comparison of repeated D N A sequences from the human filarial parasite Brugia maluvi and the animal parasite Brugia pahangi, Proc. Natl. Acad. Sci. U S A 83, 797-801. Simpson, A . J . G . , Sher. A. and McCutchan, T.F. (19821 The genome of Schistosoma mansoni: isolation of D N A ,
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25
26
27
its size, bases and repetitive sequences. Mol. Biochem. Parasitol. 6, 125-137. McLachlan, A . D . and Karn, J. (1982) Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in nmscle. Nature 299, 226-231. Lanar. D.E., Pearce, E.J., James. S.k. and Sher, A. (1986) Identification of paramyosin as schistosomc antigen recognized by intradermally vaccinated mice. Science 234, 593-596. Karn, J., Brenner, S. and Barnett, k. (1983) Protein structural domains in the Caenorhabditis ele#ans unc-54 myosin heavy chain gcne are not separated by introns. Proc. Natl. Acad. Sci. U S A 8tl, 4253-4257. Perler, F.B. and Karam, M. (1986) Cloning and characterization of two Onchocerca volvulus repeated D N A sequences. Mol. Biochem. Parasitol. 21, 171-178.
28 Pcarcc, E.J., James, S.L., Dalton, J., Barrall, A., Ramos, C., Strand, M. and Sher, A. (1986) I m m u n o c h e m i cal characterization and puriiication of Sm-97, a Schistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine. J. Immunol. 137, 3593-3600. 29 O'Farrell, P.Z., Goodman, H.M. and O'Farrell. P.J. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins, Cell 12, 1133-1142. 30 Cohen. C. and Holmes, K.C. (1963) X-ray diffraction evidence for q-helical coiled-coils in native muscle. J. Mol. Biol. 6, 423-432. 31 Matsumoto, Y., Perry. G., I, evine, R.J.C., Blanton, R., Mahmoud, A.A.F. and Aikawa, M. (1988) Paramyosin and actin in schistosomal teguments. Nature 333, 7(-,-78.
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Elsevier MBP 011)46
Construction of Onchocerca volvulus c D N A libraries and partial characterization of the c D N A for a major antigen John E. D o n e l s o n 1, B . O . L . D u k e 2, David Moser 1, Wenlin Zeng ~, Ngozi E. Erondu l, Richard Lucius 3, Alphons Renz 4, Marc Karam 5 and Guillermo Zea Flores 6 IDepartment of Biochernistrv, University of Iowa, lowa City, 1A, U.S.A. : :Dept. oJ" Infectious and Parasitic Diseases Pathology, Armed Forces Institute of Pathology, Washington, DC, U.S.A. : 3b~stitut fl~r Tropenhygiene, Universitiit Heidelberg, Heidelberg, F. R. G. ; aTropenmedizinisches lnstitut. Universitiit Tiibingen, Ti~bingen, F.R. G. ; SOnchocerciasis Control Programme, Ouagadougou, Burkina Faso: 6Ministerio de Salud Publica Asistencia Social, Dept. de EnCermaedad de Robles (Oncocercosis), Guatemala City, Guatemala (Received 9 May 1988; accepted 27 June 1988)
Adult Onchocerca volvulus were isolated from nodules removed from onchocerciasis patients at four locations - two in the West African Sudan-savanna region (near B a m a k o , Mali, and Touboro, Cameroon), one in a West African forest region ( K u m b a , Cameroon) and one near G u a t e m a l a City, Guatemala. Four different c D N A expression libraries were constructed in bacteriophage ~gt 11 using poly(A) + R N A from the adult female worms. Individual c D N A clones of single copy genes were used to compare the g e n o m e s of parasites from the different locales and to show that the haploid genome of O. volvulus is 1.5 × l/I ~ base pairs. A b o u t 1 in 700 recombinant clones in each of the four amplified c D N A libraries produces a fusion protein recognized by pooled h u m a n anti-O, volvulus antisera. Partial sequence determination of a 2.0 kb c D N A clone for an O. volvulus protein that induces an imm u n o d o m i n a n t response in rabbits revealed that this antigen has sequence similarities with Caenorhabditis elegans myosin and with schistosome paramyosin (which confers partial protection against schistosome infection). The four c D N A libraries have been deposited with American Type Culture Collection ( A T C C ) , 12301 Parklawn Drive, Rockville, MD 20852, U . S . A . , for general distribution u n d e r A T C C N u m b e r 37509. Key words: Onchocerca volvulus; c D N A library; G e n o m e size: Immunoscreening: I m m u n o d o m i n a n t antigen
Introduction An estimated 17.8 million people in sub-Saharan Africa and Latin America suffer from onchocerciasis, or river blindness [1]. The disease is caused by Onchocerca volvulus, a filarial nematode transmitted by blackflies of the genus Simulium which live near rapidly flowing rivers and streams. The infection is associated with clinical manifestations ranging from pruritis and various skin changes to severe lymphatic involvement and
Correspondence address: J.E. Donelson, Dept. of Biochemistry, University of Iowa, Iowa City, IA 52242, U.S.A. Note: Nucleotide sequence data reported in this paper have been submitted to the G e n B a n k T M Data Bank with accession n u m b e r J03995.
ocular disease. The most extreme cases occur in people of the Sudano-Guinean savanna zone of Africa, where microfilariae frequently enter the eyes and cause lesions that can lead to impaired vision and blindness [2]. In some highly endemic savanna areas children that survive beyond the age of 15 have a 40-50% chance of eventually becoming blind [3]. In contrast, the parasite burden of individuals living in the forest regions of West Africa can be as high as in the savanna but the incidence of blindness is much lower. The reason for this difference is not known but a commonly expressed possibility is that different genetic forms of the parasite may exist in the forest and savanna regions [4-9]. A major problem in the study of O. volvulus and onchocerciasis is the lack of a good laboratory model for the disease. The parasite infects
11166-6851/88/$03.51/© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
242
only humans, gorillas and chimpanzees [10]. None of the traditional laboratory m a m m a l s has been shown to harbor a productive infection of O. volvulus. Therefore, the main source of worm material comes from nodules removed from human patients, most of whom live in remote areas. This greatly limits the n u m b e r of investigators that can study the parasite and has led to large gaps in our understanding of the genetics of the worm and the immunology of the infection. For example, very little is known about the w o r m ' s metabolism or how the microfilariae evade the immune response of their host. To provide w o r m material for a larger n u m b e r of investigators and to examine the basic molecular biology of the parasite, we isolated adult worms from onchocerciasis patients in four widely spaced geographical areas and p r e p a r e d c D N A libraries of their poly(A) + R N A . In vitro translations and Southern blots show that the parasites from the four areas are very similar in their genetic make-up. When the c D N A libraries were immunoscreened with pooled patient antisera or with a rabbit antisera against an adult worm homogenate, m a n y positive clones in all four libraries were detected. Several c D N A clones identified during these immunoscreenings were found to cross-hybridize and one was characterized further. Materials and Methods
Collection of parasites. As summarized in Table I, nodulectomies were p e r f o r m e d under local anesthesia on 20-41 onchocerciasis patients at each of the four locales. I m m e d i a t e l y after their surgical removal, the nodules were submerged in a sterile solution of R P M I 1654 medium containing 20 m M H e p e s buffer, p H 7.5, 0.2 mg gentamicin ml 1 and 3 mg collagenase ml -~ (isolated from Clostridiurn histolyticum and purchased from Boehringer-Mannheim Biochemicals). The nodules were digested in this solution as described [11] for 12 - 48 h at t e m p e r a t u r e s between 30 and 37°C (equipment for maintaining a constant temperature was not available). Periodically, the nodules were placed in a Petri dish under a dissecting microscope and partially digested human tissue r e m o v e d with a small forceps. The diges-
tion solution was replaced with fresh solution if the incubation continued for more than 24 h. As soon as intact adult worms were sufficiently free of nodular tissue, they were removed with forceps and extensively rinsed with R P M I medium dispensed from a Pasteur pipette. Special care was taken during this step to remove all of the visible human tissue from the worms. Female worms and the separated smaller male worms were sorted into groups of 1 to 15, placed in cryopreservation tubes and immediately frozen in liquid nitrogen. As Table I shows, 1-3 nodules were obtained from each patient and 50-209 female worms were isolated at each collection site. Similar numbers of male worms were also obtained. Often the larger nodules contained calcified and dead worms that were discarded. One intermediate-sized nodule, from a patient in the forest area near K u m b a in southwestern C a m e r o o n , contained 31 live female worms, all of which were about the same size and could be separated intact from each other. These worms from the same nodule were used to make the K u m b a c D N A library.
Isolation of RNA and DNA. A porcelain mortar. was placed in a solid CO2-metfianol bath and filled with liquid nitrogen. Frozen female worms (23-33 in number) were placed in the mortar and ground for about 10 rain to a fine powder with a pestle. Liquid nitrogen in the mortar was periodically replaced during the grinding. The powder was transferred to 11 ml of a solution containing 8 M guanidinium thiocyanate, 100 mM Tris-HC1, pH 7.6, 20 mM E D T A , 2% Sarkosyl and 5 mM [3mercaptoethanol [12] and subjected to two 10 s
TABLE I Collection of adult Onchocerca volvulus Location
No. of patients
No. of nodules
-20 -20 41
21 -25 62
No. of live female worms
Guatemala Mali Touboro, C a me roon Kumba, Cameroon
(forest) (savanna)
50 54 156
(savanna) 28
71 ~'
(forest)
:' One nodule contained 31 live female worms.
209
243 T A B L E I1 D N A and R N A isolation from adult female worms
Kumba Touboro Mall Guatemala
No. of female worms
DNA (p.g)
Total RNA (~g)
% Poly(A) ÷ RNA
31 33 23 30
200 135 156 198
270 191 178 172
6.2 7.3 7.9 9.0
Yield/female worm
5.1
7.(l
bursts in a tissue mincer (Tekmar Co., Cincinnati, OH). This solution was centrifuged at 4000 rpm for 10 rain. The pellet was resuspended in 1 ml of the guanidinium thiocyanate solution and the centrifugation repeated. The 1 ml of supernatant from the second centrifugation was added to the original 11 ml of supernatant. The 12 ml were layered on top of step gradients of 5.7 M CsC1 (2 ml) and 3.0 M CsC1 (2 ml) containing 0.1 M E D T A in Beckman SW41 tubes and centrifuged at 29000 rpm for 20 h. The R N A was recovered in a pellet at the bottom of the tube and the D N A from the interface between the 5.7 M and 3.0 M CsC1 layers. The R N A and D N A were then treated as described [12]. Table II summarizes the recoveries. An average of 5.1 p,g D N A and 7.0 p,g total R N A was obtained from each female worm.
Construction of cDNA libraries. Poly(A) + R N A was purified from total female R N A by two passages through oligo(dT) cellulose (Collaborative Research, Type 3) [13]. Between 6% and 9% of the total R N A was recovered as poly(A) + R N A (Table II). First strand c D N A was prepared from
5 ~xg of poly(A) + R N A using AMV reverse transcriptase and oligo(dT)12_18 as primer [14]. Following alkaline hydrolysis of R N A in the RNAD N A hybrid, second strand cDNA was synthesized by hairpin priming using the Klenow fragment of D N A polymerase I (Boehringer Mannheim Biochemicals). The hairpin was cleaved with S1 nuclease. The c D N A was then treated with T4 D N A polymerase and all four dNTPs to ensure the presence of blunt ends. After methylation of the cDNA with EcoRI methylase, EcoRI linkers were ligated to the ends of the molecules and digested with EcoRI. The c D N A was purified from linker fragments and size fractionated on a Sepharose CL-4B column. After ligating the cDNA to phosphatase-treated, EcoRI-cleaved ~.gtll D N A (Strategene) and incubating with ;~ packaging extracts (GigaPack Gold, Strategene), libraries containing 27-61 × lff' phage were obtained as summarized in Table III. ?
Amplification and distribution of the libraries. Between 4 and 10 million phage in each of the four unamplified cDNA libraries were incubated with Escherichia coli strain Y1088 and plated onto 100 Petri dishes (150 cm 2) for amplification as described [15]. A summary of these amplified libraries is given in Table IV. Note that the background of phage without c D N A inserts (yielding blue plaques) increased during the amplification step, probably because phage without inserts have a larger burst size. These amplified libraries are available from the American Type Culture Collection (ATCC). lmmunoscreening of the cDNA libraries. The libraries were introduced into E. coli strain Y1090 and immunoscreened [14,16] at a density of about
T A B L E III Construction of primary c D N A libraries Poly(A)- R N A (Ixg)
No. of )~gtll recombinant clones
% without cDNA inserts (blue plaques)
No. of positive clones with human patient antisera
< l 2.5 7 8
1 out 1 out 1 out 1 out
(x 106) Kumba Touboro Mali Guatemala
5 5 5 5
61 27 30 38
of of of of
50(1 500 500 700
244
10 000 phage per Petri dish (150 cm 2) with a 1:100 dilution of pooled h u m a n anti-O, volvulus antisera or with a 1:1000 dilution of rabbit antiserum against an O. volvulus homogenate. The human antiserum was obtained from chronically infected patients living in a forest region of Liberia, West Africa. The h o m o g e n a t e used to generate the rabbit antiserum was prepared by sonicating sliced adult female worms for 2 rain at 400 W and was injected into the rabbit subcutaneously, with Freund's complete adjuvant. Subsequent boosts were made with the h o m o g e n a t e plus Freund's incomplete adjuvant. Other methods and materials. In vitro translations using rabbit reticulocyte lysates (Promega Biotec, Madison, WI) [17] and Southern blots of genomic D N A [18] were conducted as described. D N A fragments were radiolabeled in vitro for hybridizations according to the random priming procedure [19], D N A sequences were determined according to the M a x a m and Gilbert procedure [20] on fragments that were 3' end-labeled with [o~-32p]dNTPs and Klenow fragment of D N A polymerase I. Subcloning of c D N A fragments from ,~gtll to p U C plasmids and other general procedures were as described [15]. Unless otherwise noted, enzymes and other recombinant materials were from New England Biolabs, Beverly, M A , or Bethesda Research Labs, Gaithersburg, M D , and were used under conditions r e c o m m e n d e d by the supplier.
Fig. 1. Autoradiogram of two dimensional gels [29] containing the [35S]methionine-labeled in vitro translation products of total RNA isolated from adult female O. volvulus collected in Guatemala (forest region of Central America) and near Touboro. East Cameroon (savanna region of West Africa). Arrows on the right side of each panel indicate the positions of molecular weight markers of (top to bottom) 97, 75, 50, 42 and 20 kDa. respectively.
Results
In vitro translations o f O. volvulus R N A . Since the parasite-containing nodules were incubated with collagenase for 12-48 h prior to isolating the adult worms, it was necessary to determine if degradation of the parasite R N A occurred during this digestion period. In vitro translations of the isolated R N A s with rabbit reticulocyte lysates and [35S]methionine were used for this evaluation. The translation products were displayed on two-dimensional acrylamide gels, one example of which is shown in Fig. 1. The larger proteins synthesized during these in vitro translations are greater than 80 k D a , suggesting that little, if any, degradation of parasite m R N A occurred during the
collection of the parasites and isolation of the RNA. In addition, no differences, other than some changes in relative intensity, were detected among the 200-300 major translation products from the RNAs of the parasites from G u a t e m a l a (Central American forest) and from T o u b o r o (African savanna). Identical patterns of in vitro translation products were also obtained when R N A s from Mall (African savanna) and K u m b a (African forest) parasites were used (not shown). Thus, at this level of gene expression, there are no detectable differences between parasites from the forest and the savanna regions.
245
Construction and preliminary characterizations of cDNA libraries. To provide reagents to investigate parasite antigens that elicit an immune response, c D N A expression libraries in bacteriophage )~gtll were constructed from each of the four parasite R N A s as described in Materials and Methods. These R N A s , isolated from adult females, were derived from both adult tissue and the developing stages of microfilariae in the fertilized female. In addition a few adult male worms may have contaminated the preparations of the much larger females. As summarized in Table III, between 27 and 61 million primary c D N A clones were obtained from 5 Ixg of starting poly(A) ~ R N A . Examination of over 40 randomly, or specifically, selected c D N A clones has revealed that the average insert size of the libraries is 1200 bp with a range of 3(X) to 35(X) bp. Some phage in the K u m b a library may have multiple c D N A inserts since a higher percentage of the inserts in this library have internal E c o R I sites than would be Hind III GMT
K4"4
expected for their insert sizes (on average, an E c o R I site would be expected about every 4000 bp). The other three libraries appear to have fewer of these multiple cloning events although this has not been rigorously examined. Since the parasites were isolated from human tissue, it was important to determine whether human R N A and D N A contaminated the preparations of parasite R N A and D N A . The middle panel of Fig. 2 shows a test of this possibility. A Southern blot of restricted D N A from humans and from each of the four parasite preparations was probed with radiolabeled total human D N A . As expected, a strong signal occurred in the lanes containing the restricted human D N A . In the lanes that contain parasite D N A , hybridization could only be observed to the D N A from the G u a t e m a l a parasites (lanes G). Densitometer tracings of the autoradiogram revealed that this contamination is about 6% for the G u a t e m a l a n parasite D N A and less than 1% for the others.
Eco RI
HUMAN
3 2 1GMTKS GM
T K
GM
T
K GMT
K 4'4
3 2
1 GMT
KS
Fig. 2. Southern blots to determine the contamination of parasite D N A w i t h h u m a n D N A and to determine the g e n o m c s i z e of O. voh, ulus. (Left panel) Ethidium bromide stain of an 0.Sff'~ agarose gel containing 5 p,g of genomic D N A from parasites from G u a t e m a l a (G), Mall (M), T o u b o r o (T) and K u m b a (K) digested with either H i n d l l l or E c o R | . Lanes 1-4 contain E e o R | digests of 5 p,g of h u m a n D N A and. respectively, 11, 22, 33, and 44 pg of the purified 840 bp c D N A insert in a randomly selected recombinant phage of the K u m b a c D N A library. Lane 4' is the same as lane 4 except that the D N A is digested with HindIll. Lane S contains trace a m o u n t s of ~2p-labeled H i n d l l l fragments of X D N A . (Middle panel) Autoradiogram of a Southern blot of the D N A s shown in the left panel probed with labeled total h u m a n D N A . (Right panel) Autoradiogram of the D N A s in the left panel probed with the same labeled 840 bp c D N A fragment that was mixed with the h u m a n D N A in lanes 1-4'.
246
Dot blot hybridizations using human D N A as the probe seemed to be less sensitive than the Southern analysis (data not shown). By analogy, the extent of contamination of the R N A preparations is probably about the same and may be reflected in the c D N A libraries as well. The higher level of contamination in the G u a t e m a l a n parasites probably occurred because the collagenase treatment of these nodules, which were collected first, was conducted before the optimal digestion conditions had been determined.
Determination o f the genome size of O. volvulus. Since the genome size of O. volvulus has not been reported, several randomly selected c D N A clones from the K u m b a c D N A library were used as probes to determine this haploid size. The right panel of Fig. 2 shows one such experiment. The single 840 bp E c o R I c D N A insert of a randomly chosen recombinant ;~gtll phage was used as a probe for this Southern blot. It contains two internal H i n d I I I sites separated by about 200 bp. In all four genomic D N A s this c D N A hybridizes to a single E c o R I D N A fragment of 3.8 kb and to three H i n d I I I fragments of 1.0 kb, 2.0 kb and the 200 bp of internal sequence (which migrated too far to be seen in Fig. 2), as would be expected for a single copy gene in the genome. This gene is not tandemly repeated in the genome because additional H i n d I I I fragments containing sequences flanking the potential terminal gene repeats were not observed. Lanes 1-4 show the hybridization of this probe to increasing amounts of its own 840 bp sequence, which via densitometer tracings were used to correlate the signal intensity to a known amount of the sequence. From this analysis, and
by averaging the signals in the four lanes containing EcoRI-digested parasite D N A , it was determined that about 29 pg of the 840 bp sequence are present in 5 ~g of genomic D N A , which is equivalent to a haploid genome size of about 1.5 × 10~ bp. An analogous calculation was made by adding the signals of the two bands in the HindIII lanes and a similar value for the genome size was obtained. In addition, a similar result was obtained when a second unrelated e D N A insert from another recombinant phage was used as the probe (not shown). A third c D N A probe, however, yielded a hybridization pattern that was consistent with the presence of three distinct hybridizing regions in the genome (not shown). Still other e D N A probes yielded more complex hybridization patterns, suggesting that at least portions of their genomic sequences are repetitive. The value of 1.5 x 1(1~ bp for the O. volvulus genome is within a factor of two of those determined for other nematodes and trematodes such as Caenorhabditis elegans (0.8 x 1()~ bp) [21], Brugia malayi (0.8 x 1()~ bp) [22] and Schistosoma mansoni (2.7 x 10a bp) [23]. It should also be noted that all of the c D N A s that have been used to date as hybridization probes yield the same Southern blot pattern of fragments in all four O. volvulus genomes; a result that, although admittedly a small sample size, is consistent with the occurrence of relatively little coding sequence variation among parasites at different geographic locations.
Characterization of a cDNA for an O. volvulus antigen that induces an immunodominant response. A b o u t 30000 clones of each of the four c D N A libraries were immunoscreened with a
T A B L E IV Amplification of the c D N A libraries
Kumba Touboro Mall Guatemala
No. of clones that were amplified ( x lff')
No. of phage ~xl ~ after amplification (x 10'~)
?i without e D N A insert (blue plaques)
No. of positive chines with h u m a n patient antisera
4 10 10 l0
0.8 4.0 4.8 3.0
4 ~* 9 12 14
1 out 1 out 1 out 1 out
~' Note that c>~.b a c k g r o u n d increased during the amplification step.
of of of of
700 700 700 7tl0
247
rabbit antiserum against an adult O. volvulus homogenate and with pooled antisera from onchocerciasis patients in a forest area of Liberia. About 1 in 500 clones in each of the libraries produced a fusion protein recognized by the rabbit antisera and about 1 in 700 clones were detected by antibodies in the h u m a n antisera (Tables III and IV). Pre-immune sera from a rabbit or from a non-infected h u m a n did not give a positive signal with recombinant clones in any of the libraries. The fact that both the rabbit and h u m a n antisera recognize the same proportion of clones in each of the libraries supports the conclusion that most antigens and their coding sequences are very similar in parasites from all four collection sites. Small-scale D N A preparations were p e r f o r m e d on 16 plaque-purified phage from the unamplified K u m b a and T o u b o r o libraries that were positive with the rabbit antiserum. Hybridization of
Southern blots containing E c o R I digests of these phage D N A s using different phage D N A s as the labeled probe revealed that 7 out of the 16 cDNAs (44%) cross-hybridized. Each of these 7 independent recombinant phage contained a c D N A insert of 2.0-2.3 kb. The 2.0 kb c D N A insert of one of the cross-hybridizing phage was chosen for further characterization and subcloned into the E c o R I site of pUC19. The nucleotide sequences at the ends of this insert were determined by M a x a m and Gilbert sequencing of end-labeled fragments generated by cleavage with E c o R I or HindlII (in the polylinker of p U C I 9 ) . The 5' sequence at one end of the insert was found to have termination codons in all three translation reading frames, suggesting that the other strand contains the sequence appearing in the antigen's mRNA. The 400 nucleotides determined at the 5' end
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Fig. 3. (A) The 5' nucleotide, and deduced amino acid, sequence of a 2.0 kb c D N A clone encoding an i m m u n o d o m i n a n t protein of O. volvulus recognized by a rabbit antiserum. (B) The deduced amino acid sequence shown in panel A arranged as heptad repeats (a-b-c-d-e-f-g), in which the boxed positions 'a' and 'd" (asterisks) are predominantly hydrophobic residues while the other positions are often hydrophilic residues. In this sequence organization the hydrophobic residues are packed between the two strands of an s-helical coiled-coil while most of the polar groups point out [30]. (C) Comparison of amino acid positions 79-106 in the O. voh'ulus sequence shown in panel A with amino acids 292-318 in the partial paramyosin sequence of Schistosoma mansoni [25] and amino acids 853-879 in the unc-54 myosin of Caenorhabditis elegans [26]. Solid boxes surround identical amino acids and dashed boxes enclose functionally similar residues.
248 of the other strand contain a single open translation reading frame shown in Fig. 3A. This nucleotide sequence likely begins at an internal EcoRI site in the original c D N A since it does not have the EcoRI linker sequence of 5' GAAT-FCC 3'. It is not known whether this is the result of subcloning from Xgtll to the plasmid or lack of methylation during the c D N A library construction. The deduced amino acid sequence has a pattern of distinct periodic heptad repeats (a-b-cd-e-f-g)n in which hydrophobic residues predominate at positions a and d (Fig. 3B). This sequence organization is characteristic of c~-helical coiled-coils found in some structural proteins such as myosin [24]. In addition, a computer search of all of the nucleotide sequences in the GenBank database revealed that the sequence in Fig. 3A has the greatest similarity to the partial sequence of paramyosin of Schistosoma mansoni [25]. Slightly less sequence similarity was detected with segments of the o~-helical coiled-coil rod regions of other myofibrillar proteins, such as myosin from Caenorhabditis elegans [26]. Fig. 3C shows a comparison of a 26-27 amino acid region from each of these three proteins at which about 37% positional identity exists. Other regions within the partial sequence of O. volvulus antigen also show similar amino acid identity to segments of paramyosin and myosin. However, within the sequences currently available for all three antigens, these similarities are not uniformly distributed throughout the sequence shown in Fig. 3A but are confined to pockets such as the one shown in Fig. 3C. A more extensive sequence comparison awaits the complete nucleotide determination of the 2.0 kb cDNA but the common properties shown in Fig. 3B and C suggest that this immunodominant antigen of O. volvulus may be a myofibrillar protein. Discussion
The availability of c D N A libraries of adult O. volvulus m R N A should help overcome some of
the research problems associated with the inability to maintain this parasite in the laboratory. For example, it has not been possible to obtain a sufficient amount of any protein or antigen from this parasite for biochemical characterization. Now it
should be possible to synthesize large quantities of the peptide backbones of most 0. volvulus proteins in an appropriate recombinant vector/host system once their cDNAs have been identified in the libraries. In addition, to date only three reports on the D N A of O. volvulus have appeared [8,9~27]. All three describe the cloning and characterization of repetitive genomic D N A sequences from the organism for potential diagnostic uses. The expression cDNA libraries described here should contribute to this diagnostic objective and also facilitate the identification of genes for parasite antigens useful in both diagnostic and immunoprophylactic studies. A first step toward the latter goal is the detection of cDNA clones for the immunodominant antigen described here. The fact that this antigen has amino acid similarity to schistosome paramyosin is of both biochemical and immunological interest. It suggests that this antigen might be an O. volvulus analog to schistosome paramyosin and that eventual comparison of their complete sequences will provide information about the structural conservation and function of these invertebrate muscle proteins. In addition, vaccination with S. mansoni paramyosin has been shown to confer partial protection against challenge by infective larvae of S. rnansoni [28]. Indeed, when mice were immunized intradermally with extracts of S. rnansoni, the predominant schistosome antigen against which antibodies were raised was paramyosin [25]. Thus, it is perhaps not surprising that injection of extracts of another helminth parasite would induce an immune response to a similar protein. Paramyosin has recently been shown by immunoelectron microscopy to be present in the external tegument structure of schistosomes [31], which may account for its immunogenicity during a schistosome infection and may also suggest a similar external location for the O. volvulus antigen described here. In contrast to schistosomes, where sufficient paramyosin for vaccination studies was obtained from laboratory-reared parasites, further characterization of this O. volvulus antigen will require its production by recombinant techniques. Finally, no evidence for polymorphisms was observed in the in vitro translations or with the
249
few c D N A s used to probe Southern blots. The parasites from forest and savanna regions of Africa, and from Guatemala in Central America, all yielded the same patterns, providing no molecular insights into the clinical differences of their infections. Nevertheless, a 153 bp probe of O. volvulus repetitive DNA has been reported to be specific for forest parasites [8]. This suggests that differences a m o n g the parasites do exist at the D N A level and that it may be possible to differentially screen the forest and savanna c D N A li-
braries for transcripts or antigens that form the basis for these clinical differences. Acknowledgements We thank Kwang S. Kim, Virginia Strand and Charles Keller of the University of Iowa for technical support on the recombinant D N A portion of this project. The work was supported by a grant from The Edna McConnell Clark Foundation.
References 1 World Health Organization (1987) Third report of the Expert C o m m i t t e e on Onchocerciasis. Technical Report Series No. 752, World Health Organization, Geneva. 2 Connor, D . H . , Gibson, D . W . , Taylor, H . R . , Mackenzie, C . D . , Meyers, W.M. and Neafie, R.C. (1984) Onchocerciasis. In: H u n t e r ' s Tropical Medicine, 6th edn. (Strickland, G . T . , ed.), pp. 667-680, W.B. Saunders Co., Philadelphia. 3 Prost, A. (1986) The burden of blindness in adult males in the savanna villages of West Africa exposed to onchocerciasis. Trans. R. Soc. Trop. Med. Hyg. 80, 525-527. 4 Duke, B . O . L . , Lewis, D.J. and Moore, P.J. (1966) Onchocerca-Sirnulium complexes 1. - Transmission of forest and Sudan-savanna strains of Onchocerca voh, ulus from C a m e r o o n by Simulium damnosum from various West African bioclimatic zones. Anal. Trop. Med. Parasitol. 611, 318-336. 5 D u k e . B.O.L. and A n d e r s o n , J. (1972) A comparison of the lesions produced in the cornea of the rabbit eye by microfilariae of the forest and Sudan-savanna strains of Onchocerca voh,ulus. T r o p e n m e d . Parasitol. 22,354-368. 6 A n d e r s o n , J., Fuglsang, H., Hamilton, P.J.S. and de-C. Marshall, T.F. (1974) Studies on onchocerciasis in the United C a m e r o o n Republic, II. Comparison of onchocerciasis in rain-forest and Sudan-savanna. Trans. R. Soc. Trop. Med. Hyg. 68, 2/19-222. 7 Lobos, E. and Weiss, N. (1985) I m m u n o c h e m i c a l comparison between worm extracts of Onchocerca voh'ulus from savanna and rain forest. Parasitol, Immunol. 7, 333-347. 8 E r n m a n n , K . D . , U n n a s c h , T . R . , G r e e n e , B.M., Albiez, E.J., Boateng, J., D e n k e , A . M . , Ferraroni, J.J., Karam, M.. Schulz-Key, H. and Williams, P.N. (1987) A D N A sequence specific for forest form Onchocerca volwdus. Nature 327,415-417. 9 Shah, J.S., Karam, B., Piessens, W. and Wirth, D.F. ( 19871 Characterization of an Onchocerca-specific D N A clone from Onchocerca voh'ulus. A m . J. Trop. Med. Hyg. 37, 376-384. 10 D u k e , B.O.L. (1980) Observations on Onchocerca volvulus in experimentally infected chimpanzees. Tropenmed. Parasitol. 31, 41-54.
11 Schulz-Key, H., Albiez, E.J. and Buttner, D.W. (19771
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Isolation of living adult Onchocerca voh'ulus from nodules. T r o p e n m e d . Parasitol. 28, 428-4311. Chirgwin, J.M.. Przbyla, A . E . , MacDonald, R.J. and Rutter. W.J. (1979) Isolation of biologically active R N A from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Aviv, H. and Leder, P. (1972) Purilication of biologically active globin m R N A by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 14118-1412. Huynh, T.V., Young, R.A. and Davis, R.W. 11985) Constructing and screening cDNA libraries in Xgtl(I and kgll 1. In: D N A Cloning: A Practical Approach, Vol. I (D.M. Glover, ed.), pp, 4%78, 1RL Press Ltd., Oxford. Maniatis, T., Fritsch, E.F. and Sambrook, J. (19821 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY. Young, R.A. and Davis, R.W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. U S A 80, 1194-1198. Pelham, H . R . B . and Jackson, R.J. 11976) An efficient m R N A - d e p e n d e n t translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256. Southern, E.M. (1975) Detection of specitic sequences among D N A fragments separated by gel electrnphoresis. J. Mol. Biol, 98, 503-517. Feinberg, A.P. and Vogelstein, B. 11983) A technique for radiolabeling D N A restriction cndonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13. Maxam. A.M. and Gilbert, W. (1977) Sequencing end-labeled D N A with base-specific chemical cleavages. Methods Enzymol. 65,499-56t). Sulston, J.E. and Brenner. S. (1974) The D N A of Caenorhabditis elegans. Genetics 77.95-1114. McReynolds, L.A., DeSimone, S.M. and Williams, S.A. (1986) Cloning and comparison of repeated D N A sequences from the human filarial parasite Brugia maluvi and the animal parasite Brugia pahangi, Proc. Natl. Acad. Sci. U S A 83, 797-801. Simpson, A . J . G . , Sher. A. and McCutchan, T.F. (19821 The genome of Schistosoma mansoni: isolation of D N A ,
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its size, bases and repetitive sequences. Mol. Biochem. Parasitol. 6, 125-137. McLachlan, A . D . and Karn, J. (1982) Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in nmscle. Nature 299, 226-231. Lanar. D.E., Pearce, E.J., James. S.k. and Sher, A. (1986) Identification of paramyosin as schistosomc antigen recognized by intradermally vaccinated mice. Science 234, 593-596. Karn, J., Brenner, S. and Barnett, k. (1983) Protein structural domains in the Caenorhabditis ele#ans unc-54 myosin heavy chain gcne are not separated by introns. Proc. Natl. Acad. Sci. U S A 8tl, 4253-4257. Perler, F.B. and Karam, M. (1986) Cloning and characterization of two Onchocerca volvulus repeated D N A sequences. Mol. Biochem. Parasitol. 21, 171-178.
28 Pcarcc, E.J., James, S.L., Dalton, J., Barrall, A., Ramos, C., Strand, M. and Sher, A. (1986) I m m u n o c h e m i cal characterization and puriiication of Sm-97, a Schistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine. J. Immunol. 137, 3593-3600. 29 O'Farrell, P.Z., Goodman, H.M. and O'Farrell. P.J. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins, Cell 12, 1133-1142. 30 Cohen. C. and Holmes, K.C. (1963) X-ray diffraction evidence for q-helical coiled-coils in native muscle. J. Mol. Biol. 6, 423-432. 31 Matsumoto, Y., Perry. G., I, evine, R.J.C., Blanton, R., Mahmoud, A.A.F. and Aikawa, M. (1988) Paramyosin and actin in schistosomal teguments. Nature 333, 7(-,-78.