Cloning of Toxoplasma gondii gene fragments encoding diagnostic antigens

Gene, 99 (1991) 127-132

127

Elsevier

GENE

03950

Cloning of Toxoplasma gondii gene fragments encoding diagnostic antigens (Recombinant DNA; expression assay; poly(A)’ RNA)

library;

fusion protein;

protist;

tachyzoite;

toxoplasmosis;

enzyme-linked

immunosorbent

Alan M. Johnson and Susana Illana Department of Microbiology and Infectious Diseases, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042 (Australia) Received by P.A. Manning: 3 August Revised: 12 October 1990 Accepted: 15 October 1990

1990

SUMMARY

Two Toxoplasma gondii gene fragments, which encode polypeptides that can be used as diagnostic antigens in an enzyme-linked immunosorbent assay were cloned and their nucleotide sequence was determined. One of the fragments (derived from the H4 gene) is 682 bp long. The mRNA of the single-copy H4 gene is 1300 nt long. The fragment derived from the other gene, termed Hll, is 197 bp long. The mRNA of the single-copy HI1 gene is 1900 nt long. The native polypeptides encoded by the H4 and HI I genes are 25 and 41 kDa, respectively. Based on computer analysis of the deduced amino acid sequences of the polypeptides encoded by the gene fragments, both appear to be very hydrophilic and that encoded by the HI I fragment has a high antigenic index profile. These results are consistent with the diagnostic usefulness of the polypeptides encoded by the gene fragments.

INTRODUCTION

If first contracted during pregnancy, toxoplasmosis may cause foetal abnormality or perinatal death in humans and domestic animals. As many as 30-50% of individuals in most adult human populations show serological evidence of Correspondence Infectious

to: Dr. A.M. Johnson,

Diseases,

Flinders

Australia

5042 (Australia)

FLINDU

AA89624;

Abbreviations:

Department

Medical

Center,

Tel. (61-08)2759319;

of Microbiology Bedford

Park,

Telex

Fax (61-08)2768658.

aa, amino

acid(s);

AIDS,

acquired

immune

deficiency

syndrome; bp, base pair(s); BGal, 8-galactosidase; ELISA, linked immunosorbent assay; GST, glutathione-S-transferase 2.5.1.18); H, human serum-reactive murine serum-reactive phosphate phoresis;

hydrolase PIMS,

and South

clones; kb, kilobase(s)or

clones; nt, nucleotide(s); (EC 3.6.1.3);

polyclonal

reactive clones; RDen, rabbit SDS, sodium dodecyl sulfate;

PAGE,

immune

mouse

NTPase,

1000 bp; M, nucleoside

polyacrylamide-gel serum;

antibody to denatured T., Toxoplasma.

enzyme(EC

R, rabbit

tri-

electroserum-

T. gondii antigen;

0378-l 119/91/$03.50 0 1991 Elsevier Science Publishers B.V.

previously acquired asymptomatic toxoplasmosis. Immunosuppression of these otherwise healthy patients who have previously acquired the asymptomatic form of toxoplasmosis, may cause a reactivation of the disease and subsequent death. This is becoming more important, as the immunosuppression associated with AIDS causes reactivation of toxoplasmosis in up to 40% of AIDS patients (Luft and Remington, 1988). The serological diagnosis of toxoplasmosis must be very important since it is performed in thousands of laboratories worldwide. However, the production of antigenic fractions for the serological diagnosis of toxoplasmosis is complicated by the fact that the causative parasite, T. gondii, is obligately intracellular. Hence parasite preparations used for serological diagnosis are expensive, and inevitably contaminated with at least some host material. This occurs regardless of whether the parasites are grown in tissue culture or the peritoneal cavities of mice (Abbas, 1967). In an attempt to overcome these problems we have cloned and

sequenced two gene fragments that encode polypeptides which react in the ELISA for the serological diagnosis of acute toxoplasmosis in naturally infected humans (Tenter and Johnson, 1991) and experimentally infected mice (Parker et al., 1991). We report here the results of the cloning, sequencing, and characterization of the fragments of the two genes, termed H4 and Nf 1.

EXPERIMENTAL

AND DISCUSSION

(a) Isolation of antigenic @Gal fusion proteins

A T. go&ii cDNA library using RH strain tachyzoite poly(A) + RNA was constructed in 1 gtl 1 vector as described earlier (Johnson et al., 1989). The library had a titre of 1.6 x 10” plaque-forming units/ml and contained approx. 8894 recombinant members with an average insert size of 1 kb. To identify the more commonly recognized @Gal fusion proteins, the library was immunologically screened with three different types of sera: 50000 members with RDen, identifying 15 positive clones; another 50000 with a PIMS, identifying 30 positive clones; and finally another 130000 members with a serum from a patient (A.L.) naturally infected with T. gondii, identifying 27 positive clones. @%-positive fusion proteins were identified using an alkaline phosphatase conjugate screening procedure essentially as specified by the manufacturer (Promega, Madison, WI). Positive clones recognized by one serum were then crossTABLE I Cross-screeninp of Tox~plus~~ gmzdiipositive clones originally identified with one serum, against the two other sera Type of clones”

R M H

Serum b RDen

PIMS

A.L.

15 1 1

30 26

29 27

a R denotes clones originally identified with the rabbit serum RDen: M denotes clones originally identitied with the mouse serum PIMS; and H denotes clones originally identi~ed with the human serum A.L. b RDen was prepared essentially as described by Timmins et al. (1985). The RH strain tachyzoites used to immunize the rabbit were contaminated with about 5% murine peritoneal cells. PIMS was prepared as described elsewhere (Johnson et al., 1983), except that the ovine II strain of 7’.gtindii was used to chronically infect the mice. This strain was isolated from an aborted lamb foetus in 1979 at Caveside, Tasmania, by Dr. B.L. Munday, Tasmanian Department of Agriculture. Human serum used was from a patient (A.L.) suffering from acute lymphadenopathic toxoplasmosis who was bled six months after the onset of symptoms (Johnson et al., 1987). Anti-E. cofi antibodies were adsorbed from all three sera by incubating each serum with E. coli lysate several times (Helfman et al., 1984).

checked by immunoscreening with the other two sera to test whether the recombinant antigens produced were recognized by all three sera. The results are contained in Table I. The screening with the RDen serum was included because it has been found that screening Agt11 libraries with this type of antibody increases the efftciency of the screening (Timmins et al., 1985). However, the 15 R clones were not recognized by either the PIMS or A.L. serum, and were consequently not further characterized. The lack of recognition of the H and M clones by the RDen serum, and the R clones by the PIMS and A.L. sera is perhaps not surprising, as antibodies to native proteins generally recognize conformational epitopes (Rothbard, 19861, not those present in the denatured antigen used to immunize the rabbit. Interestingly, however, the one M clone that was not recognized by the A.L. serum, and the one H clone, termed H39, that was not recognized by PIMS, were both identified by the RDen serum. Because we were interested in the diagnosis of human toxoplasmosis, only the 27 H clones were purified by limiting dilution and rescreened, for further characte~zation. (b) Nucleotide

sequences

Agarose gel (1%) electrophoresis of the 27 H clones revealed that the sizes of the DNA inserts were either about 200 bp, 670 bp or 1300 bp (not shown). To determine whether these inserts were identical, EcoRI-digested Igt I 1 preparations of the 27 H clones were probed with either a representative of the 200-bp inserts, termed Hfl gene fragment, or a representative of the 670-bp inserts, termed H4 gene fragment. These results (not shown} suggested that six H clones were of the HI 1 type and 20 were of the H4 type. The clone termed H39 was unique and to date has not been characterized further. Sequencing revealed that the H4 gene fragment is 682 bp, of which only about 40% is translated as there is a stop codon at position 290 (Fig. 1). The H11 gene fragment is 197 bp of which about 85% is translated as there is a stop codon at position 170 (Fig. 1). Both the H# and HI1 gene fra~ents and their corresponding deduced aa sequences were analyzed by computer using the MacVector sequence analysis software, version 3.02 (MaeVector Sequence Analysis Software: User’s Manual, IBI, New Haven CT, 1989) on a Macintosh IIcx. Neither the H4 gene fragment, nor the HI I gene fragment sequence had significant homology with any gene sequence in the GenBank Data Base Release 61.0. There was also no si~i~c~t homology to the six other T. got&ii genes sequenced to date and encoding: P23 antigen (Cesbron-Deiauw et al., 1989); F3G3 antigen (Prince et al., 1989); P30 antigen (Burg et al., 1988); a and /.Itubulin (Nagel and Boothroyd, 1988), or the NTPase gene fragment (Johnson et al., 1989); and Bl gene (Burg et al., 1989); or to each other. The deduced aa sequences encoded

129

H4 Glu Phe Gln Glu Glu Ile Lys Glu Gly Val Glu GAATpccAAGAGGAAA~AAAGAAGGGcpGGRGGAAcACAAGcAToAAGAC

Glu

His

Asp Pro Glu Met GATCCTGAGA'IGACG

Lys

Gin Glu CAGGAG

Thr

Arg CK%

Phe Ser Lys Met Ala Lys TICAGCAAGATGGCGAAATCC~

Leu Met Val Thr Glu Cl-CA'lGGlGACCGAGAAG

Ser Phe AGTm

Glu

Asp

17 51

Ser Lys Asn AGCAAAAAT

34 102

Leu

51 153

Gly Gly Ser Ile Ser Phe Leu Thr Glu Thr GGGGGA~A~?rc;?TTcTAA~GAAAcGGGGGIcAcAATGArrGAGTpG

Gly

Glu

Leu

68 204

Pro Ccc

Gln Leu Leu CAACTACPC

His Asp Ile CACGATATTCPC

Leu

85 255

Ala

Gln

His

Ser Thr Arg Ile Glu Glu AGCACGCGAArrGAAGRGCPC

LYS Thr AAAA~@?l’

Ser

Lys

Ser Glu His Asp Met AGPGAACATGACATGGAC

Asp

Val

Thr

Met

Ile

96 310 377 444 511 578 645 682

Hll Glu Phe Pro Ala Lys Ala Val Lys Gly Phe GAATPCCCCGCAARGGCTGITARGGGAmGGTGGCACCCGCACP~ACG

Gly

Gly

Thr

Thr

Ser

Thr

17 51

Ala Pro Ala Glu Ala Gly Lys Thr GCGCCFGCl'GAGGCTGGAAAAAcGGAGTlGGACGAC

Asp

Asp

Gly Tyr Arg GGATATCGC

Pro CCG

Pro CCC

34 102

Leu

Glu

51 153

Pro Phe Asn CCATICAAC

Pro CCX

Glu

Leu

Arg Pro Ser Pro T,'r Ala Glu Leu Le" CGACCC'l'ZACCCTACGCCGAGTl'ATIGAAGGATTEGAA

Arg

Lys

Asp

56 197

Arg Met Arg Lys Glu AGAATGCGCAAAGAGTGA--

Fig. 1. Nucleotide

sequences

of the H4 and HI 1 gene fragments

sizes of 10.9 kDa and 6.2 kDa for H4 and HlZ, respectively. directions by the dideoxy chain-termination method (Sanger accession

numbers

are M57302

(H4) and M57303

and deduced

aa sequences.

The T. gondii portion

of the fusion proteins

had deduced

The H4 and HI1 gene fragments were subcloned into M13mp18 and sequenced in both et al., 1977) using [?S]dATP and the Sequenase TM kit (USB, Cleveland, OH). GenBank

(H11).

by either the H4 or HI I gene fragments had no significant homology with any protein in the NBRF PIR Data Base Release 19.0, or to each other.

the time taken for a highly abundant mRNA to be detected (Johnson et al., 1989), suggests that neither H4 nor HI1 mRNAs are highly abundant.

(c) Analysis of the H4 and HZ2 genes To determine the copy number of the H4 and Hll genes, T. gondii genomic DNA and known molar equivalents of plasmid containing either the H4 gene fragment or the HI 1 gene fragment were transferred to nylon membranes and hybridized with the respective purified gene fragment. The intensity of the autoradiographs obtained (Fig. 2) suggested that the H4 and HI1 genes were both single copy. The mRNA for H4 and HI I were found to be 1300 and 1900 nt, respectively (Fig. 3). The time taken to obtain a discernible hybridization signal on the autoradiograph, compared with

(d) Characterization of the H4 and HII gene fragment products Because the H4/GST and Hll/GST fusion proteins appear to be such good antigens for serological diagnosis, a computer analysis of their predicted aa structure was performed to see if there was any theoretical reason for this. The results obtained (not shown) for the hydrophilicity plot, secondary structure, and antigenic index of the deduced H4 and H 11 are consistent with the observed usefulness of the H4/GST and H 1 l/GST fusion proteins as antigens in the ELISA. Interestingly, based on the computer analysis, the

130

1

5

H 11 is theoretically human

H4

more

likely to be a better

the H4, and this is consistent and murine

with our results

sera (Tenter

antigen

than

obtained

with

and Johnson,

1991; Parker

et al., 1991). (e) Western-blot To determine

analysis the sizes of the native polypeptides

by the H4 and HI 1 genes, strain

ELISA

antigen

with the appropriate

A

1

(Johnson antibodies

et al., 1987) were reacted (Fig. 4). This

of the H4 and HI 1 gene products

the sizes

showed were

that

25 kDa

5

Hll

2

1

3

4

-0 It? 7

Fig. 2. Quantitation of the H4 or HI I gene in T. go&ii genomic DNA. T. gondii DNA (5 fig; lane A) or the indicated molar equivalents of plasmid containing either the H4 or HZ1 gene fragment, plus 5 pugof salmon sperm DNA, were digested with Hind111 and resolved on a 1yO agarose gel. The DNA samples were transferred to nylon membranes and probed with either nick-translated H4 or HZ1-purified [32P]DNA inserts (Johnson et al., 1986).The H4 and HZ 1 gene fragments were purified from plasmid DNA by excision with EcoRI, separation in agarose and collection onto DEAE membranes (Schleicher & Schuell, Keene, NH).

- 43 - 30

HI1

Ii4

encoded

of T. gondii RH

immunoblots

- 20.1

- 7.41 -5.31 - 2.8,

- 14.4 -F

Fig. 4. Western-blot analysis of the H4 or HZ1 gene products. H4 and

-0.6,

HI 1 inserts were released from lgtl 1 by EcoRI digestion and subcloned

into the EcoRI site of pGEX-IN. These clones were then expressed GST fusion proteins using a modified (Helfman mentioned

in E. coli. Bacterial

(Johnson

above. Fusion proteins

thione-agarose Fusion proteins

beads as previously emulsified

into mice intradermally

Fig. 3. Northern-blot analysis of the H4 or HI1 genes. T. gondii poly(A)+RNA (1 pg) was electrophoresed on a 1.5% formaldehydeagarose gel (Sambrook et al., 1989) and transferred to a nylon membrane in duplicate. The membranes were probed with either nick-translated H4 or HI Z-purified [32P]DNA inserts. Numbers indicate RNA M, standards (kb).

et al., 1989) procedure

et al., 1983) and the alkaline

at several

bled four weeks later. Samples antigen were resolved ferred to nitrocellulose I), mouse

anti-H4/GST

described

phosphatase

previously

conjugate

were purified by adsorption described

in Freund’s

as

colonies were immunoscreened system to gluta-

(Smith and Johnson,

complete

sites (Russo

adjuvant

1988).

were injected

et al., 1985). Mice were

(300 pg) of T. gondii RH strain

ELISA

on 0.1% SDS-II y0 polyacrylamide gels, transand reacted with 1: 100 dilutions of PIMS (lane fusion protein

(lane 2), mouse

fusion protein (lane 3) and mouse anti-GST antibody indicate M, standards (kDa). 0, origin; F, front.

anti-H1 l/GST

(lane 4). Numbers

131 (lane 2) and 41 kDa (lane 3), respectively. Lane 4 shows that anti-GST antibody does not react with any T. gondii polypeptide which is consistent with the fact that the GST component of the fusion proteins does not play a part in the recognition of the native polypeptides. (f) Conclusions Recombinant fusion proteins have recently been used as antigens in the ELISA to diagnose syphilis (Radolf et al., 1986), alveolar echinococcosis (Muller et al., 1989), and malaria (Srivastava et al., 1989). The infectious agents causing these diseases are di~cult to obtain pure and in quantity, and such immunoselection of diagnostic fusion proteins from cDNA libraries is likely to have much greater use in the future. In an attempt to clone T. go~dii genes which encode polypeptides likely to be important diagnostically, we have followed two different strategies. The first was to clone genes for antigens that constituted a large part of the tachyzoite, the actively dividing stage of the parasite. To that end we cloned and sequenced a gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of 7’. go~d~i(Johnson et al., 1989). However, antibodies to this antigen have since been found to be present in only a low percentage of patients with toxoplasmosis (Tenter and Johnson, 1990). Our second strategy was to screen a cDNA library with several different types of antibody to T. gondii and to test the antigens commonly identified for their usefulness in a diagnostic ELISA for toxoplasmosis. Using this second strategy we identified fragments of two genes termed H4 and HI 1) which encode polypeptides that can be used as antigens in the ELISA to measure antibodies to T. gondii in the sera of acutely infected humans and mice. Such fusion proteins are likely to replace the requirement for T. gondii to be grown in tissue culture or the peritoneal cavities of mice to prepare diagnostic ELISA antigen.

ACKNOWLEDGEMENTS

We wish to thank Dr. Rhys Willies, Dep~tment of Haematology, Flinders University, for assistance with computer analysis, and Dr. Susan Parker, Department of Microbiology and Infectious Diseases, Flinders University, for production of the fusion-protein antibodies in mice. This work was supported by a grant from the National Health and Medical Research Council of Australia. The material contained in this publication is covered by patent number PK1679.

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