Stage specific gene expression in the post-infective L3 of the filarial nematode, Brugia pahangi

MOLECULAR

iiL%HEMIc PARASITOLDGY Molecular and Biochemical

Parasitology

79 ( 1996) lO9- I I2

Short communication

Stage specific gene expression in the post-infective L3 of the filarial nematode, Brugia pahangi ’ Samuel

A.M.

Martin,

Sarah J. Hunter,

Fiona

J. Thompson,

Eileen Devaney”

Received I8 March 1996: revised 9 April 1996; accepted I5 April 1996

_ Ke~~~~~ords: Filarial nematode; leader

Brugiu

pahangi;

Gene expression; Differential

Lymphatic filariasis is a mosquito-borne disease of the tropics caused by infection with the nematode parasites Wuchereria bancrofti, Brugiu maluyi and Brugiu timori. An estimated 120 million people are infected worldwide [I]. The critical life cycle stage for the infection of the human host is the third stage larva (L3), which is deposited in the tissues by a feeding mosquito. The L3 localize to the lymphatics and develop through two moults to the adult stage. Upon transfer from the mosquito vector to the mammalian host, the L3 must re-initiate the developmental cycle and unAhhrettatiom:

L3. third

stage larva;

L4, fourth

pi., post-infective: RT-PCR, reverse transcriptase

stage larva: PCR: SLI,

spliced leader. * Corresponding author. Tel.: + 44 I41 3305751; fax: + 44 I41 3305603: e-mail: [email protected] ’ Norr: Nucleotide sequence data reported in this paper are available in the EMBL, GenBankTM and DDJB data bases under the accession numbers: Sl, X91063: S3. X91064; S3, X87903; S5. X91065: EE4. X91066.

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screening: Cytidine deaminase;

Spliced

dergo a number of adaptive changes to ensure survival. These include adaptations to deal with the new physiological environment and the avoidance of the host immune system. Changes in the pattern of expression of proteins, carbohydrates and lipids [2&4] have been described following the transfer of the L3 from mosquito to mammalian host, but almost nothing is known of the molecular events associated with the establishment of infection. Genes which are expressed to coincide with the developmental switch between hosts may have important functional roles in the parasite life cycle and, in addition, may present novel targets for chemotherapeutic or immunological intervention. Consequently, we have attempted to identify CDNAs which are differentially expressed by the L3 of the filarial nematode Brugiu pahatzgi following infection of the mammalian host. In this communication we report the isolation and partial characterisation of four cDNAs expressed by the L3 within 24 h of infection of the mammalian host.

110

S.A.M. Martin et ul. / A4oleculur md Biochemical Parasito1og.v 79 (1996) 109-l 12

A cDNA library was constructed from RNA isolated from mammalian derived L3 obtained from infected jirds, Meriones unguiculatus, at 3 days post-infection. Due to the small amounts of material available, the library was constructed by reverse transcriptase PCR (RT-PCR), using the conserved nematode spliced leader (SLl) [5] and oligo dT as primers. Approximately 10 ,~g of total RNA was extracted from 2000 post-infective (pi.) L3, of which 2 ,ug was used for reverse transcription with an oligo dT primer, (S’GCCGCTCGAGT,, 3’) at a concentration of 200 ng ml-’ [6]. The first strand cDNA was stored in H,O at - 20°C. Amplification by PCR was performed on 2 ~11 of a 1:20 dilution of the first strand cDNA using the oligo dT primer (above) and a primer corresponding to the 22 nucleotides of the nematode SLl (upper case) (S’gccggaattcGGTTTAATTACCCAAGTTTGAG 3’), essentially as described previously [6]. cDNA was digested with EcoRI and X/z01 (Promega) and ligated into the corresponding sites of 2 uniZAP (Stratagene) according to the manufacturers protocol. The titre of the library was 4.6 x lo6 pfu ml - ’ with 94% recombinants. The library was screened by differential hybridization with cDNA from mosquito derived L3 and 3 day p.i. L3 in order to identify genes which were exclusively expressed in the pi. L3. Duplicate filter lifts of 1 x IO4 plaques were screened with -12P labelled cDNA prepared by RT-PCR from both life cycle stages. Fig. 1 shows the results of a differential screen; the arrows in (a) indicate plaques which are preferentially recognized by the pi. L3 probe, those in (b) show the position of the same plaques on the duplicate filter. Twenty-seven plaques, which hybridized only to the p.i. L3 cDNA, were selected for secondary screening and cross-hybridization analysis. This yielded four independent cDNA species, termed Sl, S2, S3 and S5. The Bluescript plasmids containing cDNA inserts were removed from the i, molecule by in vivo excision. Insert sizes, as determined by restriction digestion with EcoRI and XlzoI, were 380 bp (Sl), 350 bp (S2), 490 bp (S3) and 470 bp (S5). To confirm that the selected mRNAs were in fact differentially expressed, Northern blot analy-

sis was performed on different life cycle stages. RNA was obtained from mosquito derived L3 and from parasites obtained from the mammalian host at various time points post-infection: 24 and 48 h, 3 days, 15 days (L4) and adult worms. Fig. 2 shows a representative Northern blot profile for each of the clones isolated. Each blot was repeated on at least two occasions to confirm the results obtained. None of the genes were expressed in the mosquito derived L3 but each was expressed within 24 h of entering the mammalian host (Fig. 2, lane 2, a-d). Clone Sl hybridized to a 0.8 kb transcript weakly expressed in 24 and 48 h p.i. L3, with an increasing level of expression in later life cycle stages (a), while clone S2, which

a *

rr

Fig. 1. Primary screening of approximately 2000 pfu of the 3 day p.i. B. pahangi cDNA library. Filter lifts were taken onto nylon membranes (Amersham) and the DNA was denatured, neutralized and crosslinked to the membrane. Filters were prehybridized at 65°C in 5 x SSC, 0.1% SDS, 5 x Denhardt’s solution containing 100 pg ml- ’ single stranded salmon sperm DNA and then hybridized overnight with the radioactive probe. Probes were prepared by RT-PCR on total RNA from 3 day p.i. L3 (panel a) or mosquito derived L3 (panel b). 50 ng cDNA was used as the template for random priming with 50 PCi “P-dCTP (3000 mCi ml ‘) using the ‘High Prime’ method (Boehringer). Both probes were labelled to approximately equal specific activity. Following hybridization the filters were washed to 0.5 x SSC, 0.1% SDS at 65°C and then autoradiographed. Arrowheads in panel (a) indicate plaques that hybridized differentially to the p.i. L3 probe. Arrowheads in panel (b) indicate the position of the same plaques on the duplicate filter probed with mosquito derived L3 cDNA.

123456

b

C

e Fig. 2. Northern blot analysis of total B. puh~i RNA from different life cycle stages. Approximately 2000 mosquito or mammalian derived L3 (24 h p.i., 48 h p.i. or 3 days p.i.). 750 L4 (day I5 p.i.) or 50 adults were used for RNA isolation. exactly as described previously [6]. RNA (2 11g) was resuspended in ~Pw formamide. electrophoresed on a 1.2%denaturing formaldehyde agarose gel and transferred to Nylon membrane (Amersham) with 20 x SSC. The RNA was fixed to the membrane by baking for 2 h at 80°C and UV crosslinked in a Strata-linker. Hybridization conditions were the same as described for plaque screening (see legend to Fig. I). The blots were hybridized overnight with “I’ labelled insert, gel purified from digested plasmid (specific activity - I x IOh cpm /cg-- ‘) corresponding to the differentially expressed CDNAs. Blots were then washed to 0.2 x SSC. 0.1% SDS at 65°C. The life cycle stages were us follows: lane I: mosquito derived L3. lane 3: 14 h p.i. L3. lane 3: 48 h p.i. L3. lane 4: 3 day p.i. L3. lane 5: I5 day p.i. L4 and lane 6: adult. The probes used were panel a. clone Sl; panel b, clone S2: panel c. clone S3: panel d, clone S5 and panel e. clone EE4.

hybridized to a transcript of 0.6 kb was expressed from 24 h p.i. through to the adult stage at approximately equal levels (b). Clones S3 and S5 showed particularly interesting patterns of expresssion. S3 (c) hybridized to a 0.49 kb transcript. the level of which increased over the first 48 h of infection and appeared to peak at 3 days post-infection. A weaker hybridization signal was obtained from the L4 parasites (day 15) and from

the adult. For clone S5 (d), a transcript of 0.47 kb was observed in each of the p.i. L3 stages and the L4, but no detectable hybridisation was observed in the adult. Each gel used for Northern blot analysis was stained with ethidium bromide and photographed to confirm approximately equal loading of RNA in different lanes. The blot shown in (d) was re-probed with a cDNA encoding a Bmgio ribosomal protein gene EE4 (e), which is expressed in all life cycle stages (Hunter et al., unpublished data), in order to confirm the transfer of RNA from each lane. The four differentially expressed cDNAs were used to probe Southern blots of Brugiu genomic DNA. The hybridization pattern obtained was suggestive of a single copy gene for all four CDNAs (data not shown). Double stranded sequencing was performed by cycle sequencing using Taq DNA polymerase (Cambio) and T3 and T7 fluorescently labelled primers (Hybaid) on an automatic sequencer (Licor). Sequence analysis demonstrated that two of the clones, S3 and S5. were full length containing the SLl sequence and a 3’ polyadenylated tail. Clones Sl and S2 were found not to possess the spliced leader sequence. due to the presence of an internal EcoRI site, which has resulted in the cleavage of the 5’ end of these cDNAs; both cDNAs contained a 3’ poly A tail. The sequences obtained were used to search the EMBL and SWISS-PROT data bases in order to identify homologous genes using both FASTA and BLAST search programs. Clone S5 showed significant identity to bacterial cytidine deaminase (crlcl) (45% identity) [7] and to a human c& (42% identity with S52873) [S]. None of the other differentially expressed cDNAs showed homology at either the nucleotide or the amino acid levels. This study demonstrates that changes in gene expression can be observed between mosquito derived L3 and mammalian derived L3 and presents a novel approach to isolating differentially expressed cDNAs from hlarial nematodes. Each of the four selected genes was expressed within 24 h of infection, implying that these genes may play a role in the establishment of the infection. The pattern of expression of individual genes varied in later life cycle stages. Clones Sl and S:! were expressed in each of the parasite stages examined,

S.A.M.

“2

Martin et al. / Molecular and Biochemical Parasitology 79 (1996) 109-l 12

and these may represent ‘housekeeping’ genes which are essential for survival in the mammalian host. By Northern blot analysis, S.5 appeared to be restricted to the larval stages in the mammalian host. In contrast to S5, the expression of clone S3 peaked at day 3 post-infection with a reduced level of expression in the L4 and adult parasite. S3 was isolated on more than one occasion and further analysis revealed that S3 represented 3”/0 of all clones. The relative abundance of S3 transcripts probably reflects the level of transcription of this gene at day 3 post-infection, as shown by Northern blot analysis, and the fact that differential screening tends to identify relatively abundant transcripts. A similar phenomenon was described by Scott et al. [9] who isolated a BrUgia muluyi collagen gene from a spliced leader cDNA library which represented 7% of all inserts. The aim of this study was to clone cDNAs which may have functional roles in the life cycle, as the parasite makes the developmental switch between vector and definitive host. To date only S5 shows any homology with other sequences in the data base. This cDNA is the filarial homologue of cytidine deaminase, an enzyme involved in the salvage pathway for pyrimidine nucleotides and in a novel process of site-specific RNA editing in mammals. In this latter process, a cytidine is deaminated to a uridine in the mRNA which encodes apolipoprotein B. resulting in the modification of a glutamine residue (CAA) to a translation stop codon (UAA) [lO,l 11, allowing one mRNA transcript to code for two different mature peptides with differing functions [12]. As the infective form for the human host, the L3 presents an ideal target for control, as blocking the further development of this life cycle stage would prevent disease in man. The identification and functional analysis of p.i. L3 cDNAs will provide a useful experimental system for studies aimed at defining the mechanisms used by the parasite to adapt to life in the mammalian host.

Acknowledgements This work UNDP/World

was supported by Bank/WHO Special

grants from Program for

Research and Training in Tropical Diseases, the Medical Research Council, and the Wellcome Trust. ED is a Wellcome Trust University Lecturer. We thank Alan Purvis for technical assistance.

References Ottesen. E.A. and Ramachandran, C.P. (1995) Lymphatic filariasis infection and disease: control strategies. Parasitol. Today 11, ‘29-162. [2] Devaney, E. and Jecock, R.M. (1991) The expression of the Mr 30000 antigen in the third stage larvae of Brugia pahangi. Parasite Immunol. 13, 75587. [3] Apfel, H.. Eisenbeiss, W.F. and Meyer, T.F. (1992) Changes in the surface composition after transmission of Acanthocheilonema uiteae third stage larvae into the jird. Mol. Biochem. Parasito’. 52, 63-74. L., Kusel, J.R.. Smith, H.V., Harnett, W.. ]41 Proudfoot, Worms, M.J. and Kennedy, M.W. (1993) Rapid changes in the surface of parasitic nematodes during transition from pre-parasitic to post-parasitic forms. Parasitology ‘07. 107-l ‘7. I51 Nilsen, T.W. (1993) Trans-splicing of nematode premessenger RNA. Annu. Rev. Microbial. 47, 413-440. 161Martin, S.A.M., Thompson, F.J. and Devaney, E. (1995) The construction of spliced leader cDNA libraries from the filarial nematode Brugia pahangi. Mol. Biochem. Parasitol. 70, 241-245. ‘oca[71 Song. B.H. and Neuhard. J. (1989) Chromosomal tion, cloning and nucleotide-sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase. Mol. Gen. Genet. 2’6, 462-468. F. (1993) PI Kuhn, K.. Bertling, W.M. and Emmrich, Cloning of a functional cDNA for human cytidine deaminase (cdd) and its use as a marker of monocyte macrophage differentiation. Biochem. Biophys. Res. Commun. 190, l-7. [91 Scott, A.L., Yenbutr, P., Eisinger. S.W. and Raghavan. N. (1995) Molecular cloning of the cuticular collagen gene bmcol-2 from Brugia makayi. Mol. Biochem. Parasitol. 70. 221-225. HOI Chen. S.H., Habib, G.. Yang, C.Y., Gu. Z.W., Lee, B.R., Weng, S.A.. Silberman, S.R., Cai, S.J., Desiypere. J.P., Rosseneu. M.. Gotto, A.M., Li, W.H. and Chan, L. (I 987) Apolipoprotein B-48 is the product of a messengerRNA with an organ-specific in-frame stop codon. Science 283, 363 -366. N., Morrison, J.R.. Bhattacharya, S., Pate’, [“I Navaratnam, D. Funahashi, T.. Giannoni. F., Teng. B., Davidson, N.O. and Scott, J. (1993) The p27 catalytic subunit of the apolipoprotein-B messenger-RNA editing enzyme is a cytidine deaminase. J. Biol. Chem. 268, 20709920712. CF. and Davidson, N.O. (1993) 1’21 Teng, B., Burrant, Molecular cloning of an Apolipoprotein B messenger RNA editing protein. Science 260, 1816- 1819. [l]