Contents lists available atScienceDirect
Acta Tropica
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a
Short communication
Alterations in the expression level of a putative aspartic protease in the
development of Angiostrongylus cantonensis
Kao-Pin Hwang
a,1, Shih-Hsin Chang
b,1, Lian-Chen Wang
c,∗aDepartment of Pediatrics, Chang-Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung and College of Medicine, Chang-Gung University, Kwei-Shan, Tao-Yuan, Taiwan bGraduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan
cDepartment of Parasitology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan
a r t i c l e i n f o
Article history:Received 27 May 2009 Received in revised form 19 November 2009 Accepted 20 November 2009 Available online 27 November 2009 Keywords: Angiostrongylus cantonensis Aspartic protease Developmental stages Expressional level
a b s t r a c t
Aspartic proteases are a family of proteinases with catalytic aspartate residues in the active site. These enzymes have been reported to initialize the degradation of host hemoglobin in blood-feeding helminths. After identifying an expressed sequence tag representing an aspartic protease from an Angiostrongylus cantonensis young adult dataset, this sequence was found to encode a protein with a predicted molecular mass of 46 kDa. It also showed good homologies to aspartic proteases from Caenorhabditis elegans (50.7% identity), Haemonchus contortus (43.0% identity), Necator americanus (41.5% identity), Strongyloides ster-coralis (35.9% identity), and Burgia malayi (29.6% identity). This putative aspartic protease was determined to be expressed in the infective larvae, young adults, and adult worms of A. cantonensis by quantitative real-time PCR. Among male worms, the expression level was determined to increase by 223.0± 24.2 fold in young adults relative to the infective larvae and then decreased to 7.1± 0.2 fold in adult worms. In female worms, the expression level was observed to increase by 118.5± 10.1 fold in young adults and by 277.5± 29.2 fold in the adults, when compared with infective larvae. These findings not only indicate that the expression level of aspartic protease gene in A. cantonensis changes with development but also has a sexual difference in individual developmental stages in the final host.
© 2009 Elsevier B.V. All rights reserved.
Proteases are digestive enzymes that catalyze the cleavage of peptide bonds in proteins. According to their substrates, cat-alytic center residues, catcat-alytic mechanisms and co-factors for activity, they are classified into four major classes: serine pro-teases, cysteine propro-teases, metalloproteases and aspartic proteases (Berger and Schechter, 1970). In parasitic helminths, they play essential roles in digestion of food proteins, immunomodula-tion, and host tissue invasion (McKerrow et al., 2006). Moreover, the nutrition of blood-feeding nematodes depends on a multi-enzyme synergistic proteolytic cascade in the intestine cleaving the ingested host hemoglobin tetramer into successively small frag-ments (Williamson et al., 2003b). Aspartic proteases have been shown to play a crucial role in the degradation of host hemoglobin in schistosomes (Brindley et al., 2001), hookworms (Williamson et al., 2004) and Onchocerca volvulus (Jolodar et al., 2004). More-over, a putative aspartic protease in Haemonchus contortus has been shown to be almost exclusively expressed by the blood-feeding stages (Longbottom et al., 1997).
∗ Corresponding author. Tel.: +886 3 211 8800x5092; fax: +886 3 211 8385. E-mail address:wanglc@mail.cgu.edu.tw(L.-C. Wang).
1These authors have equally contributed to this study.
Angiostrongylus cantonensis is a blood-feeding nematode usu-ally inhabiting the pulmonary arteries and right ventricle of rats. Human is a non-permissive host for this parasite. Infection of this parasite in humans leads to eosinophilic meningi-tis and eosinophilic meningoencephalimeningi-tis (Ramirez-Avila et al., 2009). Recently, we have cloned expressed sequence tags from the young adults of A. cantonensis (dbEST accession no. DN190143–DN191368). After analyzing this dataset, a sequence with a poly(A) tail was identified to represent an aspartic protease. In this study, we cloned this putative aspartic protease and deter-mined its expression levels in different developmental stages of A. cantonensis.
A life cycle of A. cantonensis (Taiwan strain) has been maintained in our laboratory by cycling through Biomphalaria glabrata snails and Sprague–Dawley rats (Wang et al., 1989). Three weeks after infection with first-stage larvae, infective (third-stage) larvae were recovered from the infected snails by digesting the tissues with 0.6% (w/v) pepsin–HCl (pH 2–3) for 1 h. Each rat was infected with 200 infective larvae by stomach intubation. Adult and young adult worms were respectively collected from the lung and brain tissues of the rats 50 and 21 days post-infection. The sex of these worms was determined by their morphological characteristics according toBhaibulaya (1979). After washing with distilled water, the worms were freeze-dried and stored at−80◦C for further experiments.
0001-706X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2009.11.008
sis and then cloned into the TOPO-TA Cloning Kit (Invitrogen) for sequencing.
To determine the expression levels of the putative aspar-tic protease gene in different developmental stages, quantitative real-time PCR was performed using SYBR Green I (RealQ PCR Master Mix; Ampliqon, Bie and Bernsen AS, Roedovre, Denmark) on the Stratagene Mx3000P QPCR System (Stratagene, La Jolla, CA, USA). ROX was used as a reference dye in a final concen-tration of 50 nM. A-actin gene transcript of A. cantonensis was employed as an endogenous control. Primers were designed by the Primer3 program (Rozen and Skaletsky, 2000). The forward and reverse primers for the putative aspartic protease gene were 5 -ACAATCCCTTGTGCAGGAAC-3and 5 -TGTCGTCCATCAACCGAATA-3and those for-actin gene were 5
-TCATCACTGTGGGAAACGAA-3and 5-GTGTTGGCGTACCAATCCTT-3respectively. After treating RNA samples with RQ1 RNase-Free DNase (Promega, Madison WI, USA), cDNA was synthesized using SuperScripTM III RNase H−
Reverse Transcriptase (Invitrogen) with oligo (dT)12primer
accord-ing to the instructions of the manufacturer. For real-time PCR, an initial denaturation was carried out at 95◦C for 10 min, followed by 40 cycles of amplification with denaturation at 95◦C for 30 s, primer annealing at 58◦C for 1 min and elongation at 72◦C for 30 s. After 40 cycles, specificities of the amplification products from each primer pair were determined by melting curve analysis and confirmed by 2% (w/v) agarose gel electrophoresis.
The relative abundances of the transcripts were determined using the MxPro real-time QPCR software v. 3.00 (Stratagene) according to the 2−Ct method (Livak and Schmittgen, 2001). Baselines and thresholds were automatically set by the software. The-actin amplification signals were employed as the internal normalyzer and samples of the infective larvae as the calibrator. The crossing point of the amplification curve with the threshold represented the cycle threshold (Ct). Four replicate runs per sam-ple were performed and negative controls without template were also included.
The gene encoding the putative aspartic protease from infec-tive larvae, young adults and adult worms of A. cantonensis was successfully amplified by quantitative real-time PCR using the designed primer pair. The amplicon was sequenced and its amino acid sequence was deduced. The full length of this clone was deter-mined to be 1602 bp (Fig. 1). There is a conserved nematode spliced leader SL1 (5-GGTTTAATTACCCAAGTTTGAG-3) at the 5end. It is a non-coding 22-nucleotide sequence. Although the phenomenon of trans-splicing of mRNA maturation has been demonstrated in many organisms (Bonen, 1993), spliced leader sequences were first identified in nematodes (Krause and Hirsh, 1987; Bektesh et al., 1988). Moreover, there is a spliced leader at the 5 end of 80% of Caenorhabditis elegans mRNAs (Nilsen, 1993). Conserved or variant SL1 sequences have been identified in the mRNA of parasitic nema-todes, including Strongyloides ratti (Guiliano and Blaxter, 2006),
tide of 21 residues. The cleavage site is between Gln21 and Ile22. Without this signal peptide, the mature protein has 404 amino acids with a predicted molecular mass of 46 kDa and a pI of 5.8 (Fig. 1). After searching the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM), no transmembrane helices were found in the deduced amino acid sequence. This finding suggests that the putative aspartic protease is a secretory enzyme.
Aspartic proteases have been cloned from blood-feeding nema-todes. Necator americanus Na-APR-2 (Necepsin I) (Williamson et al., 2003a) and H. contortus PEP2 (Smith et al., 2003) have been demon-strated to be expressed in the intestinal lumen of these parasites and catalyze the cleavage of host hemoglobin. Using the deduced amino acid sequence, the putative aspartic protease was found to have 50.7% identity with C. elegans aspartyl protease family mem-ber (asp-2) (GenBank accession no. NP 872129), 43.0% identity with H. contortus aspartyl protease precursor (GenBank accession no. CAE12199), 41.5% identity with N. americanus necepsin I (GenBank accession no. CAC00542), 35.9% identity with S. stercoralis aspartic protease precursor (GenBank accession no. AAD09345), and 29.6% identity with B. malayi aspartic protease BmAsp-1 (GenBank acces-sion no. BAC05688) (Fig. 2). These good homologies suggest the catalytic activity of the putative aspartic protease.
The presence of catalytic aspartic acid residues in the active site is a common characteristic of aspartic protease (Szecsi, 1992). In the putative aspartic protease of A. cantonensis, these residues are located in the evolutionary conserved consensus DTGS (Asp93) and DTGTS (Asp315). It also contains four conserved cysteine residues (aa 106, 277, 350 and 408). These residues may form two pairs of conserved, intrastrand disulfide bonds (Cooper et al., 1990). Moreover, searching through the NetNGync 1.0 Server (http://www.cbs.dtu.dk/services/NetNGync) revealed a potential N-linked glycosylation site at Asn78. This glycosylation site is not conserved with the other aspartic proteases (Fig. 2). This finding is consistent with the variability in aspartic protease N-linked glyco-sylation sites (Tang and Wong, 1987).
The expression levels of the putative aspartic protease in different developmental stages of A. cantonensis were analyzed by quantitative real-time PCR. The specificity of this assay was assessed by agarose gel electrophoresis and melting curve analysis. The agarose gel electrophoresis showed a single band of 151 bp for the protease and-actin genes (Fig. 3). There was also a single dis-sociation peak for each primer pair and the melting temperatures of the genes were 86◦C and 84◦C respectively (data not shown). These findings indicate that the results were gene-specific and not confused by non-specific amplification or primer dimer.
Changes in the expression levels of the putative aspartic pro-tease in young adults and adult worms were calculated relative to that of the infective larvae. For male worms, the relative expression level increased to 223.0± 24.2 (mean ± standard deviation) folds in
Fig. 1. The full length cDNA and deduced amino acid sequence of a putative aspartic protease from the young adults of Angiostrongylus cantonensis. In the cDNA sequence,
the open reading frame is shown in uppercase letters and the 5- and 3-untranslated sequences in lowercase letters. The spliced leader sequence SL1 is underlined. In
the deduced amino acid sequences, 21 amino acids in the signal peptide are gray boxed. A eukaryote polyadenylation signal polyadenylation site in the 3UTR region is double-underlined. Numbering for the nucleotides (upper) and deduced amino acid residues (lower) are shown at the left margin.
young adults and then decreased to only 7.1± 0.2 in the adult stage. For female worms, this level increased to 118.5± 10.1 folds in the young adults and further to 277.5± 29.2 folds in the adult stage (Fig. 3).
A. cantonensis has a complex life cycle. Many species of mollusks can serve as intermediate hosts for this parasite after ingesting the first-stage larvae in rat feces (Alicata, 1965). In the giant African snail Achatina fulica, the ingested first-stage larvae penetrate the intestinal wall into the hemocoel and then migrate to the man-tle, where they undergo two molts and develop into the infective larvae within 24 days. These larvae become inactive and coiled in nodules embedded within the mollusk tissues (Brockelman et
al., 1976). Although we were able to detect the expression of the putative aspartic protease in these larvae, the level was extremely low compared with those in the later stages. These findings indi-cate that the infective larvae of A. cantonensis should have a very low metabolic rate and their nutrition may not depend on the digestion of the components of the hemolymph in their mollusk host.
Rats acquired the infection by ingesting the infective larvae in intermediate hosts (snails or slugs), paratenic hosts (freshwater crustaceans, frogs, fish, or planarians), or contaminated vegetables. These larvae then penetrate the intestinal tract and migrate to the central nervous system where they molt twice and develop into
Fig. 2. Multiple amino acid sequence alignment of the putative aspartic protease from Angiostrongylus cantonensis with homologues from other nematodes: Caenorhabditis
elegans aspartyl protease family member (asp-2) (GenBank accession no. NP 872129), Brugia malayi aspartic protease BmAsp-1 (GenBank accession no. BAC05688), Strongy-loides stercoralis aspartic protease precursor (GenBank accession no. AAD09345), Haemonchus contortus aspartyl protease precursor (GenBank accession no. CAE12199), and Necator americanus necepsin I (GenBank accession no. CAC00542). Identical residues are in black boxes, conservative residues in dark gray boxes, similar residues in light gray boxes and unrelated residues have a white background. Amino acid numbers are shown on the right. The catalytic Asp residues are indicated with arrow heads (), the conserved cysteine residues by stars (), and the potential N-linked glycosylation site by open circle ().
Fig. 3. Expression levels of a putative aspartic protease in different developmental
stages of Angiostrongylus cantonens determined by quantitative real-time PCR anal-ysis. The following cDNA templates were included: lane 1, infective larva; lane 2, male young adult; lane 3, female young adult; lane 4, male adult; lane 5, female adult.-Actin was used as an endogenous control.
the young adults. About 26 days after infection, the young adults enter the blood vessels for migration and settlement in the pul-monary arteries. At this site, the worms grow rapidly and develop into adults (Alicata, 1965). It has been reported that the intestinal epithelium of adult A. cantonensis is well developed and the sur-face of intestinal lumen is densely covered with short microvilli. Moreover, the lumen is filled with host red blood cells (Hüttemann et al., 2007). These electron microscopic observations indicate that hemoglobin from the rat host should be an important nutrient for A. cantonensis.
Aspartic proteases are a family of proteinase with catalytic aspartate residues in the active site. These enzymes have been reported to play an important role in the initial or early cleav-ages of host hemoglobin in blood-feeding nematodes (Brinkworth et al., 2001; Williamson et al., 2003b). In this study, we revealed dramatic increases in the expression levels of a putative aspartic protease gene in the young adults and adult worms of A. cantonensis. These findings agree with the results obtained in H. contortus that a putative aspartic protease is almost exclusively expressed by the fourth-stage larvae and adult worms (Longbottom et al., 1997). The indication of hemoglobin digestion by parasitic aspartic proteases was not only reported for intestinal but also for tissue-dwelling nematodes (Jolodar et al., 2004). Moreover, sexual differences in the expression level of the putative aspartic protease gene were found in the young adults and adult worms of A. cantonensis. The peak expression level in the male young adults indicates that the male worms reach sexual maturity in this developmental stage. The relative low expression level in the male adult worms suggests that these worms only maintain a low metabolic rate in the rat lung. The dramatic and continuous increase in the expression level of the aspartic protease among female worms may be due to the requirement of a large amount of nutrients for sexual maturation and egg production. However, these suggestions require further investigations.
Acknowledgements
This work was supported in part by a grant from the National Sci-ence Council, Executive Yuan, ROC (NSC95-2320-B-182-039) and Chang Gung Memorial Hospital Research Grants (CMRPD170051, CMRPD170052 and CMRPG870661). We thank Prof. Petrus Tang, Department of Parasitology, College of Medicine, Chang Gung Uni-versity, for his valuable suggestions and technical supports for this study.
References
Alicata, J.E., 1965. Biology and distribution of the rat lungworm, Angiostrongylus cantonensis, and its relationship to eosinophilic meningoencephalitis and other neurological disorders of man and animals. Adv. Parasitol. 3, 223–248. Bektesh, S., Van Doren, K., Hirsh, D., 1988. Presence of the Caenorhabditis elegans
spliced leader on different mRNAs and in different genera of nematodes. Genes Dev. 2, 1277–1283.
Berger, A., Schechter, I., 1970. Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 257, 249–264.
Bhaibulaya, M., 1979. Morphology and taxonomy of major Angiostrongylus species of Eastern Asia and Australia. In: Cross, J.H. (Ed.), Studies on Angiostrongyliasis in Eastern Asia and Australia. US Naval Medical Research Unit No. 2, Taipei, pp. 4–13.
Bonen, L., 1993. Trans-splicing of pre-mRNA in plants, animals, and protists. FASEB J. 7, 40–46.
Brindley, P.J., Kalinna, B.H., Wong, J.Y., Bogitsh, B.J., King, L.T., Smyth, D.J., Ver-ity, C.K., Abbenante, G., Brinkworth, R.I., Fairlie, D.P., Smythe, M.L., Milburn, P.J., Bielefeldt-Ohmann, H., Zheng, Y., McManus, D.P., 2001. Proteolysis of human hemoglobin by schistosome cathepsin D. Mol. Biochem. Parasitol. 112, 103–112.
Brinkworth, R.I., Prociv, P., Loukas, A., Brindley, P.J., 2001. Hemoglobin-degrading, aspartic proteases of blood-feeding parasites: substrate specificity revealed by homology models. J. Biol. Chem. 276, 38844–38851.
Brockelman, C.R., Chusatayanond, W., Baidikul, V., 1976. Growth and localization of Angiostrongylus cantonensis in the molluscan host, Achatina fulica. Southeast Asian J. Trop. Med. Public Health 1, 30–37.
Cooper, J.B., Khan, G., Taylor, G., Tickle, I.J., Blundell, T.L., 1990. X-ray analyses of aspartic proteinases. II. Three-dimensional structure of the hexago-nal crystal form of porcine pepsin at 2.3 Å resolution. J. Mol. Biol. 214, 199–222.
Dassanayake, R.S., Chandrasekharan, N.V., Karunanayake, E.H., 2001. Trans-spliced leader RNA, 5S-rRNA genes and novel variant orphan spliced-leader of the lym-phatic filarial nematode Wuchereria bancrofti, and a sensitive polymerase chain reaction based detection assay. Gene 269, 185–193.
Guiliano, D.B., Blaxter, M.L., 2006. Operon conservation and the evolution of trans-splicing in the phylum Nematoda. PLoS Genet. 2, e198.
Hüttemann, M., Schmahl, G., Mehlhorn, H., 2007. Light and electron microscopic studies on two nematodes, Angiostrongylus cantonensis and Trichuris muris, dif-fering in their mode of nutrition. Parasitol. Res. 101, S225–S232.
Jolodar, A., Fischer, P., Büttner, D.W., Miller, D.J., Schmetz, C., Brattig, N.W., 2004. Onchocerca volvulus: expression and immunolocalization of a nematode cathep-sin D-like lysosomal aspartic protease. Exp. Parasitol. 107, 145–156. Joshua, G.W., Chuang, R.Y., Cheng, S.C., Lin, S.F., Tuan, R.S., Wang, C.C., 1991. The
spliced leader gene of Angiostrongylus cantonensis. Mol. Biochem. Parasitol. 46, 209–217.
Joshua, G.W., Perler, F.B., Wang, C.C., 1995. Orphon spliced-leader sequences form part of a repetitive element in Angiostrongylus cantonensis. Nucleic Acids Res. 23, 1030–1035.
Krause, M., Hirsh, D., 1987. A trans-spliced leader sequence on actin mRNA in Caenorhabditis elegans. Cell 49, 753–761.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408.
Longbottom, D., Redmond, D.L., Russell, M., Liddell, S., Smith, W.D., Knox, D.P., 1997. Molecular cloning and characterisation of a putative aspartate proteinase asso-ciated with a gut membrane protein complex from adult Haemonchus contortus. Mol. Biochem. Parasitol. 88, 63–72.
Maroney, P.A., Denker, J.A., Darzynkiewicz, E., Laneve, R., Nilsen, T.W., 1995. Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency. RNA 1, 714–723.
McKerrow, J.H., Caffrey, C., Kelly, B., Loke, P., Sajid, M., 2006. Proteases in parasitic diseases. Annu. Rev. Pathol. 1, 497–536.
Nilsen, T.W., 1993. Trans-splicing of nematode premessenger RNA. Annu. Rev. Microbiol. 47, 413–440.
Ramirez-Avila, L., Slome, S., Schuster, F.L., Gavali, S., Schantz, P.M., Sejvar, J., Glaser, C.A., 2009. Eosinophilic meningitis due to Angiostrongylus and Gnathostoma species. Clin. Infect. Dis. 48, 322–327.
Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for biol-ogist programmers. Methods Mol. Biol. 132, 365–386.
Smith, W.D., Skuce, P.J., Newlands, G.F., Smith, S.K., Pettit, D., 2003. Aspartyl pro-teases from the intestinal brush border of Haemonchus contortus as protective antigens for sheep. Parasite Immunol. 25, 521–530.
Szecsi, P.B., 1992. The aspartic proteases. Scand. J. Clin. Lab. Invest. Suppl. 210, 5–22.
Takacs, A.M., Denker, J.A., Perrine, K.G., Maroney, P.A., Nilsen, T.W., 1988. A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 85, 7932–7936.
Tang, J., Wong, R.N., 1987. Evolution in the structure and function of aspartic pro-teases. J. Cell. Biochem. 33, 53–63.
Wang, L.C., Chao, D., Chen, E.R., 1989. Acquired immunity in rats against Angiostrongylus cantonensis infection. Int. J. Parasitol. 19, 617–620.
Williamson, A.L., Brindley, P.J., Abbenante, G., Datu, B.J., Prociv, P., Berry, C., Gird-wood, K., Pritchard, D.I., Fairlie, D.P., Hotez, P.J., Zhan, B., Loukas, A., 2003a.
