Identification and intra-specific variability analysis of secreted and membrane-bound proteins from Echinococcus granulosus

Parasitology International 55 (2006) S63 – S67 www.elsevier.com/locate/parint

Identification and intra-specific variability analysis of secreted and membrane-bound proteins from Echinococcus granulosus Mara C. Rosenzvit *, Federico Camicia, Laura Kamenetzky, Patricia M. Muzulin, Ariana M. Gutierrez Departamento de Parasitologı´a, Instituto Nacional de Enfermedades Infecciosas ANLIS ‘‘Dr. Carlos G. Malbra´n’’ Av. Velez Sarsfield 563, Buenos Aires (1281), Argentina Available online 20 December 2005

Abstract Echinococcus granulosus, the etiological agent of cystic hydatid disease, exists as a series of strains or genotypes, differing in biological features. Many of the secreted and membrane-bound proteins (S/M) from helminth parasites are involved in the host – parasite interplay and constitute potential targets for diagnosis, anti-parasitic drugs and vaccines. A number of E. granulosus S/M proteins were identified using the signal sequence trap technique. Six out of seven cDNA fragments of these newly identified proteins showed nucleotide and amino acid sequence variation. Inter-strain variation was reported for other characterized S/M proteins as the vaccine target EG95 and the major hydatid cyst fluid antigen, Antigen B (AgB). AgB is highly polymorphic, 101 different sequences related to AgB were reported so far and were grouped in 5 genes (EgB1 – EgB5) and one pseudogene (EgB2p) exclusive of G5, G6/G7 genotypes. The significance of AgB polymorphism and possible consequences in diagnostic performance are discussed. The diagnostic value of the new protein variants detected in E. granulosus strains could be determined through standardized inter-laboratory studies as the recently done by the South American Network for Hydatid Serology. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Platyhelminth parasites; Echinococcus granulosus; Secreted and membrane-bound proteins; Intra-specific variation; Antigen B

1. What do we mean when we speak of Echinococcus granulosus? The cestode Echinococcus granulosus is the causative agent of cystic hydatid disease (CHD), a major zoonosis that affects many areas around the world, especially those with rural-based economies. The variability of E. granulosus previously characterized in physiological, morphological and biochemical studies has been confirmed by several molecular techniques which allowed the identification of 9 different genotypes (G1 – 9) [1]. Most of these genotypes can be assigned to strains that differ in biological features, such as intermediate host specificity, developmental rate and infectivity to humans [1]. Some of the genotypes were differentiated in 2 groups: G1/G2/ G3 cluster and G6/G7 cluster, G5 genotype was grouped closer to the G6/G7 cluster [2].

* Corresponding author. Tel.: +54 11 4301 7437; fax: +54 11 43032382. E-mail address: [email protected] (M.C. Rosenzvit). 1383-5769/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2005.11.009

2. Weapons for the worms’ war Helminth parasites live for long periods of time in the hostile medium of the host. In their struggle for life, they have developed strategies to feed and reproduce defending themselves from host immune attacks. Proteins secreted by parasites and those expressed on their surface as membrane-bound proteins are likely to represent powerful weapons to accomplish this goal, due to their interaction with host cells and molecules. Secreted and membrane-bound (S/M) proteins of parasites participate in a wide range of parasite functions, including penetration and establishment in host tissues, modulation of the host immune response and incorporation of metabolites from the host. Because of their potential exposure to the host immune system, S/M components are candidates for improved diagnostic tests, as well as new drug and vaccine targets. The knowledge of variation in S/M proteins would help to the understanding of the fine mechanisms of adaptation of helminth parasites to their specific hosts, and is necessary for the rational design and application of

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Table 1 Examples of S/M proteins from helminth parasites and associated sequence variation Species

Protein

Putative function

Application/Interest

Variation

Reference

Cooperia oncophora

Glu Cla3 AC-1, AC-3, AC-4, AC-5, Glp-7

Involved in resistance to ivermectin Potential targets for anti-parasitic drugs and diagnosis

Haemonchus contortus

CBL family

Vaccine candidates

Brugia malayi

Bm-CPI-2

Intestinal cathepsin B-like cysteine proteases Cysteine protease inhibitor

Sequence variation associated to sensitivity – resistance to ivermectin Allelic frequencies significantly different between strains, except for locus AC-5 Extremely abundant and diverse, potential antigenic diversity Lysine/asparagine dimorphism of probable functional significance

[3]

Haemonchus contortus

Glutamate-gated chloride channel Cysteine proteases

Blocks proteinases of the MHC class II antigen processing

[4]

[5] [6]

Table 2 Examples of putative secreted and membrane-bound (S/M) proteins of Echinococcus granulosus, their sequence homology, activity, life cycle stages where expression was detected and sequence variation when known S/M protein

Sequence homology/activity

Application

Stage

Sequence variation

Reference

Ag5

Sequence homology to serine proteases, cell adhesive and calcium binding motifs and activity Inhibition of serin protease activity and neutrophil chemotaxis, induction of TH-2 immune response, lipid binding activity Fibronectin type III domain

Diagnosis

Larval

Not analysed

[22]

Diagnosis

Larval

High intra-, inter-cyst and inter-strain polymorphism

[7 – 15], a

Vaccination

14-3-3.2: related with metabolic (excretory/secretory) processes

Vaccine candidates

EgP8C7

–

Protoscoleces, adult worms

EgP22B4 EgP35H4

Sequence homology to amino acid transporters Novel Vacuolar protein transporter

Gene family. EG95: No intra-strain variation (G1), inter-strain variation1 5 isoforms (adult worms, protoscoleces, metacestode tissue) Inter-strain variation not analysed Inter-strain2

[16]

14-3-3

Eg95: oncospheres, other members: oncospheres, gravid adult worms, protoscoleces 14-3-3.2: adult worms (secreted isoform)

– –

Protoscoleces, adult worms, oncospheres Protoscoleces, adult worms, oncospheres

EgP41C6 EgP74E7 EgP78F4 EgP38D7

novel novel E. granulosus EST without homologs cyclophilin

– – – –

Protoscoleces, Protoscoleces, Protoscoleces, Protoscoleces,

AgB

EG95

adult adult adult adult

worms, worms, worms, worms,

oncospheres oncospheres oncosphere oncosphere

Inter-strain2 No inter-strain variation observed2 Inter-strain2 Inter-strain2 Inter-strain2 Inter-strain2

[17]

[19], b [19], b [19], b [19], [19], [19], [19],

b b b b

a: Muzulin P., Kamenetzky L. and Rosenzvit M., unpublished results. b: Rosenzvit MC, and McManus, DP., unpublished results. 1 G1 vs. G6/G7. 2 Inter-strain variation was analysed by SSCP – PCR and sequencing for G1 and G6 E. granulosus genotypes.

diagnostic tests, antihelminthic drugs and immunoprophylaxis reagents. In spite of this, there is no extensive literature on the subject, especially for cestodes. Exceptions are a number of secreted proteins from nematodes of medical or veterinary relevance (Table 1). 3. Are S/M E. granulosus proteins variable? Secreted and membrane-bound proteins of important function in E. granulosus biology and/or application in CHD control include the major antigens Antigen 5 (Ag5) and Antigen B

(AgB), both secreted into the cyst hydatid fluid; the EG95 protein, bearing secretory signal sequence and GPI anchor motif, one of the E. granulosus 14-3-3 isoforms, secreted by the adult worm and E. granulosus cyclophilin secreted by protoscoleces (Table 2). A transcriptomic survey of E. granulosus revealed that 11 – 27% of protoscoleces and cyst wall cDNAs included potential N-terminal signal sequences [18]. The signal sequence trap (SST) technique was applied to RNA from E. granulosus protoscoleces in order to functionally identify new genes coding for S/M proteins [19]. The deduced amino acid sequences of the cDNAs identified showed significant similarity with amino acid

Fig. 1. Neighbor-joining phylogenetic tree of coding regions from AgB sequences found in Echinococcus sp. AgB sequence variants were clustered in five groups corresponding to previously described genes and one pseudogen. Each sequence variant is indicated by taxa (Eg, Em, Ev or Eo), gene subunit (B1 – B5) and a number representing the variant after a ‘‘v’’. The presence and number of each subunit in E. granulosus strains (G1 – G7) and in other Echinococcus sp. is indicated. The represented taxa are: Eg, E. ganulosus; Ev, E. vogeli; Em, E. multilocularis; Eo, E. oligarthrus. Taenia-related AgB genes were used as outgroups: Ts, T. solium; Tc, T. crassiceps (GenBank accession nos. AF076609 and U07150, respectively). ND: not determined. Numbers at nodes represent percentage occurrence of clades in 1000 bootstrap replications of the data. The phylogenetic tree was made using MEGA 2.1 program.

M.C. Rosenzvit et al. / Parasitology International 55 (2006) S63 – S67

99

66 67

98

58

100 82 56 65 100

76 100 56

100 90 99

63 52 98

58 66

100

99

54

96

68

69 92 87 80 100

98 70 72

72 99 62

100

100

64 100 63 59

100

100

EgB2v1 EgB2v2 EgB2v3 EgB2v4 EgB2v5 EgB2v6 EgB2v7 EgB2v8 EgBv9 EgB2v10 EgB2v11 EgB2v12 EgB2v13 EgB2v14 EgB2v15 EgB2v16 EgB2v17 EgB2v18 EgB2v19 EmB2v20 EmB2v12 EvB2v22 EoB2v23 EoB2v24 EoB2v25 EoB2v26 EoB2v27 EgB2pv29 EgB2pv30 EgB2pv31 EgB2pv32 EgB2pv33 EgB2pv34 EvB4v35 EvB4v36 EvB4v37 EvB4v38 EvB4v39 EvB4v40 EvB4v41 EgB4v42 EgB4v43 EgB4v44 EgB4v45 EgB4v46 EgB4v47 EgB4v48 EmB4v49 EmB4v50 EmB4v51 EgB4v52 EgB4v53 EgB4v54 EgB4v55 EgB4v56 EgB4v57 EgB4v58 EgB4v59 EgB4v60 EgB4v61 EgB4v62 EgB4v63 EgB4v64 EgB4v65 EgB4v66 EgB1v67 EgB1v68 EgB1v69 EgB1v70 EgB1v71 EgB1v72 EgB1v73 EgB1v74 EgB1v75 EmB1v76 EgB5v77 EgB5v77 EgB5v78 EgB5v79 EgB5v80 EgB5v81 EgB5v82 EgB5v83 EgB3v84 EgB3v85 EgB3v86 EgB3v87 EgB3v88 EgB3v89 EgB3v90 EgB3v91 EgB3v92 EgB3v93 EgB3v94 EgB3v95 EgB3v96 EgB3v97 EgB3v98 EgB3v99 EgB3v100 EgB3v101 EgB3v102 EgB3v103 EgB3v104 EgB3v105 EgB3v106 EgB3v107 Ts Tc

AgB subunit (n)

B2 (19)

B2p (6)

E. granulosus strain

G1/G2

G5 G6/G7

S65

Other Echinococcus species

Em Ev Eo

ND

G1/G2 G5 G6/G7

Em Ev

B1-like (5)

G5 G6/G7

Em

B1 (4)

G1/G2

ND

B5 (8)

G1/G2

ND

B3- like (3)

G5 G6/G7

ND

B3 (21)

G1/G2

ND

B4 (22)

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transporters, Krebs cycle intermediate transporters, presenilins, vacuolar protein sorter proteins, E. granulosus cyclophilin and secreted proteins without homologues. One of the putative amino acid transporters identified was shown to be localized to the tegument and subtegumental parenchyma of protoscoleces and another one to isolated cells in the parenchyma and to brood capsules wall (Camicia et al., unpublished results). The sequence variation of fragments of the cDNAs was analysed for some of the SST identified clones in two E. granulosus strains that infect humans in significant proportions (Table 2). Nucleotide and amino acid sequence variation was detected in 6 out of 7 proteins studied (Rosenzvit and Mc Manus, unpublished results). For EG95, no variation has been found in the predicted proteins encoded by eg95-1 gene in isolates from G1 genotype, but significant variation has been found with G6/G7 genotype parasites. If this has any consequence on the effectiveness of the vaccine for the G6/G7 parasites is to date unknown [16]. With Ag5, there are no systematic studies on amino acid variation, although it was suggested that it has no polymorphic nature. Since its antigenicity is determined by a combination of posttranslational (mainly glucosidic) and discontinuous proteinous epitopes (Lorenzo et al., submitted for publication), it may be hard to directly correlate differences in sequences (if they exist) with the antigenic behavior of Ag5. AgB variation has been the subject of several studies which are discussed in the following section. 4. AgB: a Pandora’s box AgB is an abundant antigen of cyst hydatid fluid, highly immunogenic in human infections and used for serological diagnosis of CHD. However, a wide variation in sensitivity and specificity was found for native AgB (reviewed in Zhang et al. [20]). Two recombinant subunits and one synthetic peptide of AgB have been tested in a standardized study [21]; their diagnostic sensitivity did not surpass that of the native AgB (80%). AgB was suggested to be involved in the evasion of host immune response due to its ability to inhibit elastase activity and neutrophil chemotaxis [10] and to elicit a non-protective

A EGB1G1 EGB3G1 EGB5G1 EGB2G1 EGB4G1

Th2 cell response [11]. Recently, the ability of native and recombinant forms of AgB to bind hydrophobic compounds has been showed [12]. However, its role(s) in E. granulosus biology has yet to be determined. Analysing the AgB variation may help to elucidate its biological function and to determine how it would be better applied to diagnosis. The polymorphic nature of AgB was noticed in the early studies of Shepherd et al. [10] and Frosch et al. [13]. A recent study showed that AgB is constituted by at least 5 gene loci (B1 –B5), each of them presenting several minor variants or alleles which could have arisen by differences between protoscoleces of the same cyst and/or cells of the same protoscolex or by generation of tandemly repeated copies of each gene within a genome [7]. The extremely polymorphic nature of AgB in E. granulosus is reflected in the number of different AgB-related sequences reported, totaling 101 sequences ([7 – 9,13– 15]; Muzulin et al., unpublished results; cDNA sequences deposited in http://zeldia.cap.ed.ac.uk/Lopho/ LophDB.php). Since several variants differ only in the intron region, these sequences represent 88 different cDNAs which were used to construct a phylogenetic tree by Neighbor-Joining method. The tree obtained (Fig. 1) shows that all the sequences can be assigned either to B1/B3/B5 or to B2/B4 clusters. Variation in AgB-related sequences is also observed for Echinococcus multilocularis, Echinococcus vogeli and Echinococcus oligarthrus (Fig. 1). In recent studies ([9]; Muzulin et al., unpublished results), the genetic variation of AgB-coding genes and their expression was analysed for five human infecting E. granulosus strains. It was observed that E. granulosus strains differ in the type and abundance of some of the AgB subunits. For example, EgB2 gene found in G1/G2 cluster is present in G5 and G6/G7 genomic DNAs as a pseudogen (EgB2p in Fig. 1), because of a mutation in the splicing site. Also, the EgB1 and EgB3 variants from G5 and G6/G7 were grouped in separate clusters with respect to G1/G2 variants (B1-like and B3-like in Fig. 1). G1 and G7 also differed in their AgB expression pattern; while G1 transcribed all the AgB subunits (B1 –5) (Fig. 2A), G7 did not show neither AgB2- nor AgB5-expressed variants ([9], Muzulin et al., unpublished results). The diversity of AgB found in E.

10 20 30 40 50 60 70 80 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

FVVVTQA---DDGLTSTSRSVMKMFGEVKYFFERDPLGQKVVDLLKELEEVFQLLRKKLRMALRSHLRGLIAEGE----FVVVAHADDDDDEVTKTKKGVMKAISEIKHFFQSDPLGKKLVEVMKDVASVCEMVRKKARMALKEYVRKLVKEDE----FVVVARAECDDDEVTKTKKGVMKAISEIKDFFRRDPLGKKLVEVMKEVASVCEMVRKKARMALKAYVRKLIEEAE----FVAVVQAK--DEPKAHMGQVVKKRWGELRDFFRNDPLGQRLVALGNDLTAICQKLQLKIREVLKKYVKNLVEEKD-DDSK FVAVVQAK--AEPERCKCLIMRK-LSEIRDFFRSDPLGQKLAALGRDLTAICQKLQLKVHEVLKKYVKDLLEEEDEDDLK

B EGB1G1 EGB3G1 EGB5G1 EGB2G1

EGB1G1

EGB3G1

EGB5G1

EGB2G1

EGB4G1

---

0.466

0.506

0.350

0.294

---

0.853

0.329

0.325

---

0.354

0.337

---

0.666

Fig. 2. Transcriptional analysis of E. granulosus G1 strain. (A) Alignment of AgB deduced amino acidic sequences obtained from RT – PCR analysis. Identical residues are presented in black, and conserved substitutions are presented in grey. Alignment was done with Clustal X 1.81. (B) Pairwise identity amino acidic matrix of the sequences showed in panel A.

M.C. Rosenzvit et al. / Parasitology International 55 (2006) S63 – S67

granulosus may reflect a need for diversity in AgB biochemical function(s) or antigenic diversity to evade the immune response of each specific host. Switching between subunits, especially from a subunit from B1/B3/B5 cluster to one from the B2/B4 cluster might imply different immune recognition by the host immune system, since few stretches of conserved amino acids are present (Fig. 2A) and the amino acid identities between different subunits, specially those from different clusters, is low (Fig. 2B). Alternatively, gene amplification may have arisen to provide high levels of AgB activity, and the diversity observed might reflect changes that are neutral for AgB functions. This last hypothesis is likely for many genomic variants of each AgB subunit that code for the same protein [9]. Combinations of these explanations are also possible. The diagnostic value of the new protein variants detected in E. granulosus strains could be determined through inter-laboratory studies as the recently done by the South American Network for Hydatid Serology [21]. 5. Conclusions Many of the S/M proteins from helminth parasites are likely to be exposed to the host, being pivotal for the host –parasite relationship. This makes these molecules potential targets for control strategies. However, precisely the contact with host molecules and cells makes S/M proteins prone to variation, which can affect their practical application. High genetic diversity was implied for E. granulosus by analysis of repetitive, mitochondrial, microsatellite, ribosomal and randomly amplified DNA markers. Analysing the variation of S/M proteins coding genes may provide a specific correlation of strain genetic diversity with functional and practical significance. Inter-strain variation was detected for several E. granulosus S/M proteins. Particularly challenging is the understanding of variation in the AgB family. Potential for antigenic variation was suggested [7,9] but has not yet been demonstrated. Whether the high intraspecific polymorphism of AgB affects its diagnostic performance also remains to be established. Acknowledgments Research supported by CABBIO, CONICET, Instituto Nacional de Enfermedades Infecciosas, ANLIS ‘‘Dr. Carlos G. Malbra´n and ‘‘Fundacio´n Alberto J. Roemmers’’ and the Australian Centre for International and Tropical Health and Nutrition, the National Health and Medical Research Council of Australia. References [1] McManus DP, Thompson RC. Molecular epidemiology of cystic echinococcosis. Parasitology 2003;127:S37 – 51. [2] Obwaller A, Schneider R, Walochnik J, Gollackner B, Deutz A, Janitschke K, et al. Echinococcus granulosus strain differentiation based on sequence heterogeneity in mitochondrial genes of cytochrome c oxidase 1 and NADH dehydrogenase-1. Parasitology 2004;128:569 – 75. [3] Njue AI, Prichard RK. Genetic variability of glutamate-gated chloride channel genes in ivermectin-susceptible and -resistant strains of Cooperia oncophora. Parasitology 2004;129:741 – 51.

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