Parasitology International 64 (2015) 37–42

Contents lists available at ScienceDirect

Parasitology International
journal homepage: www.elsevier.com/locate/parint

Expression characteristics and specific antibody reactivity of diverse

cathepsin F members of Paragonimus westermani
Chun-Seob Ahn a, Byoung-Kuk Na b, Dong-ll Chung c, Jeong-Geun Kim a, Jin-Taek Kim a, Yoon Kong a,⁎
a
Department of Molecular Parasitology, Sungkyunkwan University School of Medicine and Center for Molecular Medicine, Samsung Biomedical Research Institute,
Suwon 440-746, Republic of Korea
b
Department of Parasitology and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 660-751, Republic of Korea
c
Department of Parasitology, Kyungpook National University, School of Medicine, Taegu 700-842, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Paragonimiasis, caused by the lung fluke Paragonimus, is a major food-borne helminthic disease. Differential
Received 5 May 2014 diagnosis of paragonimiasis from tuberculosis and other infectious granulomas in the lung is a prerequisite to
Received in revised form 29 August 2014 proper management of patients. Cysteine proteases of Paragonimus westermani (PwCPs) invoke specific antibody
Accepted 23 September 2014
responses against patient sera, while antibody capturing activity of different PwCPs has not been comparatively
Available online 2 October 2014
analyzed. In this study, we observed the expressional regulation of 11 species of different PwCPs (PwCP1-11).
Keywords:
We expressed recombinant PwCPs and assessed diagnostic reliability employing sera from patients with
Paragonimus westermani P. westermani (n = 138), other trematodiases (n = 80), cestodiases (n = 60) and pulmonary tuberculosis
Paragonimiasis (n = 20), and those of normal controls (n = 20). PwCPs formed a monophyletic clade into cathepsin F and
Cysteine protease showed differential expression patterns along with developmental stages of worm. Bacterially expressed recom-
Cathepsin F binant PwCPs (rPwCPs) exhibited variable sensitivity of 38.4–84.5% and specificity of 87.2–100% in diagnosing
Developmental expression homologous infection. rPwCPs recognized specific antibodies of experimental cat sera as early as 3 or 6 weeks
Serodiagnosis after infection. Patient sera of fascioliasis, Schistosomiasis japonicum and clonorchiasis demonstrated weak
cross-reactions. Our results demonstrate that diverse PwCPs of the cathepsin F family participate in inducing spe-
cific antibody responses. Most P. westermani cathepsin F, except for PwCP2 (AAF21461), which showed negligible
antibody responses, might be applicable for paragonimiasis serodiagnosis.
© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction disease progresses to subacute/chronic stages, during which the para-

Paragonimiasis is an inflammatory lung disease caused by the genus
Paragonimus. The disease exemplifies a typical food-borne zoonotic hel-
minthiasis. Human infections occur by consumption of improperly
cooked freshwater crustaceans. Wild boars and rodents also play impor-
tant roles in human infection [1]. Paragonimus westermani, a prototype
species of the genus Paragonimus, is widely distributed in Asia. In
addition, several Paragonimus species, such as P. skrjabini (China),
P. heterotremus (Southeast Asia), P. uterobilateralis and P. africanus
(Africa), P. mexicanus (Latin America) and P. kellicotti (North America)
evoke pleuropulmonary and subcutaneous infections in humans [2].
Paragonimiasis is locally endemic in some restricted areas in conjunc-
tion with ethnic food consumption practices. Its prevalence has waned
in recent years, but approximately 20 million people are infected world-
wide and another 300 million people are at risk of infection [3].
In the early invasive stage, during which the juvenile Paragonimus
migrate in the abdominal and thoracic cavities (3–6 weeks after infec-
tion), most patients do not recognize discernible symptoms. As the
⁎ Corresponding author. Tel.: +82 31 299 6251; fax: +82 31 299 6269.
E-mail address: kongy@skku.edu (Y. Kong).
site formed a granulomatous lesions in the lung (later than 7–9 weeks
after infection), patients complain of chest/pleural pain, cough and
rusty sputum. Eosinophilia may also be present [1]. In addition,
Paragonimus migrates everywhere in the body other than lungs,
resulting in typical granulomatous lesions in the affected organs/tissues
(ectopic paragonimiasis). Nodular lesions caused by pulmonary
paragonimiasis should be differentiated from tuberculosis, cancer, fun-
gus ball and bacterial granulomas, where these diseases are prevalent
[4,5].
Detection of characteristic eggs in the sputum or stool samples is
a confirmatory diagnosis for paragonimiasis. However, egg detection
has a low sensitivity (b50%) and is not applicable to ectopic
paragonimiasis [6]. A combination of clinical and epidemiological data
of the patients together with radiological and laboratory findings
can provide a diagnostic clue to proper diagnosis. Positive antibody
(IgG/IgE) reaction by serological tests is highly sensitive and specific
[7,8]. Demonstration of elevated levels of specific IgM antibodies in
serum/pleural fluid is also diagnostic in early paragonimiasis [1].
P. westermani cysteine proteases (PwCP) exert their primary roles in
tissue invasion and migration by hydrolyzing extracellular matrices, im-
mune evasion by attenuation of host immune effector system, as well as

http://dx.doi.org/10.1016/j.parint.2014.09.012
1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.
38 C.-S. Ahn et al. / Parasitology International 64 (2015) 37–42
in nutrient uptake and metabolic turnover of other bioactive molecules
[9,10]. In addition, these molecules induce specific antibody responses
against patient sera of paragonimiasis [8,11–13]. At least 13 different
species of PwCPs are expressed in different developmental stages
including egg, metacercaria and adults [14–16]. However, immune re-
activity of individual PwCPs has not been comparatively determined
in regard to their sensitivity and specificity, and the temporal onset of
the specific antibody responses.
In this study, we observed differential transcriptional patterns of 11
species of PwCP genes in accordance with worm development. We
expressed recombinant proteins and analyzed their immunoreactivity
profile employing a panel of serum samples from homologous and het-
erologous infections. P. westermani expressed several species/isoforms
of cathepsin F family members during the developmental stages. Most
of these PwCPs showed high sensitivity and specificity in diagnosing
paragonimiasis from early infection stage, except for one molecule
(PwCP2, AAF21461). Patient sera of clonorchiasis, fascioliasis and schis-
tosomiasis japonicum showed a weak cross reactivity.
2. Materials and methods
2.1. Serum samples
A total of 138 serum samples from patients with pulmonary
paragonimiasis westermani (male, 54.3%; mean ages, 46 ± 12.8),
whose diagnosis was made by both positive antibody reactions against
adult extracts [7] and typical findings of chest high-resolution computed
tomography [17], were evaluated. Clinical presentation of the patients
included eosinophilia ranging from 3 to 65% (71/138, 51.4%), rusty
sputum (56/138, 40.6%), cough (43/138, 31.2%) and chest/pleuritic
pain (29/138, 21%). Twenty-three patients (16.7%) did not show any dis-
cernable symptoms. Eighteen patients (13%) were treated for pulmo-
nary tuberculosis for 3–6 months during which they did not show any
responsiveness. Sera from clonorchiasis (n = 35), Schistosomiasis
japonicum (n = 15), fascioliasis (n = 30), cystic echinococcosis (CE;
n = 20), cysticercosis (n = 20) and sparganosis (n = 20) were tested.
Clonorchiasis and schistosomiasis were diagnosed by stool examination.
Fascioliasis, cystic echinococcosis, cysticercosis and sparganosis were
diagnosed by positive antibody reactions, together with relevant radio-
logical findings. Patient sera of pulmonary tuberculosis (n = 20),
whose diagnosis was confirmed by sputum microscopy for acid-fast ba-
cillus were evaluated. Sera from 20 healthy blood donors who denied
any possible exposure to helminthic infections or protozoan infections
were also included. Informed consent was obtained from the patients.
Sera from cats (n = 4), experimentally infected with 50 metacercariae
were serially collected 1-, 2-, 3-, 4-, 6-, 8-, 10- and 13-week post-
infection and used in this study [18].
2.2. Cloning and sequence analysis of PwCP genes
We and other investigators previously deposited some genes
putatively encoding PwCP in the NCBI database (http://www.ncbi.
nlm.nih.gov). We retrieved these sequences and designed specific
primers for cloning the genes, which included PwCP1 (AAW28151),
2 (AAF21461), 3 (AAY81942), 4 (AAB93494), 5 (AAY81943), 6
(AAY81944), 7 (AAY81945), 8 (AAY81946), 9 (AAY81947), 10
(AAW28152) and 11 (AAY81948) (Supplementary Table S1). Each
forward and reverse primer was combined with T3 and T7 promoter
primer to PCR-amplify target genes from a P. westermani cDNA library
synthesized using mRNAs extracted either from adult worms (500 μg
dry weight) or from metacercariae (200 μg dry weight). Amplification
reactions were done at 94 °C for 4 min, 35 cycles at 94 °C for 1 min,
50 °C for 1 min, and 72 °C for 1 min, followed by a final extension at
72 °C for 10 min. The PCR-products were gel-purified, ligated into the
pGEM-T easy vector (Promega, Madison, WI, USA) and transformed
into competent Escherichia coli DH5α (Life Technologies, Grand Island,
NY, USA). The nucleotide sequences determined from both strands
using the BigDye Terminator cycle Sequencing Ready Reaction kit
(PerkinElmer, Foster City, CA, USA) and an ABI PRISM 377 DNA sequenc-
er (Applied Biosystems, Foster City, CA, USA) were deduced into amino
acid sequences. The homology patterns were determined by ClustalW2
(http://www.ebi.ac.uk/Tools/msa/clustalw2/). Signal peptides were
searched with Signal-BLAST (http://sigpep.services.came.sbg.ac.at/
signalblast.html). Structural and functional domain(s) were detected
by NCBI conserved domains (http://www.ncbi.nlm.nih.gov/Structure/
cdd/wrpsb.cgi) and InterProScan (http://www.ebi.ac.uk/Tools/pfa/
iprscan/).
2.3. Expression of recombinant proteins
The mature domains of the respective PwCPs were PCR-amplified
from the cDNA libraries using gene-specific primers containing BamHI
and HindIII sites (Supplementary Table S1). PCR products were ligated
into the pET-28a(+) vector (Novagen, Madison, WI, USA) and trans-
formed into E. coli DH5α. Their expression fidelity was confirmed by
sequencing. The constructs were transfected into E. coli BL21 (DE3)
and cultured in Luria–Bertani medium supplemented with Kanamycin
(25 μg/ml). Protein expression was induced by 0.5 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) for 4 h at 37 oC. The cells were harvested,
sonicated and recombinant PwCPs (rPwCP1-11) were purified using
nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen, Valencia,
CA, USA). Purity of each recombinant protein was monitored by 15%
reducing SDS-PAGE.
2.4. Semi-quantitative reverse transcription (RT)-PCR
To observe the transcriptional patterns of PwCP genes according
to worm development, total RNAs were extracted from eggs,
metacercariae, juveniles and adults using TriZol reagent (Life technolo-
gies, Grand Island, NY, USA). The 1- and 3-week-old worms were har-
vested from the peritoneal cavity and those of 7- and 13-week-old
worms were collected from the thoracic cavity and lungs of infected
cats. Eggs liberated from 20 adult worms incubated in physiological
saline overnight at 37 °C were individually purified under dissecting
microscope (purity N 99%) [7,18]. The RNAs were treated with RNase-
free DNase (GIBCO BRL, Rockville, MD, USA). The PwCP transcripts in
the RNA solutions (5 μg) were reverse transcribed into the first-strand
cDNAs using SuperScript First-Strand Synthesis System for RT-PCR
according to the manufacturer's instructions (Life Technologies). The
cDNAs were amplified with each of PwCP-specific primers (Supplemen-
tary Table S2). PCR conditions were as described above, except for the
PCR cycles, which were modified to 30 cycles. A primer pair for the
P. westermani β-actin gene (forward; 5′-GGCCATGTACGTTGCTATCC-3′
and reverse; 5′-CAGAGAGAACAGTGTTGGCG-3′), which was shown to
be constitutively expressed throughout developmental stages, was
used as a control. The amplified PCR products were analyzed on a 1.2%
agarose gel with ethidium bromide staining.
2.5. Immunoblotting
The rPwCP1-11 were separated by 15% reducing SDS-PAGE and
electrotransferred to polyvinylidene fluoride (PVDF) membranes
(Millipore, Bedford, MA, USA). The membranes were cut into strips.
Eleven pieces of each blot, each containing an individual recombinant
protein, were simultaneously incubated overnight with 1:200 diluted
individual patient serum or pooled cat serum (n = 4) in casein buffer
and subsequently with 1:1000 diluted horseradish peroxidase (HRP)-
conjugated host-specific anti-IgG (Cappel, Solon, OH, USA) for 4 h at
room temperature. The reactions were developed by 4-chloro-1-
naphthol (4C1N) chromogen (Sigma-Aldrich, St. Louis, MO, USA) sup-
plemented with 0.03% H2O2. Positive predictive values were calculated
as (no. true positive/[no. true positive + no. false positive]) × 100.
 C.-S. Ahn et al. / Parasitology International 64 (2015) 37–42 39

Negative predictive value was calculated as (no. true negative/[no. true
negative + no. false negative]) × 100.
3. Results
3.1. Molecular characteristics of PwCP genes
Multiple sequence alignment of several PwCP genes retrieved from
the GenBank database revealed that these molecules shared character-
istic features of family C1 members within clan CA, which was
composed of prepro-, pro- and mature-domains. The prepro-domain
contained signal peptide (SP) sequences composed of 10–18 amino
acids. In the pro-domain, inhibitor I29 domain and ERFNAQ motif
were tightly conserved. Mature peptidase C1A domain harbored
amino acid residues composing active sites for enzymatic catalysis (Q ,
C, H and N), as well as a substrate-binding residue (G) within the
GCNGG motif. Seven cysteine residues (C), which might be involved in
formation of tertiary structure by disulfide linkage, were also recog-
nized (Supplementary Fig. S1). These PwCPs shared 44–96% sequence
identity. PwCP2 had the most divergent amino acid sequences among
11 PwCPs and shared only 44% identity with other PwCPs, due mainly
to the presence of a long pro-region (Fig. 1A). The phylogenetic tree
was constructed employing amino acid sequences of family C1 protease
members of the trematode and cestode parasites. As shown in Fig. 1B,
cathepsins B, L and F formed a well-separated clade, in which PwCPs
clustered into cathepsin F. PwCPs displayed close relationships with
one another, except for PwCP2, although PwCP2 belonged to the same
clade, cathepsin F.
3.2. Expression profile of PwCP genes according to worm development
We observed developmental expression patterns of the PwCP
transcripts during the maturation of the parasite including eggs,
metacercariae, juveniles and adults (Fig. 2). PwCP3, 5, 6, 7 and 8 were
constitutively expressed throughout all stages from egg to adult in an
upregulated fashion, among which the PwCP8 transcript was rapidly
increased during the maturation of the worm in the definitive host.
PwCP1, 2, 4 and 11 appeared to be expressed from the metacercarial
to adult stages, but were not detected in eggs. PwCP1 showed preferen-
tial expression in early juvenile stages, such as 1- and 3-week-old
worms. PwCP2, 4 and 11 revealed gradually augmented expression
patterns according to worm development. PwCP9 and 10 were predom-
inant in juvenile stages, but were not expressed in adult and egg stages.
The β-actin gene used as an internal control was constitutively
expressed throughout the parasitic life stages examined. The reaction
that contained neither Taq DNA polymerase nor P. westermani cDNAs
revealed no amplified bands. This result demonstrated the differential
expression of multiple isoforms of P. westermani cathepsin F during
the developmental stages.
3.3. Expression of recombinant proteins and evaluation of diagnostic
property
We designed specific primers and isolated PwCP genes by PCR
screening of the metacercaria and adult P. westermani cDNA libraries.
We expressed their mature proteins in E. coli and purified the proteins
using Ni-NTA column. These proteins migrated to 26–27.5 kDa by

A 1 18 27 83 109 320
% identity
CP1 (AAW28151) 322

1 22 38 127 182 212 423

CP2 (AAF21461) 427 (44)
1 18 27 83 109 318
CP3 (AAY81942) 321 (88)
1 19 32 88 113 316
CP4 (AAB93494) 325 (66)
1 6 22 32 88 113 323
CP5 (AAY81943) 325 (77)
1 9 22 32 88 113 323
CP6 (AAY81944) 325 (66)
1 9 22 32 88 113 316
CP7 (AAY81945) 325 (67)
1 9 19 32 88 113 316
CP8 (AAY81946) 325 (67)
1 18 32 83 109 319
CP9 (AAY81947) 322 (92)
1 6 22 32 88 114 324
CP10 (AAW28152) 327 (96)
1 18 27 83 109 319
CP11 (AAY81948) SP Inhibitor I29 Peptidases C1A 322 (92)

B 76
100 PwCP1 (AAW28151)
PwCP10 (AAW28152)
96
PwCP9 (AAY81947)
100
PwCP11 (AAY81948)
94
PwCP3 (AAY81942)
99 PwCP5 (AAY81943)
PwCP6 (AAY81944) Cathepsin F
72 63 PwCP8 (AAY81946)
100 PwCP4 (AAB93494)
100 PwCP7 (AAY81945)
99
0.1
Clonorchis sinensis (AAF21471)
96 C. sinensis (AAD29130)
PwCP2 (AAF21461)
100 Fasciola hepatica (AAB41670)
F. gigantica (AAD23996)
66 Schistosoma japonicum (AAA87849)
Cathepsin L
100 S. mansoni (CAA83538)
Trichinella spiralis (XP_003373289)
T. spiralis (XP_003379650)
100 100 Echinococcus granulosus (CDJ16205)
100 E. multilocularis (BAJ83490)
Cathepsin B
84 S. japonicum (CAX71086)
99 S. mansoni (XP_002574974)

Fig. 1. Schematic presentation of molecular structure and phylogenetic analysis of the PwCP genes. (A) Comparison of functional domain organization. Preprodomains bearing signal
peptide (SP) are shown by dark-gray. Prodomains containing inhibitor I29 domain with ERFNAQ motif (light-gray boxes) are demonstrated by dashed boxes. Mature proteins harbored
peptidase C1A domain (black-boxes). Numerals at the top represent amino acid positions. Numerals in parenthesis denote % sequence identity based on amino acid level. (B) Phylogenetic
analysis of PwCP genes. The tree was derived from a neighbor-joining tree of the amino acid alignment, which was constructed by the Mega4 program (http://www.megasoftware.net/)
and was unrooted. Numerals at branching nodes indicate their percentages of appearance in 1000 bootstrap replicates.
40 C.-S. Ahn et al. / Parasitology International 64 (2015) 37–42
Size (bp)
CP1 756
CP2 767
CP3 741
CP4 625
CP5 687
CP6 588
CP7 528
CP8 515
CP9 691
CP10 759
CP11 783
β-actin 512
Taq (-)
cDNA (-)
Fig. 2. Differential transcription profile of PwCP genes during the developmental stages.
The cDNAs that were reverse transcribed from total RNAs (5 μg each) extracted from
each developmental stage were amplified with respective specific primers. The β-actin
gene was used for internal control. Size (bp) of the individual amplified products is indi-
cated. MC, metacercaria; 1-, 3- and 7-wk, 1-week, 3-week and 7-week-old-juvenile
worm, respectively; adult, 13-week-old adult worm. Taq (−), PCR was done without
Taq DNA polymerase (negative control). cDNA (−), PCR was done without respective
cDNAs (negative control).
SDS-PAGE analysis due to the His-tag sequence and matched well with
the deduced molecular weights of the respective proteins (Fig. 3A).
Each of the rPwCPs was electroblotted to PVDF membranes, after
which the membranes were cut into strips. Eleven pieces of blot
containing individual recombinant protein (rPwCP1-11) were simulta-
neously incubated with the patient sera of Paragonimus westermani,
other trematodiases, cestodiases and tuberculosis, together with those
of normal controls. The 11 species of P. westermani cathepsin F exhibited
relatively comparable immunoreactive patterns with minor differences,
except for PwCP2. Of the 138 paragonimiasis cases examined, 96–117
samples showed positive reactions (69.6–84.5%), while PwCP2 revealed
positive reactions only against 53 cases (38.4%) (Table 1). Representa-
tive immunoblot outcomes with PwCP7, which showed the highest di-
agnostic performance (sensitivity and specificity of 84.5% and 97.8%,
respectively, with positive predictive and negative predictive values of
96.7% and 89.3%, respectively), and that of PwCP2, which showed the
lowest immunoreactivity (sensitivity and specificity of 38.4% and
100%, respectively, with positive predictive and negative predictive
values of 100% and 67.9%, respectively) are shown in Fig. 3B. The rPwCPs
exhibited prominent antibody responses against IgG4 subclass (data not
shown). The rPwCPs demonstrated a relatively low-level of false posi-
tive reactions against 180 serum samples from other infections and
normal controls (specificity of 87.2%–100%). As shown in Table 1, pa-
tient sera of fascioliasis, schistosomiasis and clonorchiasis revealed
weak cross-reactions against several rPwCPs. Diagnostic parameters
are summarized in Table 2.
We observed the first appearance of specific antibodies against
respective recombinant proteins employing experimental cat sera
chronologically collected after infection with 50 metacercariae. As
shown in Fig. 3C and Table 2, rPwCP1, 3 and 7–11 reacted with these
sera as early as 3 weeks postinfection. Specific antibodies against
rPwCP4-6 appeared to be detected from 6 weeks after infection. Con-
versely, rPwCP2 did not show any detectable antibody responses until
13 weeks postinfection (part of data not shown).
4. Discussion
In the present study, we comparatively analyzed molecular structure
of the 11 species of PwCPs demonstrating characteristic features of
family C1 members within clan CA. Phylogenetic analysis indicated
that these molecules formed a monophylectic clade in trematode
cathepsin F. We also determined their prowess in diagnosing homolo-
gous infections, except for PwCP2 (AAF21461).
Individual PwCPs have been shown to be reliable serodiagnostic an-
tigens, while global comparison of immune reactivity of respective
PwCPs has not been addressed [8,11–13]. Most of the rPwCPs character-
ized in this study exhibited sensitive antibody responses as early as 3–6
weeks postinfection in experimental cat paragonimiasis, in addition to
their strong responsiveness against patient sera. This result implies
that these PwCPs might be able to diagnose early paragonimiasis even
before worm maturation in the hosts. In order to increase diagnostic
sensitivity, we mixed some rPwCPs and prepared an antigen cocktail.
However, the immunoblot outcome with this cocktail was similar to
that observed with single protein (data not shown). This result showed
that rPwCPs might bear the same/similar epitope in detecting specific
antibodies circulating in patient sera. We thought that paragonimiasis
caused by other species, such as P. heterotremus, P. miyazaki and
P. skrjabini may also exhibit similar diagnostic reliability with
these PwCPs. Our previous studies demonstrated that PwCP7 shows
highly sensitive and specific responses against patient sera of these
paragonimiases [8,19].
In this study, rPwCPs revealed some degree of cross-reaction with
patient sera of other trematodiases, such as fascioliasis, schistosomiasis
and clonorchiasis. However, clinical symptoms and signs are quite
different from one another in most clinical settings. Furthermore, con-
comitant imaging studies might contribute to the easy differentiation
of paragonimiasis from these infections [17,20]. However, antibody
responses against sera of other parasitic diseases, especially those of tis-
sue invasive nematodiases, such as gnathostomiasis, angiostrongyliasis,
strogyloidiasis and trichinellosis should be clarified to ensure diagnostic
applicability of PwCPs. These infections often coexist in tropical/
subtropical communities [21].
Abundant expression of diverse cysteine proteases is one of the
critical differences found in tissue invasive trematodes, such as
P. westermani and Fasciola hepatica, compared to the lumen dwellers
including Clonorchis sinensis and Opisthorchis viverrini [22,23]. Our ob-
servation demonstrates that diverse isoforms of cathepsin F, which
might be expressed as multiple paralogs/polymorphic alleles, are differ-
entially expressed during the maturation stage of the worm. When we
observed expressional regulation of PwCP transcripts following worm
maturation, two distinct patterns could be recognized. PwCP2-8 and
11 showed relatively upregulated expressions along with worm devel-
opment. This result suggested that the primary roles of these PwCPs
might be involved in nutrient digestion and remodeling of other physi-
ologically active molecules. On the other hand, expression of PwCP1, 9
and 10 appeared to be temporally augmented during which the parasite
penetrated the intestinal wall, and actively invaded and migrated
throughout the abdominal and thoracic cavities (1- to 7-week-old im-
mature stages). These enzymes might participate in metacercarial
excystment, tissue invasion/migration and immune evasion [9,10,24].
These collective data strongly suggest that PwCPs might exert different
or harmonized activity to ensure adaptation and survival within host
environments. Further studies are warranted to elucidate biological
 C.-S. Ahn et al. / Parasitology International 64 (2015) 37–42 41

CP10

CP11
CP-6
CP1
CP2
CP3
CP4

CP5

CP7
CP8
CP9
Mr
55
35
25

B Pw Cs Sj Fh CE TsM Sp Tbc Nor

abcdefghijk abc abc abc abc abc abc abc abc

PwCP2

PwCP7
C PwCP1 PwCP2 PwCP4

Post-infection (weeks)

Fig. 3. Expression and detection of specific antibodies against individual rPwCPs employing patient sera of homologue and heterologous infections, and pooled serum of experimental cat
paragonimiasis. (A) The mature domain of the respective PwCPs were expressed in E. coli BL21(DE3), purified using Ni-NTA column and monitored by 15% reducing SDS-PAGE with
Coomassie blue G-250 staining. (B) Antibody reactivity of rPwCP2 and PwCP7 against patient sera of Paragonimiasis westermani (lane Pw), clonorchiasis (Cs), Schistosomiasis japonicum
(Sj), fascioliasis (Fh), cystic echinococcosis (CE), cysticercosis (TsM), sparganosis (Sp) and pulmonary tuberculosis (Tbc), and sera from normal controls (Nor). Blots containing rPwCP2
and 7 were simultaneously incubated overnight with respective patient sera (1:200 dilutions), subsequently with HRP-conjugated anti-human IgG (1:1000 dilutions) for 4 h. Immuno-
reactive band was developed with 4C1N chromogen. (C) Antibody responses of rPwCP1, 2 and 4 against pooled cat serum (n = 4) collected chronologically after experimental infections,
which had been infected each with 50 metacercariae. Cat serum was diluted to 1:200 and HRP-conjugated anti-cat IgG was used in dilution of 1:1000. The blots were developed with 4C1N
chromogen.

reactivity and molecular mechanisms implicated in the specific expres-
sion of individual PwCPs.
P. westermani eggs harbored more than five species of cathepsin Fs.
This finding is difficult to properly explain. However, maturation of
egg including eggshell formation might be a complicated process,
which requires activation of many cofactors including proteolytic
enzymes as judged by expression profile of the PwCPs. In this respect,
cysteine proteases are critically involved in activation of phenol oxi-
dase/tyrosinase [25], which play pivotal roles in egg shell sclerotization
[26].
In this study, PwCP2 (AAF21461) was categorized in the same ca-
thepsin F clade, but showed minimal antibody reactivity against sera
from patients and cats infected with P. westermani. This molecule is
mainly localized in the vitelline gland of the adult worm [16]. RT-PCR

Table 1
Antibody reactivity of respective PwCPs against serum samples from paragonimiasis, other helminthic infections and normal controls.

Categories No. tested No. positive

CP1 CP2 CP3 CP4 CP5 CP6 CP7 CP8 CP9 CP10 CP11

Paragonimiasis 138 113 53 97 108 105 107 117 96 110 99 99

Clonorchiasis 35 5 0 4 2 7 2 0 0 5 1 4
Schistosomiasisa 15 4 0 2 2 4 1 0 0 2 0 3
Fascioliasis 30 10 0 5 2 9 3 2 0 1 2 8
Echinococcosisb 20 0 0 0 0 1 0 0 0 0 0 0
Cysticercosis 20 0 0 0 0 0 0 0 0 1 0 0
Sparganosis 20 2 0 0 0 2 0 2 0 0 0 0
Tbcc 20 0 0 0 0 0 0 0 0 0 0 0
Normal controls 20 0 0 0 0 0 0 0 0 0 0 0
a
Schistosoma japonicum infection.
b
Cystic echinococcosis.
c
Pulmonary tuberculosis.
42 C.-S. Ahn et al. / Parasitology International 64 (2015) 37–42

Table 2 [4] Barennes H, Slesak G, Buisson Y, Odermatt P. Paragonimiasis as an important alter-

Diagnostic sensitivity and specificity of the respective rPwCPs. native misdiagnosed disease for suspected acid-fast bacilli sputum smear-negative
tuberculosis. Am J Trop Med Hyg 2014;90:384–5.
Antigens % sensitivity % specificity PPV/NPVa Cat infectionb [5] Hanagiri T, Tsuda I, Tsukamoto T, Nagasako T, Kobayashi H, Hattori M, et al. Primary
lung cancer occurring concomitantly with the cicatrized and calcified ova of a
CP1 81.9 88.3 84.3/86.4 3 week parasite: report of a case. Surg Today 2001;31:443–5.
CP2 38.4 100 100/67.9 Not-detected [6] Cho SY, Lee DK, Kang SY, Kim SI. An epidemiological study of human paragonimiasis
CP3 70.3 93.9 89.8/80.8 3 week by means of micro-ELISA. Korean J Parasitol 1983;21:246–56.
CP4 78.1 96.7 94.7/85.3 6 week [7] Kong Y, Ito A, Yang HJ, Chung YB, Kasuya S, Kobayashi M, et al. Immunoglobulin G
CP5 75.8 87.2 82/82.6 6 week (IgG) subclass and IgE responses in human paragonimiases caused by three different
CP6 77.5 96.7 94.7/84.9 6 week species. Clin Diagn Lab Immunol 1998;5:474–8.
CP7 84.5 97.8 96.7/89.3 3 week [8] Yun DH, Chung JY, Chung YB, Bahk YY, Kang SY, Kong Y, et al. Structural and immu-
CP8 69.6 100 100/81.1 3 week nological characteristics of a 28-kilodalton cruzipain-like cysteine protease of
CP9 79.7 95 85.9/85.9 3 week Paragonimus westermani expressed in the definitive host stage. Clin Diagn Lab
Immunol 2000;7:932–9.
CP10 71.5 98.3 97.1/81.9 3 week
[9] Shin MH, Seoh JY, Park HY, Kita H. Excretory–secretory products secreted by
CP11 71.5 91.7 86.8/80.9 3 week
Paragonimus westermani delay the spontaneous cell death of human eosinophils
a through autocrine production of GM-CSF. Int Arch Allergy Immunol 2003;132:
Positive predictive value/negative predictive value
b
First appearance of specific antibodies against respective PwCPs in experimental 48–57.
cat paragonimiasis, which had each been infected with 50 metacercariae. [10] Na BK, Kim SH, Lee EG, Kim TS, Bae YA, Kang I, et al. Critical roles for excretory–
secretory cysteine proteases during tissue invasion of Paragonimus westermani
newly excysted metacercariae. Cell Microbiol 2006;8:1034–46.
[11] Ikeda T. Antibody responses to fluke cysteine proteinases in Paragonimus- and
data demonstrated that transcription levels of this gene were rapidly in- Fasciola-infected rats. J Helminthol 1998;72:187–91.
creased in later stages of worm development, coincident with active [12] Ikeda T. Protein A, immunocapture assay detecting antibodies to fluke cysteine
production of eggs (Fig. 2). These results suggest that the primary role proteinases for immunodiagnosis of human paragonimiasis and fascioliasis. J
Helminthol 2001;75:245–9.
of PwCP2 might be confined to protein remodeling during growth and [13] Lee EG, Na BK, Bae YA, Kim SH, Je EY, Ju JW, et al. Identification of immunodominant
development of vitelline follicles (vitellogenesis), rather than secretion excretory–secretory cysteine proteases of adult Paragonimus westermani by prote-
and stimulation of host immune system to produce specific antibodies. ome analysis. Proteomics 2006;6:1290–300.
[14] Kang SY, Cho MS, Chung YB, Kong Y, Cho SY. A cysteine protease of Paragonimus
Future studies are needed to address this intriguing issue.
westermani eggs. Korean J Parasitol 1995;33:323–30.
In conclusion, serological tests are important for the diagnosis of pul- [15] Chung YB, Kong Y, Yang HJ, Kang SY, Cho SY. Cysteine protease activities during
monary and extrapulmonary paragonimiasis, and also for differential maturation stages of Paragonimus westermani. J Parasitol 1997;83:902–7.
[16] Park H, Hong KM, Sakanari JA, Choi JH, Park SK, Kim KY, et al. Paragonimus
diagnosis from tuberculosis and cystic/nodular lesions caused by other
westermani: cloning of a cathepsin F-like cysteine proteinase from the adult
conditions [4,5]. Our result clearly indicates that P. westermani cathepsin worm. Exp Parasitol 2001;98:223–7.
F family members demonstrate excellent performance in diagnosing [17] Im JG, Kong Y, Shin YM, Yang SO, Song JG, Han MC, et al. Pulmonary paragonimiasis:
paragonimiasis. Especially, these molecules might be applicable in clinical and experimental studies. RadioGraphics 1993;13:575–86.
[18] Kong Y, Chung YJ, Yun DH, Kim LS, Kang SY, Ito A, et al. Variation of antigenic
serodiagnosis of early paragonimiasis in endemic areas. proteins of eggs and developmental stages of Paragonimus westermani. Korean J
Parasitol 1997;35:197–202.
Acknowledgment [19] Intapan PM, Sanpool O, Janwan P, Laummaunwai P, Morakote N, Kong Y, et al. Eval-
uation of IgG4 subclass antibody detection by peptide-based ELISA for the diagnosis
of human paragonimiasis heterotrema. Korean J Parasitol 2013;51:763–6.
This work was supported by a grant from the National Research [20] Henry TS, Lane MA, Weil GJ, Bailey TC, Bhalla S. Chest CT features of North American
Foundation (2012 0057 08). paragonimiasis. Am J Roentgenol 2012;198:1076–83.
[21] Murrell KD, Fried B. World Class Parasites (Volume 11): Food-borne parasitic zoonoses
(fish and plant-borne parasites). New York: Springer; 2007.
Appendix A. Supplementary data [22] Pinlaor P, Kaewpitoon N, Laha T, Sripa B, Kaewkes S, Morales ME, et al. Cathepsin F
cysteine protease of the human liver fluke, Opisthorchis viverrini. PLoS Negl Trop Dis
2009;3:e398.
Supplementary data to this article can be found online at http://dx. [23] Bae YA, Ahn DW, Lee EG, Kim SH, Cai GB, Kang I, et al. Differential activation of
doi.org/10.1016/j.parint.2014.09.012. diverse glutathione transferases of Clonorchis sinensis in response to the host bile
and oxidative stressors. PLoS Negl Trop Dis 2013;7:e2211.
[24] Chung YB, Kong Y, Joo IJ, Cho SY, Kang SY. Excystment of Paragonimus westermani
References
metacercariae by endogenous cysteine protease. J Parasitol 1995;81:137–42.
[25] Satoh D, Horii A, Ochiai M, Ashida M. Prophenoloxidase-activating enzyme of the
[1] Nakamura-Uchiyama F, Mukae H, Nawa Y. Paragonimiasis: a Japanese perspective.
silkworm, Bombyx mori: purification, characterization and cDNA cloning. J Biol
Clin Chest Med 2002;23:409–20.
Chem 1999;274:7441–53.
[2] Blair D, Xu ZB, Agatsuma T. Paragonimiasis and the genus Paragonimus. Adv Parasitol
[26] Bae YA, Cai GB, Kim SH, Sohn WM, Kong Y. Expression pattern and substrate
1999;42:113–222.
specificity of Clonorchis sinensis tyrosinases. Int J Parasitol 2013;43:891–900.
[3] Keiser J, Utzinger J. Emerging foodborne trematodiasis. Emerg Infect Dis 2005;11:
1507–14.