ISSN (Print) 0023-4001
ISSN (Online) 1738-0006
Korean J Parasitol Vol. 54, No. 6: 743-750, December 2016
https://doi.org/10.3347/kjp.2016.54.6.743
▣ ORIGINAL ARTICLE
Molecular Characterization of Nippostrongylus brasiliensis
(Nematoda: Heligmosomatidae) from Mus musculus in
India
Anshu Chaudhary, Urvashi Goswami*, Hridaya Shanker Singh
1
Molecular Taxonomy Laboratory, Department of Zoology, Chaudhary Charan Singh University, Meerut (U.P.), 250004, India
Abstract: Mus musculus (Rodentia: Muridae) has generally been infected with a rodent hookworm Nippostrongylus
brasiliensis. In this report, we present morphological and molecular identification of N. brasiliensis by light and scanning
electron microscopy and PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the protein
sequences encoded by cox1 gene, respectively. Despite the use of N. brasiliensis in many biochemistry studies from India, their taxonomic identification was not fully understood, especially at the species level, and no molecular data is available in GenBank from India. Sequence analysis of cox1 gene in this study revealed that the present specimen showed
close identity with the same species available in GenBank, confirming that the species is N. brasiliensis. This study represents the first record of molecular identification of N. brasiliensis from India and the protein structure to better understand
the comparative phylogenetic characteristics.
Key words: Nippostrongylus brasiliensis, Mus musculus, nematode, Meerut, Uttar Pradesh, mitochondrial cox1, protein
phosphorylation. Little information is available regarding parasitic nematodes protein structures and their comparison with
isolates of same or closely related species.
During a general survey of the nematode fauna of Mus musculus in the Meerut, U.P., India, several nematodes belonging
to Nippostrongylus were collected from the gastrointestinal tract.
Their examination using light and scanning electron microscopy revealed that these parasites represented the species N.
brasiliensis. Moreover, the specimens were also characterized by
using molecular approaches. The mitochondrial cox1 gene was
sequenced and analyzed in order to molecularly identify and
estimate the validity of N. brasiliensis from Indian region. We
also summarized the identification and prediction of cox1 protein structures with comparison of isolates for taxonomic
identification with a special focus on the structural aspects
through bioinformatics approach.
INTRODUCTION
Nippostrongylus Lane, 1923, a heligmonellid genus, is commonly parasitic in the digestive tract of murines. Geographically, Nippostrongylus has been known for a wide range throughout
the world [1-5]. Nippostrongylus brasiliensis Travassos, 1914, a
gastrointestinal nematode, is a cosmopolitan parasite of a commensal mouse Mus musculus. Our present knowledge of identification of N. brasiliensis nematode is still fragmented in India.
Till date, no molecular studies have been reported in India. Recently, mitochondrial genes have been successfully employed
as a molecular marker for accurate identification of nematodes
[6-8]. The mitochondrial cytochrome c oxidase subunit 1 (cox1)
gene has been widely used for identification and phylogenetic
studies, and enabled the discrimination of closely related species in nematode phyla [7-9]. The mitochondrial cox1 gene
(also known as mtCO1) is a key enzyme of aerobic metabolism, which is located in the inner mitochondrial membrane
and a major site for regulation of mitochondrial oxidative
MATERIALS AND METHODS
A total of 20 M. musculus caught from Meerut (29°01' N,
77°45' E), U.P., India were examined for parasitic infections
after dissection under chloroform or ether anesthesia. Their
gastrointestinal tracts were removed and examined under a
stereomicroscope. Total 20 male and 5 female nematodes were
recovered from the intestine of M. musculus. They were washed
• Received 28 June 2016, revised 24 August 2016, accepted 26 September 2016.
* Corresponding author (goswamiurvashi12@gmail.com)
© 2016, Korean Society for Parasitology and Tropical Medicine
This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
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Korean J Parasitol Vol. 54, No. 6: 743-750, December 2016
Table 1. Morphometric data of N. brasiliensis parasitising Mus musculus
Body length
Male
Female
Body width
Buccal cavity
length
Buccal cavity
width
Esophagus
length
Spicule
length
4.11 (4.05-4.16) 2.88 (2.85-2.90) 0.15 (0.12-0.18) 0.6 (0.4-0.9) 1.20 (1.0-1.5) 0.54 (0.53-0.55)
5.44 (5.30-5.60) 0.11 (0.10-0.12) 0.11 (0.10-0.12) 0.4 (0.3-0.6) 1.74 (1.6-1.9)
-
Anus length from
posterior end
Tail
length
0.74 (0.60-0.90) 0.52 (0.40-0.60)
All measurements are in millimeter (mm).
in saline (0.6%) and then fixed in 70% ethanol and stored until studied. For the light microscopy study, the nematodes were
mounted in glycerin. A light microscope (Motic SMZ-168, Xiamen, China) equipped with digital image analysis system
(Motic Image Plus 2.0 for Windows) and drawing attachment
was used for line drawings and morphometric analysis. For
scanning electron microscopic studies, parasites were fixed in
70% ethanol, dried by critical point-drier, mounted on SEM
studs, and finally coated with a thin layer of gold before being
examined with a JOEL Neoscope JCM5000 SEM (Nikon Instruments, Melville, New York, USA) at an accelerating voltage of
10 kV. Prepared slides of male and female N. brasiliensis were
deposited in the Museum, Department of Zoology, Chaudhary
Charan Singh University, Meerut, (U.P.), India, under the
voucher no. Nem/2015/01. Measurements are given in Table 1.
For molecular analysis, genomic DNA was extracted using a
DNeasyTM Tissue Kit (Qiagen, Hilden, Germany), according to
the manufacturer’s instructions. Eluted DNA was kept at -20˚C
until further use. The partial mitochondrial cox1 gene was amplified by PCR using the primers LCO1490 (5´-GGTCAACAAATCATAAAGATATTGG-3´) and HC02198 (5´-TAAACT
TCAGGGTGACCAAAAAATCA-3´) [10] with cycling profile described previously [10]. PCR products were checked on ethidium bromide stained 1% TAE buffer gel and purified by the
PurelinkTM Quick Gel Extraction and PCR Purification Combo
kit (Invitrogen, Carlsbad, California, USA) following the manufacturer’s instruction. Sequencing was carried out by the same
primers using an ABI Big Dye Terminator version 3.1 cycle sequencing kit with an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, California, USA). Sequences were aligned
using Clustal W [11] and manually adjusted. Using the BLASTn
algorithm, the obtained sequence was compared with those
available in the NCBI database (National Centre for Biotechnology Information; http://www.ncbi.nlm.nih.gov).
The phylogenetic tree was built using the maximum likelihood (ML) and Bayesian inference (BI) analyses. DNA pairwise distances were calculated using the Kimura 2 parameter
model with the MEGA 6 software [12]. For ML analysis, GTR +
G + I model was chosen based on the best fitting substitution
model using the Akaike Information Criterion in MEGA 6
[12]. The tree topology was tested by using bootstrapping over
1,000 replications. TOPALi 2.5 [13] was used to construct the
tree for BI analysis. For BI analysis, substitution model was
tested by the Bayesian Information Criterion and GTR+I+G
was chosen. BI analysis was run for 1,000,000 generations,
sampling every 100th tree and discarding ‘burn in’ first 25% of
the sampled tree. Oesophagostomum columbianum (KC715827)
was used as an outgroup for analysis.
For study of protein sequence, a primary sequence analysis
of the N. brasiliensis isolates was performed using the ProtParam [14]. The cox1 protein secondary structure analysis of
the N. brasiliensis isolates was obtained using the program
SOPMA [15]. The cox1 protein sequence of N. brasiliensis and
most related isolate sequence alignment were generated by ESPript 3.0 [16]. To carry out the cox1 homology search for N.
brasiliensis against Protein Data Bank (PDB) was performed by
using SWISS-MODEL [17]. The same model was employed to
generate the 3D structure of the N. brasiliensis for cox1. The
model with high score was validated by the Phyre 2 [18] and ITESSER [19]. The model was refined by energy minimization
using the NAMD package [20] and subjected to quality evaluation. MEMSAT-SVM and MEMPACK in PSIPRED workbench
[21] were used for the prediction of transmembrane helices
and topology of N. brasiliensis protein sequence. RAMPAGE
[22] was used for quantitative protein structure evaluation of
N. Brasiliensis, and the Ramachandran plot was utilized for
geometric assessment. To evaluate the quality of the model
and study the energy of residue–residue interactions using a
distance-based pair potential ProSA program [23] was employed. TM-align [24] was used for the superimposition between Indian and USA isolates of N. brasiliensis protein sequences for comparison.
RESULTS
The male and female nematodes collected in this study were
Chaudhary et al.: Molecular characterization of N. brasiliensis in India
diagnosed on the basis of morphological characteristics and
preliminarily identified as N. brasiliensis. The male’s bursa with
asymmetrical lateral and small dorsal lobes, each branch ending in out of the 3 tips, spicules almost equal, filiform and gubernaculum present (Fig. 1A). Female’s tail conical, pointed,
vulva present near anus, and oviparous (Fig. 1B). Measurements were taken and presented in the table (Table 1). SEM
images were also provided for the topological view of N. brasiliensis (Fig. 2).
The sequence obtained for the mitochondrial cox1 region was
705 bp in length and deposited in the GenBank database under
the accession no. KX146839. There was no N. brasiliensis sequence registered from India till date, thus, comparison was possible with 3 N. brasiliensis sequence (nos. U57035, AF096235,
and AF263480) available in GenBank. The available 3 isolates of
N. brasiliensis showed a pairwise comparison, 0.14% (U57035),
1.75% (AF096235), and 1.97% (AF263480) nucleotide difference with Indian isolate, respectively. Sequence analysis showed
that in all, an isolate from USA (no. U57035) was found the
closest to Indian isolate based on the cox1 sequence. Surprisingly,
phylogenetic analysis of data showed that 2 isolates of N. brasiliensis (nos. KX146839 and U57035) were located on the same
A
B
Fig. 1. Line drawings of N. brasiliensis. (A) Male. (B) Female.
Scale bars (A)= 0.4 mm; (B) 0.2 mm.
A
D
745
B
C
E
F
Fig. 2. Scanning electron microscopy of N. brasiliensis. (A, B) Anterior portion of male mouth. (C) Tail portion of a male with copulatory bursa. (D, E) Anterior portion of female. (F) Striations on the female body. Scale bars (A) and (D)= 10 µm; (B), (C) and (F)= 20 µm; (E) 2 µm.
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Korean J Parasitol Vol. 54, No. 6: 743-750, December 2016
Fig. 3. A phylogenetic tree is based on the mitochondrial cox1 sequence demonstrating the position of N. brasiliensis isolate with other
nematode species. The tree was generated by maximum likelihood (ML) method. Numbers of nodes indicate bootstrap values (1,000
replications) of ML and posterior probabilities (BI), respectively. GenBank accession nos. are listed along the species names. Species examined in this study are indicated in bold.
Table 2. Results of primary sequence analysis of N. brasiliensis isolates of cox1 protein available in NCBI database from various geographical regions
N. brasiliensis
GenBank no.
Length
Molecular wt. (Da)
pI
-R
+R
E.C.
I.I.
A.I.
GRAVY
India
USA
Korea
UK
KX146839
U57035
AF096235
AF263480
235
215
147
131
25825.5
23423.8
16232.3
14289.1
6.37
6.17
7.18
7.01
10
8
6
5
8
6
6
5
44460
31970
28420
25440
27.52
31.47
33.24
31.51
107.83
115.16
124.56
131.60
-0.691
-0.830
-0.978
1.141
-R, negatively charged residues; +R, positively charged residues; E.C., extinction coefficients; I.I., instability index; A.I., aliphatic index; GRAVY, grand
average of hydropathicity.
clade, whereas, the other 2 from Korea and UK (nos. AF096235
and AF263480) formed a separate major clade (Fig. 3). This result demonstrated the paraphyly of Nippostrongylus as its isolates
were segregated into 2 lineages. According to the phylogenetic
tree, N. brasiliensis formed a clade along with Trichostrongylus and
Heligmosomoides as the sister group. In addition, all the species
that formed the tree belonged to the superfamily Trichostrongyloidea based on mitochondrial cox1 analysis (Fig. 3).
Further, we studied the cox1 protein sequence to predict the
3D structure and comparative modeling to show the well conserved structural elements. Results of primary and secondary
sequence analysis of cox1 protein sequences of N. brasiliensis
isolates available on the NCBI database from various geographical regions are shown in Tables 1 and 2. Sequence alignment and tertiary structure analysis of protein sequence of N.
brasiliensis Indian isolate showed the closest similarity with an
isolate from USA (Table 3), and template sample (PDB Id:
3abm) against PDB search was presented in Figs. 4 and 5. In
Fig. 4A, sequence alignment comparison of the isolate from
India and USA with the template sequence is shown. Fig. 4B
specifically represents the sequence comparison between Indian and USA isolates of N. brasiliensis with template sequence
along with the consensus sequence where except few sites, the
nucleotide sequence from Indian isolate showed the highest
similarity with USA isolate (Fig. 4C). Fig. 4D depicts about Indian and USA isolate with secondary structure elements on top
of the sequences as helices with squiggles, arrows present
β-strands, and TT letters show the turns. Below the sequence
solvent accessibility is provided by a bar (blue is accessible,
cyan is intermediate, white is buried) (Fig. 4D).
The resulting 3D model of the cox1 protein structure was
sorted according to the scores evaluated by protein energy, and
the validation of the model was checked by assessing the quality of protein backbone conformation by RAMPAGE for reli-
Chaudhary et al.: Molecular characterization of N. brasiliensis in India
A
B
C
D
747
Fig. 4. Alignment of the cox1 protein sequence. (A) N. brasiliensis from India and its isolates available on Genbank database from other
regions. (B) N. brasiliensis isolate from India and USA along with the template search against the PDB database. (C) N. brasiliensis isolate from India and USA and their consensus sequence (D) showing the N. brasiliensis isolate from India and USA with secondary structure elements presented on top.
Table 3. Results of secondary sequence analysis of cox1 data of
N. brasiliensis isolates available in the NCBI database from various geographical regions
N. brasiliensis
India
USA
Korea
UK
α-helix
Extended
strand (%)
β-turn
(%)
(%)
Random coil
(%)
32.77
37.67
31.29
35.88
27.66
26.51
35.37
37.40
14.47
11.16
10.88
6.87
25.11
24.65
22.45
19.85
ability. Rampage ϕ and ψ regions were used for validation of
experimental protein structures. The obtained Ramachandran
plot (Psi-Phi) pairs for Indian isolate had 96.6% of residues in
most favored regions, while 3.4% residues in generously allowed regions (Fig. 5) whereas for USA isolate was 96.2%. The
3D model of cox1 protein sequence of isolate from India and
USA along with sample template (PDB Id: 3abm) were built
(Fig. 6). The ProsA analysis showed the overall interaction energy of the model was -3.71 kcal/mol, which is relatively similar to the USA isolate Z score -3.61 kcal/mol. Hydropathy
analysis of cox1 protein sequence of isolate from India by
MEMSAT-SVM and MEMPACK suggested the presence of 5
transmembrane (TM) helix (Fig. 7). Comparative analysis of
cox1 protein structures of isolates from India and USA was performed using TM-align; it compared the 3D structures of pro-
Fig. 5. The Ramachandran plot of the structure of N. brasiliensis
isolate from India showing residue predicted by RAMPAGE in favored and allowed outer regions.
teins and computation structural alignments between 2 protein structures showing the structural similarity. Fig. 8 shows
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Korean J Parasitol Vol. 54, No. 6: 743-750, December 2016
A
B
A
C
B
D
E
Fig. 6. The 3D structure of cox1 showing homology in helices,
β-strands, and turns. (A) N. brasiliensis isolate from India. (B) N.
brasiliensis isolate from USA. (C) The template (PDB Id: 3abm). (D)
N. brasiliensis isolate from Korea. (E) N. brasiliensis isolate from UK.
Fig. 7. Predicted transmembrane regions of N. brasiliensis isolate
from India by (A) MEMSAT-SVM showing TM helices and loop regions and (B) helix orientation cartoon and the predicted helical
packing arrangement by MEMPACK, colors in the cartoon indicate hydrophobic residues (blue), polar residues (red), and
charged residues (green for negative, purple for positive). The
lines between residues indicated a predicted interaction.
DISCUSSION
Fig. 8. The superimposition of 3D model of cox1 protein of N.
brasiliensis isolate from India and USA. Indian isolate sequenced
shows in blue while the USA isolate in red. Superimposition clearly states the similarity between the 2 isolates.
the superimposition of Indian and USA isolates; the 2 isolates
were almost similar except few bases that might be due to different geographical regions. The results shown above gave sufficient information of structurally conserved regions in a predicted protein structure.
The present study adds to our knowledge and provides the
strong molecular based evidence for the existence of N. brasiliensis from India parasitizing M. musculus as a common parasite of rodents. Male and female parasites were identified
based on morphological diagnostic characteristics but molecular data supplemented more confirmation and validation to
the study. However, sometimes species identification based
only on morphology cannot be considered reliable especially
in the cases where species are morphologically indistinguishable. In India, little attention has been paid to molecular studies of nematodes relatively to morphology that represents an
obstacle in identification. We report for the first time the use
of mitochondrial cox1 gene for molecular identification of N.
brasiliensis from India. Mitochondrial DNA evolves very quickly in genomes that is why differences in very closely related
Chaudhary et al.: Molecular characterization of N. brasiliensis in India
species can be easily identified among closely related/within
species [25,26]. Phylogenetic analysis clearly stated that in all
isolates of N. brasiliensis available on the database had closest
homology with the isolate from USA if compared with isolates
from Korea and UK as they had very short cox1 protein sequences. All N. brasiliensis isolates did not form a single clade
and showed 2 lineages, this might be due to the paraphyly of
this genus as also reported earlier, so the results were consistent with these studies [27-29]. Mitochondrial cox1 proteins
have been relatively consistent functional and structural contexts over evolutionary time [30] and have been well conserved
in their structures as also proved in the results of this study for
determining specific relationships. The protein of isolates from
India and USA confirmed 5 transmembrane helixes at the
same position indicating structural similarity. The outcome of
RAMPAGE showed the reliability, and strong acceptance of the
predicted structures of both isolates confirmed their structural
similarities and the validation of Indian isolate.
In India, there is a need of attempts to integrate molecular
analyses as well as morphology that can provide more effective
means of characterization for nematodes. The present study
added important details and indicated that the analysis of protein structural components was an important technique for
obtaining the 3D structure that will help in studying phylogeny and identification of closely related isolates. The present research offered a backbone to understand the functional and
structural insight of cox1 protein of N. brasiliensis that can be
used for future molecular studies.
ACKNOWLEDGMENTS
The authors would like to thank the Head of the Department of Zoology, Chaudhary Charan Singh University, Meerut
(U.P.), India, for laboratory facilities. Special thanks to Prof.
Jerzy M. Behnke, School of Biology, University of Nottingham,
NG7 2RD, UK, for his help and providing literature for identification of parasite species (N. brasiliensis).
CONFLICT OF INTEREST
The authors declare that they have no conflict of interests.
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