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 Table of Contents  
ORIGINAL ARTICLE
Year : 2011  |  Volume : 1  |  Issue : 2  |  Page : 76-82  

Cloning and sequence analysis of partial genomic DNA coding for HtrA-type serine protease of Wolbachia from human lymphatic filarial parasite, Wuchereria bancrofti


1 Microbiology and Molecular Biology Division, Vector Control Research Centre (ICMR), Puducherry, India
2 National Institute of Malaria Research (ICMR), Ranchi, India

Date of Web Publication31-Oct-2011

Correspondence Address:
S L Hoti
Vector Control Research Centre (ICMR), Medical Complex, Indira Nagar, Puducherry - 605 006
India
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DOI: 10.4103/2229-5070.86935

PMID: 23508470

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   Abstract 

Background: Periplasmic serine proteases of HtrA type of Wolbachia have been shown to play a role in the pathogenesis of filarial disease. Aims: This study was aimed to sequence Wb-HtrA serine protease and analyze its phylogenetic position by comparing with other filarial and non-filarial nematode homologs. Materials and Methods: Partial HtrA gene fragment was amplified from DNA isolated from periodic and sub-periodic Wuchereria bancrofti parasites collected from Pondicherry and Nicobar islands, respectively. The amplicons were sequenced, and sequence homology and phylogenetic relationship with other filarial and non-filarial nematodes were analyzed. Results: Partial orthologue of HtrA-type serine protease from Wolbachia of W. bancrofti was amplified, cloned and sequenced. The deduced amino acid sequence exhibited 87%, 81% and 74% identity with the homologous Wolbachia proteases identified from Brugia malayi, Onchocerca volvulus and Drosophila melanogaster, respectively. The Wb-HtrA has arthologues in several proteobacteria with very high homology and hence is highly conserved not only among Wolbachia of filarial parasites but also across proteobacteria. The phylogenetic tree constructed using Neighbor-Joining method showed two main clusters: cluster-I containing bacteria that dwell in diverse habitats such as soil, fresh and marine waters and plants and cluster-II comprising Anaplasma sp. and Erlichia, and Wolbachia endosymbionts of insects and nematodes, in distinct groups. Conclusions: HtrA-type serine protease from Wolbachia of W. bancrofti is highly conserved among filarial parasites. It will be of interest to know whether filarial Wolbachia HtrA type of serine protease might influence apoptosis and lymphatic epithelium, thereby playing a role in the filarial pathogenesis. Such information will be useful for identifying targets for the development of newer drugs for filariasis treatment, especially for preventing lymphatic pathology.

Keywords: HtrA serine protease, lymphatic filariasis, Wolbachia, Wuchereria bancrofti


How to cite this article:
Dhamodharan R, Hoti S L, Sivapragasam G, Das M K. Cloning and sequence analysis of partial genomic DNA coding for HtrA-type serine protease of Wolbachia from human lymphatic filarial parasite, Wuchereria bancrofti. Trop Parasitol 2011;1:76-82

How to cite this URL:
Dhamodharan R, Hoti S L, Sivapragasam G, Das M K. Cloning and sequence analysis of partial genomic DNA coding for HtrA-type serine protease of Wolbachia from human lymphatic filarial parasite, Wuchereria bancrofti. Trop Parasitol [serial online] 2011 [cited 2019 Jul 18];1:76-82. Available from: http://www.tropicalparasitology.org/text.asp?2011/1/2/76/86935


   Introduction Top


Global program for elimination of lymphatic filariasis (LF) has been launched in which a single dose of combination of diethylcarbamazine (DEC) and albendazole is administered annually to interrupt transmission of LF infection. However, DEC is only microfilaricidal and partially macrofilaricidal, thus leaving out some of the adult worms which might lead to persistent transmission. Hence, there is a need felt for drugs that can kill adult parasites in order to achieve the interruption of transmission of infection in the ongoing LF elimination program. Further, chemotherapy tools are also required for preventing the morbidity/management of lymphedema. Also, there is a risk of development of drug resistance against Albendazole, [1],[2] which is being co-administered with DEC in the LF elimination program.

Recently, the endosymbiont of filarial parasites, Wolbachia sp., has been found to be a target for anti-filarial chemotherapy and tetracycline class of compounds such as Doxycycline is being advocated for the elimination of filarial infection. [3],[4],[5],[6],[7],[8],[9] Wolbachia pipientis is a maternally transmitted bacterial endosymbiont and is associated both with filarial nematodes and their arthropod vectors. [10] They were discovered first in female filarial parasites. [11] Most of the filarial parasites, including human filarial parasites such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus are infected with the intracellular symbiont, Wo. pipientis. The Wo. pipientis of filarial nematodes appears to have evolved a mutualistic association with its hosts. [5],[12] Its presence in bancroftian filarial parasites in India has been found to be ubiquitous. [13]

Wolbachia endosymbionts of filarial parasites are targeted because of the fact that they play important roles in the development and biology of filarial parasites. [14] Evasion of immune-mediated damage by the help of Wo. pipientis also enables long-term survival of filarial nematodes in the mammalian host. Antibiotic reduction of bacteria showed that they are required for normal fertility and development of the parasite and even for the protection of the parasites from host immunity. [15],[16] Some molecules exported from Wo. pipientis of living filarial parasites are responsible for filarial disease pathogenesis. [17],[18],[19],[20] Thus, the role of Wo. pipientis in pathogenesis of filarial disease offers a new target for chemotherapy. [3],[4],[5],[6],[7],[9],[21] Several proteins have been identified from endosymbiont bacteria of filarial parasites, such as Wolbachia surface protein (WSP), Aspartate aminotransferase, Cell cycle protein (ftsZ), Heat shock protein 60 (Hsp 60), etc., and characterized.

Periplasmic serine proteases of HtrA type, also known as DegP and probably identical to "Do protease", [22] are involved in the complex regulation of signal transduction system and cellular physiology. It was first reported in  Escherichia More Details coli.[23],[24] Since then, such proteases have been found in a wide range of bacteria, including Wolbachia sp. and eukaryotes, and are found to be essential for virulence and survival under environmental stress conditions. It is suggested that HtrA is essential for mycobacterial persistence in the host. [25] Two null mutants of this enzyme - one associated with thermosensitivity [26] and another with decreased degradation of abnormal periplasmic proteins - have been reported, [24] indicating that the main role of HtrA is the degradation of abnormal proteins. HtrA of Wolbachia endosymbionts of insects such as Drosophila melanogaster has been studied extensively. [27] Similarly, HtrA of Wo. pipientis from O. volvulus has been characterized recently. [28] These studies have shown that the full-length cDNA of HtrA encodes a 494 amino acid long protein with a calculated mass of 54 kDa. HtrA is exported into the filarial host cells and functionally identified as a chaperone. It is characterized by a catalytic triad of serine proteases with one or more C-terminal PDZ domains and a signal peptide. No such studies on the HtrA type of serine protease of Wolbachia of human lymphatic filarial parasite, Wu. bancrofti, are available till date. The aim of the present study was to isolate the gene/gene fragment coding for HtrA from the endosymbiont of Wu. bancrofti, characterize it structurally, investigate its polymorphism and infer its phylogeny.


   Materials and Methods Top


Parasite materials

Blood samples were drawn from known carriers of microfilaria of Wu. bancrofti, residing in Pondicherry, between 19:00 hours and 23:00 hours and Microfilariae (Mf) were purified by membrane filtration. [29] Mf of sub-periodic form of Wu. bancrofti, isolated from blood smear on slides collected from carriers living in Car Nicobar islands, were purified by a method developed by Bisht et al. [30] Informed consent was obtained from all the carriers before the collection of blood samples.

DNA isolation and amplification of HtrA serine protease

DNA from Mf of Wu. bancrofti was extracted using DNA tissue kit (Qiagen, Germany), following the protocol provided for micro-tissue DNA extraction by the manufacturer. The final pellet of DNA was dissolved in 1× TE buffer (pH 8.0) and stored at −20°C until use. The homolog of HtrA gene fragment from Wu. bancrofti was amplified from genomic DNA using gene specific primers (WBF: 5′-ATGAAAAGTAAGGTTTTATCTAT-3′and WBR 5′-TAAACCAAATGGGTTACCTATTGC-3′) designed based on serine protease from Wolbachia of D. melanogaster. [28] The polymerase chain reaction (PCR) was performed in a gradient thermocycler in a total reaction volume of 30 μl containing 3 μl of 10× PCR buffer [750 mM Tris-HCl (pH 8.8), 200 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20], 1 mM MgCl 2 , 200 μM of each of dNTPs, 1 U of Taq DNA polymerase (Finzyme), and 100 pmol of each primer and 3 μl of DNA template. The reaction condition followed was: initial denaturation of 94°C for 5 min followed by 35 cycles of initial denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and a final extension at 72°C for 10 min.

Cloning and sequencing of HtrA

The gel purified amplicons were ligated into pGEM-T easy vector (Promega, USA) and transformed into E. coli (JM109) host cells, following the manufacturer's instructions. Plasmid DNA was isolated from recombinant colonies selected on LB plates supplemented with Ampicillin, using plasmid miniprep kit (Qiagen, Germany). The presence of insert was checked by restriction digestion and gene specific PCR amplification. The clones with inserts were sequenced using Big Dye Terminator V3.1 ready reaction sequencing mixture and analyzed in an automated 3130XL Genetic Analyzer (Applied Biosystem, Foster City, CA, USA).

Sequence analysis

The sequencing outputs were edited by using Chromas (http://www.technelysium.com.au/chromas.html) and BioEdit programs to remove vector sequences and ambiguous regions. The nucleotide sequences obtained were checked by the BLAST search in the National Center for Biotechnology Information (NCBI) site for determining similarities with previously reported sequences. The amino acid sequence of the cloned fragment of Wolbachia of Wu. bancrofti was deduced using Sequence Manipulation Suite (http://www.bioinformatics.org/sms2) and compared with the HtrA orthologous sequences of Wo. pipientis of other filarial parasites, insects and bacteria obtained from GenBank database. These sequences were aligned by using ClustalW and the annotations of possible protein coding region and domains were identified using swissprot database.

Phylogenetic analysis

Wolbachia serine protease (HtrA type) sequences retrieved as above were subjected to multiple sequence alignment using ClustalW. [31] Analysis of multiple sequence alignment of the members of the HtrA family of proteins was performed using ClustalW tool in BioEdit. The sequence similarity among HtrA sequences was calculated and Neighborhood-Joining (NJ) phylogenetic tree with bootstrap (1000 replicates) was constructed using MEGA software V4.0. [32]


   Results Top


The PCR amplification using serine protease specific primers yielded a ~924 bp fragment of the 1.5 kb ORF of the HtrA [Figure 1]a. The purified amplicons were cloned into a sequencing vector (p-GEMT easy, Promega, USA) and upon checking by colony PCR and restriction digestion with EcoRI, some of them were found to have 924 bp inserts [Figure 1]b and [Figure 2]. The inserts from the recombinant clones were sequenced and the nucleotide sequence of the gene fragment, spanning 187 to 497 amino acid residues, was found to match with the known serine protease sequences of other α-proteobacteria available in GenBank, upon Blast search, thus confirming its identity. The Wu. bancrofti Wolbachia serine protease is AT rich (66.07% AT and 33.93% GC). The deduced amino acid sequence resulted in a protein of 297 amino acids, which contained a trypsin domain and two PDZ domains [Figure 2]. However, the fragment has two amino acid residues missing (histidine at aa position 107 and asparatic acid at aa position 137) of the catalytic triad, mainly because the forward primer was designed to locate at aa position between 187 and 497. The trypsin domain has catalytic serine (of the triad) in the CNSGGP motif, which was found to be highly conserved upon comparing with the already characterized Wolbachia serine proteases of other organisms [Figure 3]. The gene fragment has no introns, thus confirming its bacterial origin.
Figure 1: PCR amplicons of HtrA from Wu. bancrofti genomic DNA: (a) 1-100 bp ladder; 2-6 genomic DNA; 7 negative control. Colony PCR with gene specific PCR showing amplicons (b) and ECoRI digestion of plasmids (c) showing the inserts (~900 bp). A-E, 2-3, 6-9 are positive clones (white colonies); 4-5 negative control (blue colonies); 1 kit control

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Figure 2: Nucleotide sequence and deduced amino acid sequence of Wb-Wolbachia HtrA-type serine protease. The deduced amino acid sequence is shown below the nucleotide sequence using single letter code. The trypsin domain (dark shade) and PDZ domains (light shades) of serine protease are marked sequentially. The HtrA-type serine protease specific motif (GNSGGP) is marked in bold

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Figure 3: Multiple sequence alignment of the amino acid sequence of Wb-Wolbachia serine protease (HtrA type) performed using ClustalW. Conserved serine protease specific motif (GNSGGP) is underlined. Variable amino acid residues are shown in bold. BM, Brugia malayi; OV, Onchocerca volvulus; DM, Drosophila melanogaster; MU, Muscidifurax uniraptor; CQ, Culex quinquefasciatus and WP, Wolbachia pipientis

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The genomic DNA encoding Wb-Wolbachia HtrA was compared with the previously published sequences of HtrA from Wolbachia of other parasites and insects (data not shown). The identity of this gene fragment was almost complete with that of Wolbachia of B. malayi (98%), while it was slightly less (97%) with that of O. volvulus. These results show that the gene is highly conserved among filarial parasites. The identity of the gene with that of Wolbachia of insects was also significantly high (88% with D. melanogastor and 82% with Culex quinquefasciatus) compared with other endosymbiotic bacteria (68%), thus indicating that it is generally conserved across α-proteobacterial group.

The phylogenetic tree was constructed using NJ and Maximum Likelihood methods employing Mega V4.0 program. [32] The two trees were congruent (data not shown), and hence, the NJ tree constructed was robust. The topology of the NJ tree showed two main clusters [Figure 4]. Cluster I contained bacteria that dwell in diverse habitats such as soil, fresh and marine waters and plants, with most of them being α-proteobacteria. Rizobium legumnosarum, the endosymbiont of leguminous plants, and Nitrosococcus oceani, a marine nitrogen fixing chemilithoautotroph, fell under this cluster. The latter bacterium formed the most recent common ancestor (MRCA) of the tree. Cluster-II comprised two sub-clusters: sub-cluster A having Anaplasma sp. and sub-cluster B having Erlichia and Wolbachia endosymbionts of insects and nematodes, each in turn forming distinct groups. The Wolbachia sub-cluster branched into a nematode Wolbachia group and an insect Wolbachia group with the former group branching early. Almost all the divisions at different levels were supported by good bootstrap values (>60).
Figure 4: Neighbor-joining (NJ) phylogenetic tree relating the Wb-Wolbachia HtrA-type serine protease with other selected serine proteases from Wolbachia of insects and parasites. The number near node represents bootstrap values and the different groups of bacterial source are differentiated with color shades

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   Discussion Top


In the present study, a partial fragment of HtrA of Wolbachia from Wu. bancrofti, the major lymphatic filarial parasite, was isolated and its phylogeny was inferred. This is the first report of HtrA from lymphatic filarial parasite, Wu. bancrofti, although partial. The derived partial protein sequence showed the presence of trypsin domain and two PDZ domains, characteristic of perisplasmic serine proteases. Since the N-terminal fragment was not obtained due to PCR primer bias in the partial protein, the information on the leader sequence and two amino acid residues, viz., histidine and aspartic acid of the catalytic triad, were missing. Upon search in the GenBank using the protein sequence of the partial gene as query, orthologues of this gene were found in several proteobacteria having high homology. Nucleotide and amino acid alignment of the HtrA showed that the gene is highly conserved among filarial parasites. Genomic sequences of HtrA were analyzed from Wolbachia of periodic and sub-periodic Wu. bancrofti populations through cloning the gene fragments and sequencing several clones. The nucleotide sequences of inserts of all these clones were identical (data not shown). This is also reflected in the striking similarity of the HtrA of Wolbachia of B. malayi and O. volvulus, which exhibited 96% and 93% match. Thus, the gene is not only highly conserved among Wolbachia filarial parasites but also across proteobacteria.

Wolbachia
species belong to the order Rickettsiales, under the subdivision of proteobacteria [33] and are closely related to the genera Ehrlichia, Cowdria and Anaplasma. [6] The genus of Wolbachia of filarial nematode and arthropods is identified based on DNA sequence data, [34],[35],[36] and sub-grouping of Wolbachia is ongoing. [37] In filarial nematodes, the phylogeny of Wolbachia matches with that of the nematode host phylogeny. [12] Earlier, studies on the phylogenetic analyses using three different Wolbachia genes (ftsZ, wsp, and 16S rDNA) generally resulted in congruent trees. [38],[39] The phylogenetic tree constructed using HtrA-type serine protease of filarial parasites also resulted in a similar tree, indicating that genes are evolving in parallel. The variation in the amino acid residues of HtrA proteins resulted in a tree that differentiated wolbachians from rest of the proteobacteria. Interestingly, in the cluster that comprised Wolbachia of nematodes as well as insects, the former branched earlier, suggesting a possibility that the Wolbachia of nematode parasites might have crossed over to insects, many of them being their vectors.

As stated earlier, Wolbachia plays an important role in the biology of the nematode hosts and in the immunopathology of filariasis in humans. [6],[7],[14],[15],[17],[20],[40] Studies on animal models of filarial parasites have shown that antibiotic targeting of Wolbachia with tetracyclines have profound effects on the development, viability, and fertility of filarial parasites, [41] and elimination of Wolbachia from worm tissues causes reproductive abnormalities in worms and affects worm's embryogenesis, resulting in sterility. [8],[21],[42],[43],[44],[ 45] Thus, filarial nematodes are essentially dependent on Wolbachia endosymbionts for their larval development and adult-worm fertility, viability and overall survival. The endosymbionts are hence focused rightly as potential targets for chemotherapy with antibiotics. Recently, Taylor et al. [46] reported complete elimination of Mf within 8-14 months following treatment with Doxycycline. Among the group of tetracycline antibiotics, Rifampicin has been shown to be more active in reducing worm motility, viability, and clearance of Mf. [31]

The roles played by Wolbachia in the pathogenesis of filariasis include: a) recognition by the host's immune system and induction of antibody responses, b) inducing inflammatory responses of the innate immune system, c) eliciting severe/systemic inflammatory responses mediated by their endotoxin after treatment with ivermectin or Diethylcarbamazine, [6],[18],[20],[40] d) inducing anti-inflammatory responses following inflammatory responses and represent major triggers for neutrophilic granulocytes. While interruption of transmission is the main objective of the LF elimination program launched globally, management of morbidity is another pillar of the program. As stated above, Wolbachia plays a very important role in the pathogenesis mediated through soluble extracts such as lipopolysaccharide (LPS) antigens. These antigens induce potent inflammatory responses, including tumor necrosis factor (TNF)-, interleukin (IL) 1-γ and nitric oxide (NO) from macrophages, through the Toll-like receptor 4 (TLR4) signaling.[6],[47] It may be possible that the filarial pathology contributed by Wolbachia could be reduced or prevented by targeting the endosymbiont. [15] In order to verify this, extensive studies on the interaction of molecules of Wolbachia such as HtrA with the host need to be carried out. HtrA-type serine protease of Wolbachia from filarial parasite is an exported protein, which will eventually be exposed to human host when the filarial parasites die naturally, or as a consequence of host defense mechanism [28] or due to anti-filarial chemotherapy. In this context, it should be noted that IgG1 antibodies reactive against serine protease of Wolbachia were detected in the sera of O. volvulus infected patients. [28] Some HtrA proteins have also been shown to influence apoptosis, which plays a major role in tissue development and homeostasis of nematodes [48],[49] and adherence of Camplylobacterium jejuni to the human epithelial cells. [50] In view of these functions of HtrA, it will be of interest to know whether filarial Wolbachia serine protease might also influence apoptosis and lymphatic epithelium, thereby influencing the filarial pathogenesis. Such studies will pave way for identifying targets for the development of newer drugs for filariasis treatment, especially for preventing lymphatic pathology.

 
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