Characterization of cofactor-independent phosphoglycerate mutase isoform-1 (Wb-iPGM) gene: A drug and diagnostic target from human lymphatic filarial parasite, Wuchereria bancrofti

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Abstract

The inter-conversion of 3-phosphoglycerate and 2-phosphoglycerate during glycolysis and gluconeogenesis in filarial nematodes, is catalyzed by a co-factor-independent phosphoglycerate mutase (iPGM). The gene encoding iPGM isoform-1 was amplified from Wuchereria bancrofti, the major causative agent of human lymphatic filariasis. Partial genomic DNA (gDNA) fragment of the gene was also amplified from periodic and sub-periodic forms of W. bancrofti and Brugia malayi and sequenced. The Wb-iPGM isoform-1 gene encodes an ORF of 515 amino acids and is found to share 99.4%, 96.0%, and 64.0% amino acid sequence identity with iPGM of B. malayi, Onchocerca volvulus, and Caenorhabditis elegans, respectively. Serine and all the other 13 amino acid residues involved in the catalytic function of iPGM are highly conserved. Further comparison of iPGM nucleotide and amino acid sequences of Wolbachia of B. malayi with Wb-iPGM showed 41% and 54.4% similarity, respectively. The analysis of partial genomic and amino acid sequences and phylogenetic tree of Wb-iPGM indicated that this gene, apart from being a potential drug target, could provide diagnostic, taxonomical, and evolutionary markers. This is the first report of the characterization of iPGM gene from W. bancrofti.

Highlights

► We characterized iPGM gene of Wuchereria bancrofti and assessed its polymorphism by comparing with nematode and other organism. ► iPGM can be focused as a good macrofilarial drug target because it is present both in filarial parasite and its endosymbiont Wolbachia but absent in human host. ► iPGM is highly conserved and can be used a good taxonomic marker.

Introduction

Lymphatic filariasis (LF) is a major tropical disease that affects nearly 120 million people worldwide. It is a serious public health problem in India, which contributes to 40% of the world burden (WHO, 2008). The etiological agents of human LF are the nematode species, Wuchereria bancrofti, Brugia malayi and Brugia timori, with the former being the major cause accounting for 90% of the infections. The World Health Organization ranks the complex clinical conditions caused by LF as second on the list of infectious diseases that lead to permanent and long-term disabilities (Molyneux and Taylor, 2001). Chemotherapy remains the mainstay for the treatment of the diseases caused by filarial nematodes, as there are no anti-filarial vaccines, and vector control being largely considered economically not feasible. However, the drugs administered are primarily microfilaricidal and partially macrofilaricidal (Noroes et al., 1997). Hence current strategies of Mass Drug Administration (MDA) for filariasis elimination are partially successful because the drugs do not eliminate all the adult worms present in a carrier. It may also be noted that a remote island where a DEC based mass chemotherapy was in place for three decades, eradication could not be achieved. Thus possibly indicating resistance to this drug (Esterre et al., 2001). The adult filarial parasites survive for long time, and hence those that survive the drug treatment continue to remain as a source of transmission in the community. The MDA has to be administered for as long as 6 years which is average life span of an adult worm. Inadequate coverage and compliance of the mass drug administration add further to the problem. Chemotherapy for LF has not been changed for the past 2–3 decades, thus raising the issue of drug resistance (Kumar et al., 2007). Hence, there is a need for new anti-filarial drugs, especially those that can kill adult parasites. Presently, the greater challenge is to identify new class of drugs that attack targets in the adult stage. Several structural and functional proteins of filarial parasites have been focused as drug targets (Gupta and Srivastava, 2006). The nematode genome sequence data recently made available from sequencing projects (http://www.nematode.net), coupled with new gene function data, provide an opportunity to identify newer drug targets. Since enzymes involved in metabolic activity of the parasites are required in all stages of their life cycle, they form appropriate targets for the development of anti-parasitic drugs. Enzymes involved in carbohydrate metabolism have generated interest as drug targets, and one such enzyme is phosphoglycerate mutase (PGM), (EC 5.4.2.1) that catalyzes the interconversion of 2- and 3-phosphoglycerate (2-PG and 3-PG) in the glycolytic and gluconeogenic pathways (Jedrzejas et al., 2000a, Jedrzejas et al., 2000b).

Two distinct PGM enzymes, viz., co-factor-independent phosphoglycerate mutase (iPGM) and the co-factor-dependent phosphoglycerate mutase (dPGM) (Grana et al., 1992, Grana et al., 1995) distinguished based on the requirement of co-factor (2,3-diphosphoglycerate) exist in different organisms. The size, amino acid sequence, three-dimensional structure, and catalytic mechanisms of these two enzymes are vastly dissimilar (Chander et al., 1999). Generally, iPGMs are metalloenzymes comprising of ∼500 amino acids, catalyze the intramolecular transfer of the phospho-group on monophosphoglycerates through a phosphoserine intermediate and does not require 2,3-diphosphoglycerate as co-factor. In contrast, dPGM is composed of ∼250 amino acids and catalyzes the intermolecular transfer of the phospho group between the monophosphoglycerates and the cofactor (2,3-diphosphoglycerate) through a phosphohistidine intermediate (Jedrzejas et al., 2000a, Jedrzejas et al., 2000b, Rigden et al., 2002). Vertebrates, including humans, are known to possess only dPGM, whereas in other organisms, distribution of the two forms has been considered complex and unpredictable because either dPGM or iPGM or both forms may be present. Most of the filarial parasites, including human parasites such as W. bancrofti, B. malayi and Onchocerca volvulus are infected with the intracellular Wolbachia (Taylor et al., 2000). Wolbachia plays an important role in the biology of the host and in the immuno-pathology of filariasis (Brattig et al., 2000). Antibiotic targeting of Wolbachia with Tetracycline can have profound effects on the filarial parasites (Hoerauf et al., 1999) and elimination of Wolbachia from worm tissues causes reproductive abnormalities in worms and affect worm’s embryogenesis, resulting in sterility (Casiraghi et al., 2002, Taylor et al., 2005). Because of the essential dependency of Wolbachia, endosymbionts of filarial nematodes it is focused as a potential target for chemotherapy. However, nematodes, their endosymbionts including the free-living nematode, Caenorhabditis elegans, have only iPGM. Inhibition of iPGM activity by RNAi leads to lethality in C. elegans (Zhang et al., 2004). In view of these facts, iPGM of Wolbachia is proposed as a candidate for anti-filarial drug development (Foster et al., 2009).

In this study, we present the complete coding sequence of W. bancrofti iPGM and a partial genomic fragment of the iPGM from periodic and sub-periodic forms of W. bancrofti and B. malayi for the first time. The aim of the study was to structurally characterize iPGM of W. bancrofti and investigate its polymorphism among populations of W. bancrofti from distant geographic locations of India. This is important because the gene may not be conserved, in which case it may not be an ideal candidate for the development of anti-filarial drug. Further, we also derived its phylogenetic relation with other organisms. Both the genomic and amino acid sequences of this enzyme were compared with the sequences of iPGM of other nematodes, especially with filarial nematodes B. malayi and its endosymbiont (Wolbachia), O. volvulus, and the free-living nematode C. elegans. The phylogenetic position of W. bancrofti iPGM was analyzed based on amino acid sequences of iPGM orthologs of several other organisms.

Section snippets

Parasite materials

Blood samples were drawn from known carriers of microfilaria of W. bancrofti, residing in Pondicherry, between 20:00 and 23:00 h. Five milliliters of venous blood was collected from volunteers in a tube containing EDTA, and the mf were purified by membrane filtration (Chandrashekar et al., 1984). Microfilariae (mf) of sub-periodic form of W. bancrofti were isolated from blood slides collected from carriers living in Car Nicobar islands during daytime. The mf from the slides were purified by a

Structural characterization of iPGM of W. bancrofti

Amplicons of ∼1.5 kb (Fig. 1a) from cDNA library of infective stage larvae of W. bancrofti and ∼900 bp fragment (Fig. 1b) from gDNA of mf of W. bancrofti were obtained by PCR amplification and sequenced. The blast search in the NCBI site yielded matching sequence of iPGM of C. elegans and B. malayi. The nucleotide (nt) sequence of iPGM of W. bancrofti (Wb-iPGM) was found to match closely (98.4%) with iPGM isoform-1 of B. malayi, which is also reported to have another form, isoform-2 (Zhang et

Discussion

Global Programme for Elimination of LF (GPELF), launched recently employs a strategy of MDA with single dose of DEC or Ivermectin, co-administered with albendazole, annually (WHO, 2007). Indications from the programme so far are that these drugs are partially successful in interrupting the transmission of infection in the endemic communities. The adult parasites have a very long life span and those that survive the drug administration continue to serve as a source of infection, thus frustrating

Acknowledgments

One of the authors, RD was a recipient of Senior Research Fellowship from Council of Scientific and Industrial research (CSIR), New Delhi. This work is part of research projects supported by Indian Council of Medical Research (ICMR). The authors are thankful to Dr. P.K. Das former Director and Dr. P. Jambulingam, the present Director, VCRC, for their critical comments.

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