TIMPs of parasitic helminths – a large-scale analysis of high-throughput sequence datasets
© Cantacessi et al.; licensee BioMed Central Ltd. 2013
Received: 5 April 2013
Accepted: 28 May 2013
Published: 30 May 2013
Tissue inhibitors of metalloproteases (TIMPs) are a multifunctional family of proteins that orchestrate extracellular matrix turnover, tissue remodelling and other cellular processes. In parasitic helminths, such as hookworms, TIMPs have been proposed to play key roles in the host-parasite interplay, including invasion of and establishment in the vertebrate animal hosts. Currently, knowledge of helminth TIMPs is limited to a small number of studies on canine hookworms, whereas no information is available on the occurrence of TIMPs in other parasitic helminths causing neglected diseases.
In the present study, we conducted a large-scale investigation of TIMP proteins of a range of neglected human parasites including the hookworm Necator americanus, the roundworm Ascaris suum, the liver flukes Clonorchis sinensis and Opisthorchis viverrini, as well as the schistosome blood flukes. This entailed mining available transcriptomic and/or genomic sequence datasets for the presence of homologues of known TIMPs, predicting secondary structures of defined protein sequences, systematic phylogenetic analyses and assessment of differential expression of genes encoding putative TIMPs in the developmental stages of A. suum , N. americanus and Schistosoma haematobium which infect the mammalian hosts.
A total of 15 protein sequences with high homology to known eukaryotic TIMPs were predicted from the complement of sequence data available for parasitic helminths and subjected to in-depth bioinformatic analyses.
Supported by the availability of gene manipulation technologies such as RNA interference and/or transgenesis, this work provides a basis for future functional explorations of helminth TIMPs and, in particular, of their role/s in fundamental biological pathways linked to long-term establishment in the vertebrate hosts, with a view towards the development of novel approaches for the control of neglected helminthiases.
KeywordsTissue inhibitors of metalloproteases Parasitic helminths Excretory/secretory products Transcriptomics Bioinformatics Protein structure Functional inferences
Parasitic helminths cause devastating diseases in humans and animals worldwide [1, 2]. Amongst these parasites, soil-transmitted helminths (STHs), including Ancylostoma duodenale and Necator americanus (hookworms), Ascaris sp. (roundworms) and Trichuris spp. (whipworms), are estimated to infect over one-sixth of all humans [1, 2], while trematodes, including the blood flukes Schistosoma spp. and the carcinogenic liver flukes Clonorchis sinensis and Opisthorchis viverrini, affect >200 million people worldwide [3–5].
Traditionally, the control of helminth infections has relied on the repeated and frequent use of anthelmintics [6, 7], which is likely to lead to the development of drug resistance against the compounds administered (cf. [8–10]). Indeed, some studies [11–15] have reported a reduction in efficacy of mebendazole and pyrantel in N. americanus and A. duodenale in areas of Mali, North-Western Australia and Zanzibar, which has been attributed to emerging anthelmintic resistance. Given the limited knowledge of the molecular mechanisms linked to the development of drug resistance in parasitic helminths , as well as the unavailability of effective vaccines, much attention is now directed towards the identification of novel targets for intervention [7, 17]. A detailed understanding of the molecular biology of parasitic helminths, and in particular of the structure and function of key genes and gene products playing essential roles in host-parasite interactions, could provide a basis for the design of novel therapeutics.
Among several groups of helminth molecules involved in the host-parasite interplay, protease inhibitors have been the subject of intense investigations due to their roles in a range of fundamental molecular processes, including regulation of host proteases and modulation of the host’s immune response . Amongst these molecules, the inhibitors of cysteine- and serine-proteases (= cystatins and serpins, respectively; MEROPS family I25 and I04, respectively) are known to participate in the cascades of molecular events leading to parasite development through the larval stages (cystatins) , as well as in the inhibition of host molecules responsible for the initiation of blood coagulation (serpins) [20, 21]. In addition, both molecular groups have been proposed to play key roles in the evasion and modulation of the immune response of the vertebrate host [19, 21, 22]. In contrast to data on cystatins and serpins, knowledge of the biological roles of parasite-derived tissue inhibitors of metalloproteases (TIMPs) is limited. Eukaryote TIMPs are a multifunctional family of inhibitors of matrix metalloproteases (MMPs), including collagenases and gelatinases, which function as important regulators of extracellular matrix (ECM) turnover, tissue remodelling and cellular behaviour . The N-terminal domain of TIMPs contains a netrin module (=‘NTR’; Prosite: PDOC50189) which, in addition to harbouring the functional site responsible for the primary metalloprotease inhibitory activity, is associated with a range of biological roles, including axon guidance, regulation of cell-cell interactions during embryogenesis, cell proliferation, angiogenesis and pro- and anti-apoptotic pathways [23, 24]. The NTR domain of TIMPs is also found in other groups of proteins, such as the frizzled-related (PDOC50038) and the laminyn-type EGF-like (PDOC00961) proteins, in which it fulfills distinct biological roles (cf. ).
In the canine hookworm Ancylostoma caninum, TIMPs are abundant components of the excretory/secretory (ES) products of the adult worm [25–27]. Ac-TMP-1 and Ac-TMP-2, two abundantly expressed TIMPs recognized by sera from dogs vaccinated with irradiated third-stage larvae (L3s) of A. caninum, were proposed to play key roles in the host-parasite interplay [25, 27]. To date, no data on the occurrence of TIMP homologues/orthologues in other parasitic helminths is available. Over the last decade, advances in next-generation sequencing (NGS) technologies and bioinformatics [28–31] have provided the infrastructure for large-scale analyses of the genomes and transcriptomes of a range of parasitic helminths of public health significance, including the nematodes N. americanus, Ascaris suum, Trichuris suis and Trichinella spiralis (gastrointestinal nematodes) [32–36] and the trematodes Schistosoma mansoni, S. japonicum, S. haematobium (blood flukes), C. sinensis and O. viverrini (liver flukes) [37–41]. These advances have resulted in an expansion of sequence data available in public databases (e.g., http://www.gasserlab.org/, http://www.genedb.org/, http://www.ncbi.nlm.nih.gov/, http://nematode.net/NN3_frontpage.cgi and http://www.sanger.ac.uk/research/projects/parasitegenomics/), which represent an invaluable resource for studies of TIMPs in parasitic helminths. In the present study, we (i) conducted the first large-scale investigation of TIMP proteins in a range of parasitic helminths of both human and veterinary health significance; (ii) inferred phylogenetic relationships between/among helminth TIMPs based on predictions of secondary structures of protein sequences; and (iii) investigated differences in the levels of transcription of genes encoding putative TIMPs in different developmental stages of A. suum (cf. ), N. americanus (cf. ) and S. haematobium (cf. ).
Sequence data, and identification and bioinformatic analyses of TIMPs
The sequence data obtained from public sequence databases (i.e. National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/; ENSEMBL Genome Browser at http://www.ensembl.org/index.html; WormBase, at http://www.wormbase.org; GeneDB at http://www.genedb.org/; http://www.gasserlab.org) [32–34, 39, 40, 42–45] and analysed herein included known TIMP amino acid sequences from Homo sapiens (GenBank accession numbers XP_010392.1, NP_003246.1, P35625.1 and Q99727.1), Mus musculus (accession numbers P12032.2, P25785.2, P39876.1 and Q9JHB3.1), Canis familiaris (AF112115.1), Gallus gallus (AAB69168.1), Oryctolagus cuniculus (AAB35920.1), Drosophila melanogaster (AAL39356.1), A. caninum (AF372651.1 and EU523698.1), A. duodenale (ABP88131.1) and Caenorhabditis elegans (NP_505113.1), as well as predicted peptides inferred from (i) the whole or draft genome sequences of S. mansoni, S. japonicum, S. haematobium (http://www.genedb.org), A. suum (http://www.wormbase.org), T. spiralis (http://www.ncbi.nlm.nih.gov/nuccore/316979833), Brugia malayi and Wuchereria bancrofti (human filarial nematodes) (http://www.sanger.ac.uk/; ), N. americanus (human hookworm; ), and (ii) the transcriptomes of T. suis (swine whipworm), Oesophagostomum dentatum (swine nodule worm) (http://www.gasserlab.org), Dictyocaulus filaria (sheep lungworm; ) and C. sinensis, O. viverrini (human liver flukes), Fasciola hepatica and F. gigantica (bovine and deer liver fluke, respectively) (http://www.gasserlab.org). The algorithms BLASTp  and InterProScan  were used to identify TIMP proteins in each of the genomic and transcriptomic datasets based on sequence homology (e-value cut-off: 10-5) with known TIMP proteins from eukaryotes . In addition, the software pScan (http://www.psc.edu/general/software/packages/emboss/appgroups/pscan.html) was used to identify regular-expression based diagnostic patterns for TIMPs (Prosite: PS00288). Signal peptides were also predicted using the program SignalP 3.0, employing both the neural network and hidden Markov Models . Putative ES TIMP proteins were identified based on the presence of a signal peptide and sequence homology to one or more known ES proteins listed in the Secreted Protein (http://spd.cbi.pku.edu.cn/; ) and the Signal Peptide (http://proline.bic.nus.edu.sg/spdb/index.html; ) databases.
Secondary structure predictions and homology modelling
Structure-based sequence alignments of TIMP proteins were computed and manually edited with SBAL  guided by secondary structure elements predicted using the PSIPRED software . Individual structure-based alignments of amino acid sequences were subjected to analysis by Bayesian inference (BI) using the program MrBayes v.3.1.2  and verified by Maximum Likelihood analysis using the program MEGA v.5  and the Jones-Taylor-Thornton substitution model with uniform rates among sites (JTT + G + I). Each BI analysis was conducted for 1,000,000 generations (ngen = 1,000,000), with every 100-th tree being saved, using the following parameters: rates = gamma, aamodelpr = mixed, and the other parameters left at the default settings. Tree and branch lengths were measured employing the parameter ‘sumt burnin = 1000’; an unrooted, consensus tree was constructed, with ‘contype = halfcompat’ nodal support being determined using consensus posterior probabilities and displayed employing the software FigTree (http://tree.bio.ed.ac.uk/software/figtree/). For selected TIMPs, homologues with known three-dimensional structures were identified using the protein-fold recognition software pGenTHREADER  and selected as templates for comparative modelling using MODELLER . Twenty independent models were generated, and the model with the lowest energy was selected, its geometry analysed using PROCHECK  and then inspected visually with PyMOL .
Assessment of levels of transcription of TIMP-encoding genes
The raw sequence reads derived from each of the non-normalized cDNA libraries from A. suum infective L3s (iL3s; from eggs), migrating L3s (from liver and lung), fourth-stage larvae (L4s, from the small intestine) and muscular and reproductive tissues from each adult male and female , N. americanus iL3s and adults (mixed males and females) , as well as S. haematobium eggs and adult male and female  were mapped to the longest contigs encoding individual putative TIMP proteins using the program SOAP2 . Briefly, raw sequence reads were aligned to the non-redundant transcriptomic data, such that each raw sequence read was uniquely mapped (i.e. to a unique transcript). Reads that mapped to more than one transcript (designated ‘multi-reads’) were randomly assigned to a unique transcript, such that they were recorded only once. To provide a relative assessment of transcript abundance, the number of raw reads that mapped to each sequence was normalized for length (i.e. reads per kilobase per million reads, RPKM) [34, 40, 63].
Results and discussion
TIMP proteins of parasitic helminths
Number of tissue inhibitor of metalloproteases (TIMP) and netrin module (NTR)-containing protein sequences, respectively, identified in each sequence dataset and listed according to taxa
TIMPs (no. with SP)
NTR-module containing proteins (no. with SP)
Of the eight genes encoding putative TIMPs in N. americanus, transcription of NECAME_13168 and NECAME_07191 was significantly up-regulated in iL3s (cf. Table 1; ), thus supporting a role for these proteins in the infection process of the human host. Conversely, NECAME_08457 and NECAME_08458 displayed high transcription levels in adult N. americanus (cf. Table 1; ), which likely reflects a diversification of function of members of this protein family in different developmental stages of this parasite. In the future, studies of differential transcription of genes encoding TIMPs in both genders and different tissues of N. americanus may help elucidate the roles that these molecules play in the fundamental molecular biology of the adult nematode. In A. suum, transcription of GS_04796 was significantly up-regulated in the adult female reproductive tissue of this nematode, whereas GS_21732 was up-regulated in the male muscle (cf. Table 1; cf. ). The putative TIMP proteins encoded by GS_04796 and GS_21732 share ~40% similarity with C. elegans CRI-2 (WBGene00019478; http://www.wormbase.org), the expression of which has been localized to the body wall musculature and to the vulval, anal and pharyngeal muscles of the adult nematode (cf. http://www.wormbase.org). In C. elegans, cri-2 is known to function in the cascade of molecular events linked to the regulation of the innate immune response to lipopolysaccharide (LPS) . In a previous study, inhibition by small interfering RNAs (siRNAs) of the M. musculus ortholog of C. elegans cri-2 in a mouse macrophage cell line stimulated with Escherichia coli LPS resulted in decreased production of interleukin-6 (IL-6) . This cytokine, in vivo, is associated with a wide range of biological activities, which include the generation of acute-phase reactions in response to infections by pathogens . The putative role/s that parasite homologs of C. elegans cri-2 play in the modulation of innate immunity in vertebrate hosts remain/s unknown. However, recent evidence that recombinant Ac-TMP-1 promotes the development of a regulatory immune response by modifying the functions of bone marrow-derived dendritic cells and subsequent development of regulatory T cells , supports a key role for this TIMP in establishing an anti-inflammatory environment.
In flatworms, the S. haematobium gene A_01727 encoded the only trematode TIMP protein that could be identified using computational methods. Analysis of transcriptional regulation of S. haematobium A_01727 in different developmental stages revealed that this molecule is up-regulated in the adult male of this parasitic trematode (Table 1; cf. ). The transcript encoding mouse TIMP-1 is up-regulated in male gonads during testis morphogenesis, while expression of the corresponding protein was restricted to the cords of foetal testes . In addition, the human and mouse genes encoding TIMP-2 are known to include the differential display clone 8 (DDC8) gene, whose transcription is enhanced during spermatogenesis . These observations, together with earlier findings of increased expression of TIMP-1 in human foetal Sertoli cells [72, 73] and testicular expression of TIMP-2 in rats , led to the hypothesis that these molecules may play specific roles during testis organogenesis and development , as well as in the migration of germ cells through the seminiferous epithelium . Therefore, it is tempting to speculate a role for S. haematobium A_01727 in biological processes linked to the reproductive activity of the adult male fluke; however, this hypothesis requires rigorous testing. In the future, genetic manipulation of N. americanus, A. suum and S. haematobium by RNA interference (RNAi) and/or transgenesis [75–78], may help elucidate the function/s of putative helminth TIMPs in the reproductive biology of these organisms, as well as in other fundamental molecular processes, for instance those linked to host invasion and modulation of the host’s innate immune response.
Structural analyses of eukaryote TIMPs
Three-dimensional structures of tissue inhibitors of metalloproteases (TIMPs) and their complexes available in the Protein Data Bank (PDB;) as of Nov 2012 http://www.rcsb.org/pdb/home/home.do
PDB accession code
The main interactions of TIMPs with their target proteases are formed by a continuous peptide at the N-terminal end (Cys1-Pro5 in human TIMP-1) and in a loop connecting two adjacent β-strands (Met66-Cys70 in human TIMP-1). The two regions are covalently linked by a disulphide bond (Cys1-Cys70 in human TIMP-1), and are located in the netrin module (N-TIMP) of the protein which adopts the fold of a five-stranded α-barrel with Greek key topology (OB-fold) flanked by two α-helices. The N-terminus of N-TIMP inserts into the active site of the target protease and the α-amino and the carbonyl group of Cys-1 (human TIMP-1) coordinate the active site zinc ion of the protease by displacing a water molecule otherwise bound to the metal . Residue 2 (Ser, Thr) projects into the specificity (S1′) pocket of the protease. Residues 3–5 interact with the protease residues in the primed subsites, which normally harbour substrate residues C-terminal of the scissile bond. Similarly, residues 66–70 of TIMP-1 occupy the non-primed subsites of the protease that otherwise interact with the residues N-terminal to the scissile bond.
As apparent from the structure-based amino acid sequence alignment (Figure 1), TIMPs from parasitic helminths are characterised by higher sequence variation than their mammalian homologues, in accordance with the results of previous analyses of invertebrate TIMPs . With respect to structure-function relationships, however, the most important feature grafted onto the netrin fold seems to be the conformation neighbouring Cys-1. In vertebrate TIMPs, 2 is either a serine or threonine that projects into the protease specificity pocket. It is important to note that neither Ac-TMP-1 nor Ac-TMP-2 have been convincingly shown (via 1:1 inhibitor:enzyme molar ratios) to possess MMP inhibitory activity. Moreover, AceES-2 produced with a flush N-terminus was screened for MMP activity at 15:1 and 115:1 molar ratios and did not display inhibitory activity (cf. ). The amino acid sequence alignment in Figure 1 highlights the general motif of TIMPs, C-X-C, in this region. It shows for the helminth TIMP with published inhibitory activity, Ac-TMP-2, that in addition to serine and threonine, lysine is a tolerated residue at position 2 for inhibition. Notably, AceES-2 and Ad-TIMP-1 from A. duodenale lack the second cysteine residue as well as a suitable residue at position 2 (Ser/Thr/Lys) able to protrude into the S1′ pocket of the protease for inhibition (cf. Figure 1). On this basis, one would predict Ad-TIMP-1 to not have any MMP-inhibitory activity. Thus, helminth TIMPs that show conservation at position 2 are likely to display inhibitory activities against human MMPs. The S. haematobium protein encoded by A_01727 possesses two residues (Arg-Ser) between the two N-terminal cysteine residues, which makes the prediction of functional effects difficult in the absence of experimental structures.
The current availability of ‘-omics’ technologies, applied to in-depth investigations of pathogens causing neglected diseases [31, 92–94], are becoming pivotal for a better understanding of the structure and function of TIMP proteins in different species and developmental stages of parasitic helminths. For instance, data from in-depth comparative structural analyses between helminth TIMPs and their vertebrate counterparts, will be crucial in future studies aimed at assessing the suitability of parasite TIMPs as novel targets for intervention. Supported by the availability of the whole-genome sequences of, for instance, schistosomes and A. suum[34, 37, 38, 40] and by current efforts to expand genomic sequencing to other neglected parasites (e.g. hookworms; ), the application of gene manipulation technologies such as RNAi and/or transgenesis [94, 96, 97], will allow the function of helminth TIMP proteins in fundamental biological pathways to be elucidated. Perhaps the most important question that is yet to be addressed in any depth is the function of helminth TIMPs. Are they inhibitors of metalloproteases? Is their primary purpose to suppress inflammation, and if so, how do they do it? We hope that the molecular information provided herein on parasitic helminth TIMPs will provide a framework on which to build intensive research activities around this intriguing family of proteins and their roles in host-parasite interactions.
This work was supported by a program grant from the National Health and Medical Research Council of Australia (NHMRC). AL and CC are supported by a NHMRC principal research fellowship and early-career research fellowship, respectively. The authors would like to thank Dr Alex Strongin (Sanford-Burnham Medical Research Institute, La Jolla, CA, USA) for helpful discussions on TIMP/MMP interactions.
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