NemChR-DB: a database of parasitic nematode chemosensory G-Protein Coupled Receptors

Abstract

Nematode Chemosensory G-Protein Coupled Receptors (GPCRs) (NemChRs) have expanded within nematodes, where they play important roles in foraging and host-seeking behavior. NemChRs are most highly expressed during free-living stages when chemosensory signaling is required for host detection and nematode activation in various parasitic nematodes, and therefore position NemChRs at the transition from infective to parasitic stages, making them important regulators to study in terms of host-seeking and host specificity. To facilitate the analysis of NemChRs, here we describe an integrative database of nematode chemoreceptors called NemChR-DB. This database enables users to study diverse parasitic nematode chemoreceptors, functionally explore sequence entries through structural and literature-based annotations, and perform cross-species comparisons.

Nematodes are a ubiquitous and diverse group of animals that comprise both free-living and parasitic species. Parasitic nematodes of humans infect more than 1 billion people globally with an estimated 4 billion at risk for infection (Bethony et al., 2006). Four species of soil-transmitted helminths (STHs), Ascaris lumbricoides, Trichuris trichiura, Necator americanus and Ancylostoma duodenale, are responsible for most cases of human infection. They are prevalent in tropical and subtropical zones with poor sanitation practices that lead to transmission from eggs present in human feces. In the case of hookworm, heavy infection can result in debilitating and sometimes fatal iron deficiency anemia caused by blood loss due to the adult nematodes that feed in the intestine. In some cases, blockage of the intestine will necessitate surgery. These heavy infections are especially devastating in children, causing stunted physical and cognitive development, which may be permanent (Lozoff et al., 1991). Pregnant women as well as elderly people are also at high risk for morbidity (Bundy et al., 1995). Patients with lighter infections may be asymptomatic or exhibit mild symptoms such as diarrhea or abdominal pain.

G-Protein Coupled Receptors (GPCRs) represent the largest and most diverse family of membrane proteins in eukaryotes. Each member of the family shares a common architecture comprised of a single polypeptide containing seven hydrophobic transmembrane domains. A subgroup of GPCRs called the Nematode Chemosensory GPCRs (NemChRs) are unique to nematodes, and therefore represent important substrates to study in order to understand and potentially treat nematode infections (Robertson and Thomas, 2006; Krishnan et al., 2014). Additionally, NemChRs and their agonists are important regulators of diverse behaviors in nematodes (Sengupta et al., 1996; Choe et al., 2012; Greene et al., 2016), and genetic deletion of their downstream effectors has been shown to block nematode activation and behavioral responses to host-emitted cues (Gang et al., 2020; Wheeler et al., 2020). Recent work from our group and others has revealed a strong correlation between NemChR expression and free-living stages of parasitic nematodes (Bernot et al., 2020; Wheeler et al., 2020), suggesting a clear role for NemChRs in host-seeking behavior. The primary sensory organ in nematodes is called the amphid and at the neuroanatomical level this organ is highly conserved across diverse nematode species (Ashton and Schad, 1996). In Caenorhabditis elegans NemChRs have been shown to localize at the dendritic cilia of the amphid sensory organ (Sengupta et al., 1996; Vidal et al., 2018), and based on sequence data NemChRs have been organized into 19 groups. Of these 19 groups, members are organized into the following family and subfamily classifications: Sra - sra, srab, srb, sre; Str - srd, srh, sri, srj; Srg - srt, sru, srv, srx srxa; and other: srbc, srsx, srw, srz (Robertson and Thomas, 2006). Interestingly, chemosensory GPCRs in eukaryotes likely evolved independently multiple times which has led to substantial diversification of chemosensory GPCRs between deuterostomes and protostomes, and subsequent expansion of ancestral chemosensory GPCRs within the nematode lineage to produce the NemChRs (Krishnan et al., 2014). In order to accelerate the study of NemChRs in terms of nematode evolution and chemosensory behavior we here describe a unified resource of NemChRs called NemChR-DB. NemChR-DB is freely available to all users and provides an intuitive and integrative platform for the study of parasitic nematode chemoreceptors. NemChR-DB is accessible at the following URL: http://ohalloranlab.net/nemchr-db.

Data contained within NemChR-DB was mined from WormBase ParaSite (ver. WBPS14) (Howe et al., 2017) (Table 1) and UniProt (UniProt Consortium, 2019) using a pipeline developed previously (Wheeler et al., 2020). Briefly, hmmsearch (Mistry et al., 2013) was used to compare proteins against a database of GPCR Pfam hidden Markov models (HMMs) (Finn et al., 2014), and then filtered to keep proteins with matches to nematode chemoreceptor HMMs and Caenorhabditis elegans chemoreceptors. We also included an additional filter to remove FMRFamide-like receptors. Sequences were then provided as input to TMHMM (ver. 2.0c) (Sonnhammer et al., 1998) and Phobius (Käll et al., 2004) in order to obtain the predicted number of transmembrane (TM) domains and their topology. The sequences were then used to retrieve additional information such as PubMed ID, taxonomy, molecular weight and more from UniProt. Next, all of this information was organized into a database of JSON objects which is served asynchronously using Ajax and jQuery DataTables. The database contains 8,790 putative chemoreceptors from 53 unique nematode species representing 20 superfamilies of nematodes and 28 nematode families. Table 2 details the summary statistics for NemChR-DB in terms of chemoreceptor counts and quality scores for genomes represented in the database. We also examined the relationship between putative chemoreceptor counts and genome quality by comparing the numbers of putative chemoreceptors from each species with Core Eukaryotic Genes Mapping Approach (CEGMA) scores, (Parra et al., 2007) and Benchmarking Universal Single Copy Orthologs (BUSCO) (Seppey et al., 2019) scores (Fig. 1). From this analysis, we did not observe a significant correlation in either case, suggesting that the numbers of predicted NemChRs in each species were not influenced significantly by genome quality (Fig. 1: BUSCO Pearson’s correlation coefficient r = 0.037 and CEGMA r = 0.156, P > 0.05 in each case). However, it is worth pointing out that BUSCO scores differed across different genomes we used to identify NemChRs (Table 2: $\overline{x}$ BUSCO score=75.25 and σ=18.5), and so improvement of BUSCO scores in some cases may alter final NemChR counts.

Table 1.

List of species and genomes used to predict chemoreceptors for NemChR-DB

Species name	Assembly	BioProject ID	Clade
Trichuris muris	TMUE3.0	PRJEB126	Clade I
Trichuris suis	Tsuis_adult_female_1.0	PRJNA208416	Clade I
Trichinella spiralis	T1_ISS3_r1.0	PRJNA257433	Clade I
Trichuris trichiura	TTRE2.1	PRJEB535	Clade I
Trichinella pseudospiralis	T4_ISS588_r1.0	PRJNA257433	Clade I
Romanomermis culicivorax	nRc.2.0	PRJEB1358	Clade I
Toxocara canis	Toxocara_canis_adult_r1.0	PRJNA248777	Clade III
Ascaris suum	AscSuum_1.0_submitted	PRJNA80881	Clade III
Brugia malayi	Bmal-4.0	PRJNA10729	Clade III
Ascaris lumbricoides	A_lumbricoides_Ecuador_v1_5_4	PRJEB4950	Clade III
Onchocerca volvulus	ASM49940v2	PRJEB513	Clade III
Brugia pahangi	B_pahangi_Glasgow_0011_upd	PRJEB497	Clade III
Dracunculus medinensis	D_medinensis_Ghana_v2_0_4	PRJEB500	Clade III
Brugia timori	B_timori_Indonesia_v1_0_4_001_upd	PRJEB4663	Clade III
Onchocerca ochengi	ASM90053720v1	PRJEB1465	Clade III
Thelazia callipaeda	T_callipaeda_Ticino_0011_upd	PRJEB1205	Clade III
Syphacia muris	S_muris_Valencia_v1_0_4	PRJEB524	Clade III
Loa loa	Loa_loa_V3	PRJNA37757	Clade III
Litomosoides sigmodontis	ASM90053727v1	PRJEB3075	Clade III
Anisakis simplex	A_simplex_0011_upd	PRJEB496	Clade III
Wuchereria bancrofti	W_bancrofti_Jakarta_0011_upd	PRJEB536	Clade III
Dirofilaria immitis	nDi.2.2	PRJEB1797	Clade III
Parascaris univalens	ASM225920v1	PRJNA386823	Clade III
Elaeophora elaphi	E_elaphi_v1_0_4	PRJEB502	Clade III
Steinernema glaseri	S_glas_v1_submitted	PRJNA204943	Clade IV
Bursaphelenchus xylophilus	ASM23113v1_submitted	PRJEA64437	Clade IV
Steinernema carpocapsae	ASM75764v3	PRJNA202318	Clade IV
Rhabditophanes sp. KR3021	Rhabditophanes_sp_KR3021	PRJEB1297	Clade IV
Strongyloides venezuelensis	S_venezuelensis_HH1	PRJEB530	Clade IV
Meloidogyne hapla	Freeze_1	PRJNA29083	Clade IV
Strongyloides stercoralis	S_stercoralis_PV0001_v2_0_4	PRJEB528	Clade IV
Strongyloides ratti	S_ratti_ED321_v5_0_4	PRJEB125	Clade IV
Strongyloides papillosus	S_papillosus_LIN_v2_1_4	PRJEB525	Clade IV
Parastrongyloides trichosuri	P_trichosuri_KNP	PRJEB515	Clade IV
Globodera pallida	GPAL001	PRJEB123	Clade IV
Panagrellus redivivus	Pred3	PRJNA186477	Clade IV
Ancylostoma ceylanicum	A_ceylanicum1.3.ec.cg.pg	PRJNA72583	Clade V
Ancylostoma caninum	A_caninum_9.3.2.ec.cg.pg	PRJNA72585	Clade V
Haemonchus contortus	Hco_v4_coding_submitted	PRJNA205202	Clade V
Ancylostoma duodenale	A_duodenale_2.2.ec.cg.pg	PRJNA72581	Clade V
Nippostrongylus brasiliensis	N_brasiliensis_RM07_v1_5_4_0011_upd	PRJEB511	Clade V
Necator americanus	N__americanus_v1	PRJNA72135	Clade V
Dictyocaulus viviparus	D_viviparus_9.2.1.ec.pg	PRJNA72587	Clade V
Angiostrongylus cantonensis	A_cantonensis_Taipei_v1_5_4	PRJEB493	Clade V
Haemonchus placei	H_placei_MHpl1_0011_upd	PRJEB509	Clade V
Diploscapter pachys	DipSp1Ass11Ann3	PRJNA280107	Clade V
Heligmosomoides polygyrus	nHp_v2.0	PRJEB15396	Clade V
Oesophagostomum dentatum	O_dentatum_10.0.ec.cg.pg	PRJNA72579	Clade V
Heterorhabditis bacteriophora	Heterorhabditis_bacteriophora-7.0	PRJNA13977	Clade V
Angiostrongylus costaricensis	A_costaricensis_Costa_Rica_0011_upd	PRJEB494	Clade V
Strongylus vulgaris	S_vulgaris_Kentucky_0011_upd	PRJEB531	Clade V
Teladorsagia circumcincta	T_circumcincta.14.0.ec.cg.pg	PRJNA72569	Clade V
Cylicostephanus goldi	C_goldi_Cheshire_0011	PRJEB498	Clade V

Table 2.

Summary of chemoreceptor counts and genome quality scores in NemChR-DB

Metric	Value (species)
Max. CR counts	811 (Ancylostoma ceylanicum)
Min. CR counts	21 (Litomosoides sigmodontis)
Avg. CR counts	165.8
Median CR counts	135
StDev CR counts	137.4
Max. BUSCO score	97.6 (Onchocerca volvulus)
Min. BUSCO score	11.4 (Cylicostephanus goldi)
Avg. BUSCO score	75.25
Median BUSCO score	79.9
StDev. BUSCO score	18.5
Max. CEGMA score	99.6 (Strongyloides ratti)
Min. CEGMA score	17.74 (Cylicostephanus goldi)
Avg. CEGMA score	90
Median CEGMA score	94.76
StDev CEGMA score	14.9

Max., maximum; Min., minimum; Avg., average; StDev, standard deviation.

Fig. 1.

Correlation plots of chemoreceptor counts versus complete BUSCO scores (A) and CEGMA scores (B). BUSCO and CEGMA scores were downloaded from WormBase ParaSite ver. WBPS14 (WS271). BUSCO: Pearson’s correlation coefficient r=0.037 and P=0.79053; CEGMA r= 0.156 and P=0.2599. Spearman’s Rho value r_s= −0.136 and P=0.323 for BUSCO correlation and r_s= 0.094 and P=0.49 for CEGMA correlation, suggesting that there is no significant evidence for correlation between genome quality and chemoreceptor counts. Plots were generated using matplotlib, and Pearson’s correlation coefficient calculated using pearsonr and Spearman’s Rho calculated with spearmanr from scipy.stats.

The database can be searched by clicking the “BROWSE” link from the top of each page (Fig. 2). Users can sort the data tables by clicking on headers, and perform searches using diverse identifiers including species or gene name. Search results can be copied or exported as an Excel file by simply clicking on the corresponding button at the top of the table after performing a search. The TM topology column was created using TMHMM (Sonnhammer et al., 1998) and Phobius (Käll et al., 2004) to provide the residue positions (starting and ending) for each TM domain. TMHMM also returns a value indicating how likely a protein is to be a TM protein (typically values >18 indicate a TM protein), and Phobius indicates whether the protein is predicted to have a signal peptide; both of these data points are also represented as columns in the database table (ExpAA and SP). Protein sequences for each entry can be viewed by clicking the ‘+’ icon next to each entry (Fig. 2). NemChR-DB can also be searched by protein sequence by clicking the “BLAST” link from the top of any page. A local BlastP executable is launched from server-side Hypertext Preprocessor (PHP) scripts and returned as Hypertext Markup Language (HTML) in the user-specified output format. Default parameters are implemented for BlastP including an initial word_size match set to 3, a minimum threshold score for adding a word to the lookup table set to 11, and the heuristic value (in bits) for the final gapped alignment equal to 25. Users can use BLAST to search for similar sequences in other parasitic NemChRs in the database organized by clade, or against the entire database by simply selecting the database type on the BLAST page. Furthermore, users may be interested in understanding the relationship between NemChRs in their parasite of interest with that of NemChRs from C. elegans, and to accommodate this, there is an option to use BLAST to search for similar sequences against all parasitic NemChRs contained within the database as well as the predicted NemChRs from C. elegans. In addition to searching the database with candidate chemoreceptors using BLAST, users can explore TM domain topology of candidate chemoreceptors by navigating to the TM FINDER page. Here, users can supply raw protein sequence to identify predicted TM domains in their protein. TM Finder uses TMHMM (Sonnhammer et al., 1998) to identify predicted TM domains and renders the results using Feature-Viewer (Garcia et al., 2014).

Fig. 2.

From the home page of NemChR-DB users can navigate various links at the top to browse the database (1), search and download results (2 and 3), or explore functional annotations as well as related literature (4). Users can also characterize candidate nematode chemoreceptors using TM Finder to identify and visualize predicted transmembrane domains (5) or query the database using BlastP to uncover related sequences (6). NemChR-DB can be accessed at the following site: http://ohalloranlab.net/nemchr-db,

NemChR-DB is the first database of parasitic nematode chemoreceptors and provides a platform for users to study NemChRs and functionally explore these receptors from very diverse nematode species. To construct NemChR-DB, data were sourced from highly curated databases so as to provide the most up-to-date information of annotated nematode chemoreceptors in diverse species. Our database is designed to permit growth and allow for new sequences and annotations to be easily included when they become available. We plan to add chemoreceptors from more species on a rolling basis based on user feedback (more details can be found on the METHODS page of the database at the following URL: http://ohalloranlab.net/nemchr-db/methods.html), and using the WormBase Application Programming Interface (API) we will track new releases to include in NemChR-DB. In conclusion, NemChR-DB will serve as a useful portal for exploration and discovery in future experiments on nematode chemoreceptor biology.