Molecular insights into the heat shock proteins of the human parasitic blood fluke Schistosoma mansoni

Aguoru, Nancy A.; Kirk, Ruth S.; Walker, Anthony J.

doi:10.1186/s13071-022-05500-7

Molecular insights into the heat shock proteins of the human parasitic blood fluke Schistosoma mansoni

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Published: 13 October 2022

Volume 15, article number 365, (2022)
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Molecular insights into the heat shock proteins of the human parasitic blood fluke Schistosoma mansoni

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Nancy A. Aguoru¹,
Ruth S. Kirk¹ &
Anthony J. Walker¹

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Abstract

Background

Heat shock proteins (HSPs) are evolutionarily conserved proteins, produced by cells in response to hostile environmental conditions, that are vital to organism homeostasis. Here, we undertook the first detailed molecular bioinformatic analysis of these important proteins and mapped their tissue expression in the human parasitic blood fluke, Schistosoma mansoni, one of the causative agents of the neglected tropical disease human schistosomiasis.

Methods

Using bioinformatic tools we classified and phylogenetically analysed HSP family members in schistosomes, and performed transcriptomic, phosphoproteomic, and interactomic analysis of the S. mansoni HSPs. In addition, S. mansoni HSP protein expression was mapped in intact parasites using immunofluorescence.

Results

Fifty-five HSPs were identified in S. mansoni across five HSP families; high conservation of HSP sequences were apparent across S. mansoni, Schistosoma haematobium and Schistosoma japonicum, with S. haematobium HSPs showing greater similarity to S. mansoni than those of S. japonicum. For S. mansoni, differential HSP gene expression was evident across the various parasite life stages, supporting varying roles for the HSPs in the different stages, and suggesting that they might confer some degree of protection during life stage transitions. Protein expression patterns of HSPs were visualised in intact S. mansoni cercariae, 3 h and 24 h somules, and adult male and female worms, revealing HSPs in the tegument, cephalic ganglia, tubercles, testes, ovaries as well as other important organs. Analysis of putative HSP protein-protein associations highlighted proteins that are involved in transcription, modification, stability, and ubiquitination; functional enrichment analysis revealed functions for HSP networks in S. mansoni including protein export for HSP 40/70, and FOXO/mTOR signalling for HSP90 networks. Finally, a total of 76 phosphorylation sites were discovered within 17 of the 55 HSPs, with 30 phosphorylation sites being conserved with those of human HSPs, highlighting their likely core functional significance.

Conclusions

This analysis highlights the fascinating biology of S. mansoni HSPs and their likely importance to schistosome function, offering a valuable and novel framework for future physiological investigations into the roles of HSPs in schistosomes, particularly in the context of survival in the host and with the aim of develo** novel anti-schistosome therapeutics.

Human TNF-α induces differential protein phosphorylation in Schistosoma mansoni adult male worms

Article 07 November 2015

Heat Shock Proteins: Role, Functions and Structure in Parasitic Helminths

Role of Hsp90 in Plasmodium falciparum Malaria

Background

Human schistosomiasis, which is caused by parasitic Schistosoma flatworms, remains one of the most important tropical diseases in terms of public health impact despite the continued deployment of control measures [1,2,3]. Globally, over 200 million people are infected with schistosomes across 78 countries, and approximately 700 million people are at risk of infection [4,5,6]. Three main Schistosoma species are responsible for endemic disease, Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobium.

Schistosomes exhibit gonochorism and have a complex life cycle that involves passage through a molluscan intermediate host and a mammalian definitive host [2, 7]. When voided in the urine or faeces, and upon contact with freshwater, schistosome eggs hatch, releasing miracidia that swim using cilia to locate a compatible snail host, which they then penetrate. Next, each miracidium transforms into a mother sporocyst and undergoes asexual reproduction, producing daughter sporocysts that have the capacity to generate large numbers of cercariae for release [8,9,10]. These non-feeding human-infective cercariae swim using their bifurcated tail to locate the definitive host; they then penetrate the skin with the help of proteolytic enzymes secreted from their acetabular glands [11,12,13]. During penetration, the cercaria loses its tail and the head transforms into a schistosomulum (aka somule). The somules transit the skin, enter the vasculature and migrate via the lungs to the hepatic portal system where they develop into sexually mature dioecious adult worms [2, 14]. The ability of the cercaria to successfully transform into a somule is critical to its establishment as a human parasite [2]. The passage through, and transfer between, two different hosts exposes the schistosome to substantial changes in local environment to which the parasite must adapt to survive, grow and develop [2, 15].

Heat shock proteins (HSPs) are evolutionarily conserved proteins that are expressed in cells constitutively and can also be induced by stress [16, 17]. Generally, HSPs can be broadly classified into two families, the small ATP-independent HSPs of molecular mass 8–28 kDa, and the larger ATP-dependent HSPs of molecular mass 40–105 kDa [17]. Initially discovered as proteins upregulated in heat-stressed Drosophila melanogaster [18], HSPs are now understood to perform vital functions that regulate cellular homeostasis both in stressed and unstressed scenarios. HSPs are involved in multiple cellular processes and function mainly as molecular chaperones, which facilitate native protein stabilization, refolding, translocation, and degradation [19, 20]. Considering that the cercaria-to-somule transformation provides a unique physiological stress involving increases in temperature and salinity as the schistosome moves from freshwater to a warm-blooded environment and loses its tail, it is plausible that HSPs play a vital role in ensuring the survival of the parasite during this transition. Moreover, HSPs are likely to be essential to the continued survival of the schistosome in the hostile environment of the host, where they must fend off immune attack. Upregulation of HSP expression during the earliest stages of intra-mammalian somule development has been observed at the schistosome tegument through proteomics [21]. Furthermore, heat shock factor 1 (a major transcriptional activator responsible for transcribing heat shock genes) was localised to the acetabular glands of S. mansoni cercariae, suggesting a potential role for HSPs in cercarial invasion and transformation [22]. However, of the few investigations on schistosome HSPs, most have studied an individual HSP family member.

In the current study, a comprehensive and comparative molecular bioinformatic analysis of all S. mansoni HSPs was carried out, and HSP family members were mapped within human infective S. mansoni life stages, providing an atlas of HSP expression within the worm. The data provide novel insights into the complexities of HSPs in schistosomes, the factors that govern their regulation, and their potential role in schistosome function.

Results and discussion

Comparative analysis of S. mansoni HSPs

A total of 69 human HSPs were mined from databases of the National Center for Biotechnology Information (NCBI) [23] across the five HSP families (HSP 10, HSP 40, HSP 60, HSP 70 and HSP 90). The J domain-containing proteins found on the NCBI and InterPro databases, which are also known as DNAJ(HSP40)C proteins, are listed here as DNAJC23-30 (as in [23]) (Additional file 1: Dataset S1). The human HSP amino acid sequences (from UniProt) were BLASTed against S. mansoni proteins on WormBase ParaSite [24]. After removal of duplicate and partial sequences, and after ensuring that all S. mansoni HSPs contained the necessary functional domains (using InterPro), 55 S. mansoni HSPs were identified across the five HSP families (Table 1; Additional file 1: Dataset S1). As in humans, the S. mansoni HSP 10 and HSP 60 families each contained one member, Smp_097380.1 and Smp_008545.1, respectively. However, 12 HSP 70 family members were identified, one less than in humans, seven of which are almost identical copies replicated in two different parts of the genome. Furthermore, in contrast to humans that have 49 HSP 40 and five HSP 90 family members, S. mansoni was found to possesses 38 HSP 40s and three HSP 90s (Table 1; Additional file 1: Dataset S1).

Table 1 Heat shock proteins (HSPs) identified in Schistosoma mansoni and the corresponding Schistosoma japonicum and Schistosoma haematobium HSPs

Full size table

Based upon currently available genomic data, the composition of the Schistosoma HSP families appears broadly similar across the Schistosoma species investigated here, with most variation in protein number seen in HSP 40 and HSP 70 families (Fig. 1). Schistosoma bovis contains the most HSP 40s and HSP 70s (40 and 17 members, respectively), whereas Schistosoma rodhani possesses the fewest HSP 40s (30 members), and Schistosoma curassoni the fewest HSP 70s (five members). Interestingly, the free-living flatworms Macrostomum lignano (a marine basal flatworm) and Schmidtea mediterranea (a freshwater planarian) have considerably more HSPs than schistosomes (Fig. 1), with 163 and 75 HSPs, respectively; the striking expansion of HSPs in M. lignano is mainly due to a very large 123-member HSP 40 family. The significance of this expansion of HSPs in the free-living flatworms, when compared to schistosomes, is unknown, but could be linked to their free-living rather than parasitic habit. In planarians, DNAJA1, a HSP 40 family member, is enriched in neoblasts [25] and is required for stem cell maintenance, regeneration and homeostasis [26]; the function of the equivalent protein in schistosomes is worthy of investigation, particularly given the importance of stem cells to schistosome survival in the host [27].

To determine the similarity of individual HSPs between the three most common human-infective Schistosoma spp., S. mansoni HSP amino acid sequences were compared against those of S. japonicum and S. haematobium. While HSP 60 was found in all three species, HSP 10 was surprisingly absent from both S. japonicum and S. haematobium (Table 1). Most of the S. mansoni HSP 40 family members showed greater similarity to S. haematobium (~ 87%) compared to S. japonicum (~ 77%) (Table 1). The S. mansoni HSP 70 and HSP 90 family members were also more similar to those of S. haematobium compared to S. japonicum, with the exception of Smp_072320 (chain A heat shock cognate 71 kDa protein-like). The high similarity between the HSP members across these three Schistosoma spp. indicates that these HSPs likely play a vital role in the parasite and might be essential for its survival. That S. mansoni HSPs show more similarity with S. haematobium HSPs than those of S. japonicum agrees with data published by Young et al. [Full size image

Gene ontology-based functional enrichment analysis revealed that the HSP family networks were mainly enriched for proteins involved in “protein processing in the endoplasmic reticulum” Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, except for HSP 10 and HSP 60 networks, which were dominated by proteins involved in “ribosome” and “metabolic pathways”, respectively (Fig. 5). As the HSP 60/HSP 10 chaperonin complex functions in the mitochondrial matrix [47], it is not surprising that most of their interacting proteins are mitochondrial matrix proteins. Proteins involved in “protein export” were enriched in the HSP 40 and HSP 70 networks; these HSPs are known to intimately engage in protein folding and trafficking mechanisms [48]. In Plasmodium falciparum, HSP 40 members are among the exportome proteins involved in the correct presentation of P. falciparum erythrocyte membrane protein 1 on the host cell surface [49,50,51]. The HSP families were also dominated by other interesting KEGG pathways important for schistosome biology [e.g. “RNA degradation”, “autophagy—animal”, “citric cycle (TCA) cycle”, “carbon metabolism”, “mitophagy—animal”, “endocytosis”, “phagosomes”, “glycolysis or gluconeogenesis”, “spliceosome”, “peroxisome”, “oxidative phosphorylation”, and “biosynthesis of amino acids”]. The HSP 90 network was the only network enriched for proteins involved in “FoxO signalling” (5/96) and “mTOR signalling” (4/96) pathways. Collectively, these data provide a comprehensive framework for further understanding the biological and functional actions of these HSPs in S. mansoni.

Phosphoproteome analysis of S. mansoni HSPs

Protein phosphorylation is an important post-translational modification, involving kinase-mediated addition of phosphate to serine, threonine, or tyrosine residues in eukaryotes, with removal enabled by protein phosphatases; such “switching” of phosphorylation status governs protein–protein interactions and activation of cellular signalling pathways [52, 53]. Given the importance of HSPs to cellular regulatory processes, experimentally discovered phosphorylation sites were mined from the recently published S. mansoni phosphoproteome [54] and were annotated. At least one bone fide phosphorylation site was found in 17 of the 55 HSPs, with 76 phosphorylation sites detected in total (Fig. 6; Additional file 4: Figure S1; Additional file 5: Dataset S4), including 20 sites within HSP functional domains. Over 80% (67/76) of the identified S. mansoni HSP phosphorylation sites were also successfully predicted by the phosphorylation site prediction tools, the Phosphorylation Site Database (PHOSIDA) and the Human Protein Reference Database (HPRD). The phosphorylation sites amongst the HSPs were 51% phosphoserine, 28% phosphothreonine and 21% phosphotyrosine. HSP 40, HSP 70 and HSP 90 family members were mainly phosphorylated on serine residues, whereas 60% of HSP 60 phosphorylation sites were on threonine (Fig. 6). No phosphorylation sites were found or predicted for HSP 10.

Protein kinase substrates contain short sequence motifs surrounding the phosphorylated residue which facilitate their recognition by the protein kinase. Therefore, using the PhosphoMotif discovery tools, HPRD and PHOSIDA, motifs surrounding the phosphorylated residue on each of the S. mansoni HSPs could be matched to known kinases. These included PKC, PKA, Akt, ERK1/2, CK1/2 GSK-3, Chk1, CAMK I, II, IV and GPCR, with the CK1/2 (e.g. SPxxS, TxxS) motif being the most represented in the dataset (Fig. 6; Additional file 4: Figure S1; Additional file 5: Dataset S4). TC-PTP and SHP1 phosphatase substrate motifs were also identified on Smp_035200.1 (HSP 40), Smp_106130.1 (HSP 70) and Smp_072330.1 (HSP 90). HPRD also identified consensus sites for protein binding, including 14-3-3, BARDI BRCT, Plk1 PBD, WW, FRIP PTB and MDC1 BRCT domain binding motifs, which function as modular protein domains that mediate protein–protein interaction between the phosphorylated protein and client protein.

Evolutionary conserved phosphorylation sites are believed to be of core functional relevance among certain species [55]. Therefore, S. mansoni HSP phosphorylation sites were compared with those of humans through manual alignment of each S. mansoni phosphorylated HSP with its human ortholog (Additional file 6: Figure S2). Thirty phosphorylation sites (out of the 76 identified) were conserved with human HSPs (Table 2), with Smp_072330 (HSP 90) containing the most (nine sites). Insight into the function(s) of such phosphorylation sites in the human HSPs could provide a comprehensive framework for understanding their role in S. mansoni HSPs. For example, CK2-mediated phosphorylation of human HSP 90α Ser²³¹/HSP 90β Ser²²⁶, equivalent to S. mansoni HSP 90α isoform 2-like (Smp_072330) Ser²²⁵, caused dissociation of the aryl hydrocarbon receptor (AhR; a transcription factor associated with cellular response to environmental stimuli such as xenobiotics)—HSP 90 complex and destabilisation of AhR protein in humans [56, 57]. Mutation of this site to a non-phosphorylatable alanine increased the transcriptional activity of AhR and stabilized its interaction with HSP 90 [56]. Also, phosphorylation of human HSPA1A (HSP 70) at Tyr⁴¹, similar to S. mansoni heat shock cognate 71 kDa protein isoform 1-like (Smp_106930) Tyr³⁹, regulates HSP 70 protein stability, and inhibition of phosphorylation of HSP 70 at this site with erlotinib (a tyrosine kinase inhibitor) results in increased degradation of HSP 70 [58]. Further understanding of the role of conserved HSP family member phosphorylation sites in humans might provide valuable insight into schistosome HSP function and reveal novel strategies for schistosome control.

Table 2 Phosphorylation sites identified in Schistosoma mansoni HSPs that are conserved with phosphorylation sites present in the human HSP orthologs

Full size table

Map** the expression of HSPs in S. mansoni

Because HSPs are evolutionarily conserved, commercially available antibodies against HSPs (mainly targeting Homo sapiens sequences) were selected based on conservation in the antibody binding region (where known) with the S. mansoni protein; antibodies recognising regions of high homology were selected (Additional file 7: Figure S3). In total, 13 antibodies were tested by western blotting of S. mansoni protein extracts, and five were found suitable (Fig. 7). A single immunoreactive band was detected with HSP 10 (~ 10 kDa), HSP 60 (~ 60 kDa), HSP 70 (~ 70 kDa) and HSP 90 (~ 90 kDa) antibodies in 24-h somules, adult male and female worms (Fig. 7), at the expected size. However, when testing anti-HSP 40 antibodies raised against human DNAJB1, which has closest homology to S. mansoni Smp_104730, a band at ~ 40 kDa was detected in the adult worms and in the 24-h in vitro cultured somules; a stronger ~ 70-kDa band was also detected, but only in the somules.

The in situ distribution of HSPs in cercariae, 3-h and 24-h in vitro cultured somules, and adult male and female S. mansoni were next determined using immunofluorescence and confocal laser scanning microscopy. In all cases, negative controls of the different life stages showed minimal background staining (Additional file 8: Figure S4). Labelling of intact cercariae, 3-h and 24-h somules, male and female adult worms with anti-HSPE1 (HSP 10) antibodies and analysis of image projections and/or individual confocal z-sections revealed prominent expression of HSP 10, an ATP-independent mitochondrial resident protein, in the oesophagus, cephalic ganglia, sub-tegument and parenchyma tissue of cercariae (Fig. 8a). Similarly, HSP 10 localised to the sub-tegument and cephalic ganglia of 3-h and 24-h in vitro cultured somules (Fig. 8b, c). In addition, HSP 10 was observed in the acetabulum and tegument of 3-h somules (Fig. 8b) and the gland duct of 24-h somules (Fig. 8c). In adults, HSP 10 was clearly evident in the testes and surface tubercles of males (Fig. 8d, e), and in the ovary and some vitelline cells of the female (Fig. 8f, g). For HSP 60, another mitochondrial resident protein and a co-chaperone of HSP 10, immunoreactivity was observed in the oesophagus, cephalic ganglia, and spines of cercariae (Fig. 9a, b) and in the acetabulum, cephalic ganglia, gland duct and tegument of 3-h (Fig. 9c) and 24-h (Fig. 9d) somules. In adults, HSP 60 was most noticeable in the testes and tubercles of males (Fig. 9e, f) and tegument/sub-tegument of females (Fig. 9g). HSP 60 was also present in the parenchymal tissue of the adult worms.

Labelling with anti-HSP 40 antibodies revealed the presence of HSP 40, a co-chaperone of HSP 70, in the cercarial gland duct, the acetabulum, cephalic ganglia (Fig. 10a) and cercarial tail tissue (Fig. 10b). In 3-h and 24-h somules, HSP 40 localised predominantly to the tegument, sub-tegument, acetabulum, and cephalic ganglia (Fig. 10c, d). HSP 40 was mainly found in the tubercles of males (Fig. 10g) and tegument of both male and female worms (Figs. 10f and h); however, it was absent from the testes of the male worms (Fig. 10e). Parenchyma tissue was also stained. Cercariae, 3-h and 24-h somules, male and female worms labelled with anti-HSP 70 antibodies displayed similar localisation patterns (Fig. 11) to that seen for its co-chaperone HSP 40. However, in addition, striking immunoreactivity was observed at the head–tail junction of cercariae (Fig. 11a) and 24-h somules (Fig. 11c), and the oral tip of the 24-h somule (Fig. 11c).

Labelling with anti-HSP90β antibodies revealed expression of HSP 90 in the cephalic ganglia and gland duct of cercariae (Fig. 12a) and 3-h somule (Fig. 12b). In addition, HSP 90 was observed in the sub-tegument, head/tail junction and tegument of 3- and 24-h somules (Fig. 12b-c). HSP 90 was also evident in the testes and tubercles of male worms (Fig. 12d–f) and the ovary and sub-tegument of the females (Fig. 12g, h). Antibody staining was broadly consistent between HSPs that are known to act as co-chaperones, supporting the idea that the anti-HSP antibodies were reacting with their intended targets in intact worms. All antibodies also reacted with proteins of the expected molecular weights (Fig. 7). However, the possibility that the antibodies might interact with other S. mansoni proteins cannot be ruled out, especially with anti-HSP 40 antibodies, where an immunoreactive band was observed at ~ 70 kDa in 24-h in vitro transformed somules only. Furthermore, unlike HSP 10 and HSP 60 that have only one family member, several members make up the HSP 40, 70 and 90 families; we are unable therefore to identify the specific HSP member(s) of these families that have been mapped within the parasite stages. Notwithstanding these caveats, the immunofluorescence profiling of multiple HSPs across several human-infective schistosome life stages provides a detailed and novel insight into the potential roles of these proteins in coordinating schistosome function.

Members of the HSP 90 family interact with steroid hormone receptors and are essential for fertility [59]. HSP 90α has been implicated to play a role in female mouse oocyte meiosis [60]; in males, the absence of HSP 90 caused the disruption of testicular development and azoospermia [61]. Similar to the data published by Xu et al. [62] on S. japonicum, HSP 90 was prominent in the ovary and testes of adult S. mansoni worms, implying a potential role in the regulation of adult fertility and differentiation of the germline stem cell population in schistosomes. Interestingly, HSP 60, unlike its co-chaperone HSP 10, was also present in the testes of the male worm, but was less prominent in the female ovary, suggesting a potential role in spermatogenesis but not in oogenesis.

The tegument proteins of schistosomes are known to play important roles in a variety of cellular processes, including nutrition, excretion, osmoregulation, and signal transduction, and key roles in host-parasite interactions, including immune evasion and modulation [2, 63]. From the data presented here, all HSPs investigated were present in the tegument of 3- and 24-h somules. This agrees with a previously published proteomic study where five HSPs (Smp_106930, Smp_148530, Smp_072330, Smp_008545 and Smp_049550) were detected in the somule tegument [21]. In addition, HSP 10, 40, 60, 70, and 90-like proteins have been identified by proteomics in tegument surface membranes of adult S. mansoni after differential extraction [64]. Collectively, these data demonstrate that HSPs likely play a role in assisting the parasite to adapt to the host immune microenvironment, supporting its transition from an immune-sensitive to an immune refractory state.

Conclusions

To the best of our knowledge, this study provides the first comprehensive and comparative analysis of schistosome HSPs, with a particular focus on those of S. mansoni. The aim of presenting these novel insights is to provide a valuable framework for future research investigating the importance of HSPs to schistosome growth, development, maturation and survival. In recent years, there has been an increased focus on HSPs in the field of disease research, with HSPs considered promising drug targets for infectious diseases and non-communicable diseases like cancer [65,66,67,68]. Several attributes of HSPs make them potential drug targets for the treatment of human schistosomiasis. Firstly, HSPs are essential regulators of protein homeostasis, and thus their direct inhibition can be lethal; the central involvement of HSPs in functional networks can also hamper downstream metabolic pathways. In addition, the interaction of HSPs, particularly HSP 90, with numerous intracellular and receptor regulated kinases makes them important gatekeepers of the signal transduction machinery that regulates cellular function. The human orthologs of some members of three S. mansoni HSP families, HSP 40 (Smp_035200, Smp_047190), HSP 70 (Smp_106930, Smp_049550, Smp_106130) and HSP 90 (Smp_072330, Smp_155740 and Smp_030300), are highlighted as druggable targets on the Tropical Disease Research (TDR) Targets database (www.tdrtargets.org). Despite the high similarity between HSPs of S. mansoni and humans, evident differences in amino acid sequence may be sufficient to allow development of inhibitors that specifically target only the HSPs of the parasite, perhaps with a focus on those that are highly expressed in the human-host resident life stages. That HSPs were detected in numerous tissues near the surface of the somules and adult worms, including the tegument, by immunofluorescence, supports the idea that delivery of drugs targeting HSPs to the worms should be achievable. Furthermore, consideration of HSP protein–protein interactions, including those involving cell signalling networks and phosphorylation of HSP residues, should facilitate a systems level understanding of such processes in the context of HSPs. Future research should also encompass investigations into the importance of HSPs during schistosome host-parasite interactions, particularly given the abundance of HSPs in schistosome extracellular vesicles [69, 70]. Because HSPs act as important gatekeepers that govern regulatory control of multiple mechanisms within cells, we anticipate that schistosome HSPs could emerge as excellent targets for the development of novel drug-based anti-schistosome therapeutics.

Methods

Schistosome material

Biomphalaria glabrata snails were infected with miracidia released from eggs isolated from livers of experimentally infected mice kept for the laboratory maintenance of the S. mansoni (Puerto Rican strain) life cycle at the Wellcome Sanger Institute, UK (courtesy of Dr Gabriel Rinaldi). All mouse procedures were performed under the approval of the Animal Welfare and Ethical Review Body of the Wellcome Sanger Institute, and in accordance with the UK Home Office approved project license P77E8A062. When patent, snails were placed in filtered tap water (Brimac filter, Silverline) and exposed to light to induce cercarial emergence. Cercariae were either fixed immediately for immunofluorescence and confocal microscopy or mechanically transformed into skin somules as previously described [71]. The somules were maintained in 48-well plates (non-tissue culture treated; 1000 somules/ml of Basal Medium Eagle (ThermoFisher Scientific) containing 1% antibiotics/antimycotic in each well) overnight at 37 °C/5% CO₂ before fixing in ice cold acetone or processing for western blotting. Adult S. mansoni worms were also supplied by Dr Gabriel Rinaldi of the Wellcome Sanger Institute.

Database mining and HSP identification

The name and nomenclature for each H. sapiens HSP were obtained from the Hugo Gene Nomenclature Committee (HGNC) (https://www.genenames.org/data/genegroup/#!/group/582). The FASTA amino acid sequence for each human HSP was retrieved from UniProt (https://www.uniprot.org/) and a protein Basic Local Alignment Search Tool (BLASTp) search against S. mansoni carried out at WormBase ParaSite (https://parasite.wormbase.org/index.html) using each human HSP sequence as a query. The best sequence matches were recorded, duplicate sequences carefully removed, and those partial in length or missing important HSP functional domains were excluded following interrogation with InterPro (https://www.ebi.ac.uk/interpro/). In addition, WormBase ParaSite was further queried with S. mansoni HSP family names. The final proposed name for each HSP was derived from reverse-BLAST of each S. mansoni HSP against non-redundant H. sapiens protein sequences at NCBI (https://blast.ncbi.nlm.nih.gov); HSPs for free-living flatworms were also obtained using this approach. S. mansoni HSP amino acid sequences were used as the query sequence for the other Schistosoma species, with searches performed at WormBase ParaSite.

Phylogenetic analysis

To investigate the phylogenetic relationship between the identified S. mansoni HSPs as well as between HSPs of S. mansoni, S. haematobium and S. japonicum, protein sequences were aligned using ClustalW [72]. The aligned sequences were then exported into Molecular Evolutionary Genetic Analysis (MEGA) X (https://www.megasoftware.net/) and the neighbour joining method of phylogenetic analysis used to infer the evolutionary relationship between the HSPs. The “model” feature on MEGA X was used to find the best model for estimating the tree with the default setting.

Retrieval of S. mansoni HSP expression data

Data on HSP expression across the various S. mansoni developmental life stages were obtained from schisto.xyz (http://schisto.xyz/) using S. mansoni Smp_identifiers. The maximum expression for each gene was assigned a value of 100% and the expression values for each life stage were then calculated as a percentage of this. A heatmap-based comparative analysis of the normalised expression levels across the various life stages was constructed using Microsoft Excel.

Interactomics

Possible associations between S. mansoni HSPs and other proteins were investigated using Cytoscape 3.6.0. running the Cytoscape plugin, StringApp, which imports data from the Search Tool for the STRING database [73]; the analysis was done at 0.70 (high) confidence with 100 additional interactors. The generated network was then analysed within Cytoscape, including the interrogation of KEGG pathway data retrieved using STRING enrichment analysis.

Phosphoproteome

Phosphorylation sites present within each of the identified S. mansoni HSPs were mined from the S. mansoni phosphoproteome dataset published by Hirst et al. [54] searching by Smp_identifier and sequence. Domain information for each HSP was retrieved from the conserved protein domain tool within NCBI BLASTp and from InterPro. Potential protein kinases and protein phosphatases that are responsible for phosphorylating/dephosphorylating identified phosphorylation sites were discovered using HPRD (https://www.hprd.org/) PhosphoMotif Finder and PHOSIDA (http://www.phosida.de/) de novo motif finder. Information on consensus sites for protein binding to phosphorylation motifs was identified using HPRD, and conserved phosphorylation sites between human and S. mansoni HSPs were identified through PhosphoSitePlus (https://www.phosphosite.org/homeAction.action).

Protein extraction and sodium dodecyl sulphate–polyacrylamide gel electrophoresis/western blotting

Adult worms were homogenised on ice in 1× radio immunoprecipitation assay buffer (Cell Signalling Technology, Leiden, The Netherlands) containing 1% Halt protease and phosphatase inhibitor cocktail (Thermo Scientific) (15 µl/worm) using a motorised microfuge tube pestle. Particulate material was pelleted by 30-s pulse centrifugation and the supernatant collected. An appropriate volume of sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis sample buffer (4×) and sample reducing agent (10×) (Life Technologies) was added and the sample heated at 95 ℃ for 5 min and sonicated for 1 min. For somules, each 24-h somule sample (~ 1000 somules) was transferred from the culture well into a microfuge tube on ice for 5 min and pulse centrifuged. Pelleted somules were then lysed in sample buffer and sample reducing agent before heating at 95 ℃ for 5 min. Protein extracts from cercariae were obtained in the same way.

The parasite protein samples (~ 10 µg) were electrophoresed using 4–12% Bolt Bis–Tris Plus gradient gels (ThermoFisher Scientific) in 2-morpholinoethanesulphonic acid monohydrate (MES) SDS Running Buffer (Life Technologies), after which they were transferred to nitrocellulose membrane (GE Healthcare Life Sciences). Blots were blocked in 1% bovine serum albumin (BSA)/Tween Tris-buffered saline (TTBS) and incubated overnight with gentle agitation at 4 °C in either anti-HSP 70 (SMC-162 C/D; StressMarq), anti-HSP 60 (SMC-111A/B; StressMarq), anti-HSP 40 (SPC-100; StressMarq), anti-HSPE1 (ARPP54651_P050; Aviva Systems Biology), or anti-HSP 90β (STJ93608; St. Johns Laboratory) antibodies (1:1000 dilution in BSA/TTBS). Blots were then washed with TTBS and incubated in the secondary antibody (1:3000 anti-mouse IgG, HRP-linked or anti-rabbit IgG, HRP-linked antibody in TTBS; Cell Signalling Technology). The wash step was repeated, and blots were then visualised using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) on a GeneGnome imager (Syngene).

Immunofluorescence

Cercariae, somules (24 h, cultured in vitro), and male and female adult worms were fixed in ice-cold acetone and placed at 4 ℃ for at least 24 h. They were then washed in phosphate-buffered saline (PBS) and treated with 1% glycine in PBS, before permeabilisation in 0.3% Triton X-100 in PBS. Samples were washed in PBS and then blocked in 10% goat serum for 2 h. For adult worms incubated with anti-mouse primary antibodies, further blocking with 0.1 mg/ml Goat F(ab) Anti-Mouse IgG H&L (ab6668; abcam) was performed. Washing was repeated and the samples were incubated in either anti-HSPE1, anti-HSP 40, anti-HSP 60, anti-HSP 70, or anti-HSP 90 primary antibodies (1:50 in 1% BSA in PBS) for 3 days with gentle agitation at 4˚C. The parasites were washed with PBS and incubated with Alexa Fluor 488 secondary antibody (1:500 in PBS) (Invitrogen, UK) for 2 days at 4 °C. Control parasites were treated similarly but without the addition of the primary antibody. After three 30-min washes, samples were carefully transferred onto Silane-Prep Slides (Sigma) and mounted under nail varnish-sealed coverslips in VECTASHIELD mounting medium (Vecta Laboratories, UK). Slides were then visualised on a Leica SP2 AOBS or Zeiss LSM 800 confocal laser-scanning microscope using 40× or 63× oil immersion objectives.

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Acknowledgements

We are extremely grateful to Gabriel Rinaldi and Matt Berriman (Wellcome Sanger Institute, Cambridge) for supplying schistosome material for this work.

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Molecular Parasitology Laboratory, School of Life Sciences, Pharmacy and Chemistry, Kingston University, Penrhyn Road, Kingston upon Thames, KT1 2EE, Surrey, UK
Nancy A. Aguoru, Ruth S. Kirk & Anthony J. Walker

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Nancy A. Aguoru
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Anthony J. Walker
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Contributions

NAA designed and carried out the experimental work, interpreted and analysed the data, and wrote the manuscript. AJW conceived and supervised the project, designed experiments, interpreted the data and wrote the manuscript. RSK conceived and supervised the project and critically revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Anthony J. Walker.

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Additional file 1

: Dataset S1. Mining Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobium HSPs. Two tabs are included, the first details the mining of S. mansoni HSPs against human HSP protein sequences using BLASPp and the second details the comparative analysis between the three Schistosoma spp.

Additional file 2

: Dataset S2. Expression of HSP genes in different developmental stages of Schistosoma mansoni. Data are presented as raw normalised gene expression and as percentages of the highest gene expression value.

Additional file 3

: Dataset S3. Putative protein-protein interactions (nodes) for HSP family members. Six tabs are included, one for each HSP family and one for all HSP families combined.

Additional file 4

: Figure S1. Phosphorylation sites within Schistosoma mansoni HSPs. Data on the phosphorylation sites of S. mansoni HSPs were mined from the S. mansoni phosphoproteome published by Hirst et al. [54]. Thirteen HSPs are shown, the remaining four can be viewed in Fig. 6. Domains within each HSP were identified using the conserved protein domain tool within InterPro and NCBI. Identification of putative upstream kinases, phosphatases and binding motifs were obtained using HPRD motif finder and PHOSIDA. Kinases are in black and are denoted by a plus or a cross symbol, phosphatases are in red and are denoted by a minus symbol, binding motifs are in blue and are denoted by an asterisk.

Additional file 5

: Dataset S4. Phosphorylation sites within Schistosoma mansoni HSPs, mined from the S. mansoni phosphoprotein dataset, and motifs identified with HPRD and PHOSIDA. Six tabs are included, representing (i) the identified phosphorylation sites; (ii) sequence (+/− six amino acids) surrounding the phosphorylation site; and (iii–vi) motifs identified within HSPs 40, 60, 70, and 90.

Additional file 6

: Figure S2. Comparative pairwise alignments of human HSP amino acid sequences against corresponding Schistosoma mansoni sequences (identifiers are provided in the figure), with homologous phosphorylation sites highlighted (in red). Phosphorylation sites from human HSPs were obtained from PhosphoSitePlus (https://www.phosphosite.org/homeAction.action) and were matched to S. mansoni sites obtained from the S. mansoni phosphoprotein dataset (Additional file 4: Figure S1).

Additional file 7

: Figure S3. Comparative pairwise alignments of human HSP amino acid sequences [HSP 10 (CAG28616), HSP 40 (NP_006136.1), HSP 60 (NP_002147), HSP 70 (NP_005336.3) and HSP 90 (NP_001258898.1)] against a corresponding Schistosoma mansoni HSP sequence {HSP 10 (Smp_097380), HSP 40 (Smp_104730), HSP 60 (Smp_008545), HSP 70 [Smp_106930 (Smp_302170)] and HSP 90 (Smp_072330)}. Highlighted areas are antibody recognition sites in the human protein. Where the antibody is raised to the whole protein sequence (such as anti-HSP 40 antibody and anti-HSP 70 antibodies), no regions are highlighted. Asterisks indicate positions that have a single, fully conserved residue; colons represent conservation between groups with strongly similar properties; periods indicate conservation between groups of weakly similar properties

Additional file 8

: Figure S4. Negative controls for the different life stages under study showing no/minimal background staining in the absence of anti-HSP antibodies. Samples were processed for immunofluorescence using Alexa Fluor 488 mouse (first slide) or rabbit (second slide) secondary antibodies and rhodamine phalloidin (in red) but without the addition of a primary antibody. Samples were mounted on slides and imaged using a Zeiss LSM 800 laser scanning confocal microscope. Representative micrographs are single z-sections through the parasites. Scale bars = 25 µm (for cercariae and somules) and 50 µm (for adult worms).

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Aguoru, N.A., Kirk, R.S. & Walker, A.J. Molecular insights into the heat shock proteins of the human parasitic blood fluke Schistosoma mansoni. Parasites Vectors 15, 365 (2022). https://doi.org/10.1186/s13071-022-05500-7

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Received: 30 May 2022
Accepted: 16 September 2022
Published: 13 October 2022
DOI: https://doi.org/10.1186/s13071-022-05500-7

Molecular insights into the heat shock proteins of the human parasitic blood fluke Schistosoma mansoni

Abstract

Background

Methods

Results

Conclusions

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Background

Results and discussion

Comparative analysis of S. mansoni HSPs

Phosphoproteome analysis of S. mansoni HSPs

Map** the expression of HSPs in S. mansoni

Conclusions

Methods

Schistosome material

Database mining and HSP identification

Phylogenetic analysis

Retrieval of S. mansoni HSP expression data

Interactomics

Phosphoproteome

Protein extraction and sodium dodecyl sulphate–polyacrylamide gel electrophoresis/western blotting

Immunofluorescence

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Acknowledgements

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Supplementary Information

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