The Schistosoma mansoni nuclear receptor FTZ-F1 maintains esophageal gland function via transcriptional regulation of meg-8.3

Schistosomes infect over 200 million of the world’s poorest people, but unfortunately treatment relies on a single drug. Nuclear hormone receptors are ligand-activated transcription factors that regulate diverse processes in metazoans, yet few have been functionally characterized in schistosomes. During a systematic analysis of nuclear receptor function, we found that an FTZ-F1-like receptor was essential for parasite survival. Using a combination of transcriptional profiling and chromatin immunoprecipitation (ChIP), we discovered that the micro-exon gene meg-8.3 is a transcriptional target of SmFTZ-F1. We found that both Smftz-f1 and meg-8.3 are required for esophageal gland maintenance as well as integrity of the worm’s head. Together, these studies define a new role for micro-exon gene function in the parasite and suggest that factors associated with the esophageal gland could represent viable therapeutic targets.


Introduction
Schistosomiasis is a disease caused by parasitic flatworms of the genus Schistosoma that affects over 200 million people. The main cause of disease pathology is the egg production of the

RNA interference
dsRNA was generated as previously described [30,31]. For both the initial NR screen and the RNA-seq "screen" RNAi experiments, RNAi experiments were performed as previously described using paired worms [32]. Subsequent experiments were performed using only male parasites. The parasites were fixed as previously described [31]. Throughout the 20-day experiment, worms were monitored for gross physical phenotypes (i.e., detachment from the plate, loss of movement, head curling, etc).

RNAseq for Smftz-f1 RNAi-treated worms
To examine gene expression changes following loss of Smp_328000 (Smftz-f1), worms were cultured as previously described [33] and only male worms were harvested after 5 or 9 days in culture. We predicted that transcriptional changes that occur at earlier timepoints following RNAi are most likely to represent direct targets of SmFTZ-F1, whereas changes at later time points would represent both the direct and indirect effects of SmFTZ-F1 loss. As controls, worms cultured in parallel were treated with a non-specific dsRNA [31]. For RNA extraction, male worms were collected, excess media removed, and 100 μL of TRIzol was added to the worms. Total cellular RNA was isolated from cells using TRIzol reagent (Invitrogen) following the manufacturer's protocol and processed for Illumina sequencing. Libraries for RNAseq analysis were mapped to the S. mansoni genome and analyzed as previously described [33]. RNAseq datasets for the Smftz-f1 (RNAi) experiments are available at NCBI through the accession number (GSE188736).

Purification of Recombinant SmFTZ-F1
To generate an anti-SmFTZ-F1 antibody, an N-terminal fragment of SmFTZ-F1 corresponding to AA 1-150 was amplified and subcloned into the pET28 vector with an N-terminal SUMO-His6 tag for expression in Escherichia coli. This fragment was purified from transformed BL21 cells grown in LB medium and induced with 1mM Isopropyl-β-D-thiogalactoside for 16 hrs at 18˚C. Cells were pelleted and resuspended into lysis buffer containing 50 mM NaH 2 PO 4 , 500 mM NaCl, 20 mM Imidazole, and protease inhibitors (Roche cOmplete, Mini, EDTA-free). The suspension was sonicated, lysate was centrifuged for 25 min at 14,000 x g, and rotated with resin for 1 hr. The resin was washed, and protein was eluted in lysis buffer containing 250mM imidazole. The sample was then dialyzed to remove the NaCl and imidazole. The SUMO-His6tag was then cleaved using purified ULP-1. The protein was then concentrated using an Amicon concentrator, 10-kDa cut-off (Millipore). Rabbit polyclonal antibodies were generated by YenZym (California).

Imaging
Confocal imaging of fluorescently labeled samples and brightfield imaging (i.e, whole mount in situ hybridizations) were performed using a Nikon A1 Laser Scanning Confocal Microscope or a Zeiss Axio Zoom V16 equipped with a transmitted light base and a Zeiss Axio Cam 105 Color camera, respectively.

Electrophoretic mobility shift assay (EMSA)
Biotin end-labeled probes were prepared using Pierce Biotin 3' End DNA Labeling Kit following manufacturer's instructions with the following modifications. Complementary oligos were end-labeled separately and then annealed before use. Oligos were annealed by mixing together equal amounts of labeled complementary oligos, denaturing the mixture at 90˚C for 1 minute, then slowly cooling and incubating at the melting temperature for 30 minutes. The oligos were then frozen and thawed on ice for use. SmFTZ-F1 protein was prepared by in vitro translation with PURExpress (NEB). EMSA was performed using the LightShift Chemiluminescent EMSA Kit (ThermoFisher Scientific, USA) according to the manufacturer's instructions. DNA binding reactions were performed in a 20 μL volume containing biotin-labeled oligonucleotides and in vitro translated protein from the PURExpress kit. Reaction products were then separated by electrophoresis. Thereafter, the protein-DNA complexes were transferred onto a positively charged nylon membrane (Millipore, USA) and detected by chemiluminescence using a GE Healthcare ImageQuant LAS 4000. For competitive binding experiments, 20-or 200-fold excesses of unlabeled DNA probes were also included in the binding reaction.

Immunofluorescence
The parasites were fixed in methacarn (60 mL of methanol, 30 mL chloroform, and 10 mL glacial acetic acid) for 1 hr. Parasites were dehydrated in MeOH and stored at −20˚C until processing. Worms were then processed similarly to colorimetric and fluorescence in situ hybridization following rehydration steps which were performed as previously described [23,29,31] with the following modifications. After post-fixation with 4% formaldehyde in PBSTx, the worms were then washed twice in PBSTx then were blocked with FISH block (0.1 M Tris pH 7.5, 0.15 M NaCl and 0.1% Tween-20 with 5% Horse Serum and 0.5% Roche Western Blocking Reagent [36]) for 1 hr and incubated overnight with 2.5 μg/mL of anti-SmFTZ-F1 antibody. After 6X PBSTx washes for 20 minutes each, the worms were then incubated in secondary antibody (1:1000 Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate) in FISH block for 4 hours at 4˚C. After several washes in PBSTx, parasites were counterstained with DAPI for 30 minutes then placed in 80% glycerol and were then mounted on slides in Vectashield (Vector Laboratories). amputated and processed for chromatin immunoprecipitation. Briefly, worms were fixed with formaldehyde (0.75%) to cross-link DNA and proteins, DNA was digested using micrococcal nuclease, chromatin was sheared using a Biorupter (8 cycles of sonication: 30s each, 0.5 sec on, 0.5 sec off; amplitude = 30%,), chromatin was incubated with antibodies (SmFTZ-F1 at 5 μl, preimmune serum negative control at 5 μl), and immunoprecipitates were bound to protein G magnetic beads (30 μl). The protein-DNA cross-linking was reversed, DNA was purified, and enrichment of DNA sequences was detected using qPCR and primers listed in S1 Table. Data were normalized and analyzed using percent input analysis [37].

FISH/WISH using RNAi treated worms
Colorimetric and fluorescence in situ hybridization analyses were performed as previously described [23,29,31]. Dextran labeling of the parasite gut was done as previously described [23]. All fluorescently labeled parasites were counterstained with DAPI (1 μg/ml), cleared in 80% glycerol, and mounted on slides with Vectashield (Vector Laboratories).

Carmine staining
Carmine red staining was performed as previously described [38]. Confocal laser scanning microscopy images were taken on a Nikon A1 Laser Scanning Confocal Microscope.

Statistical analysis
All two-way comparisons were analyzed using Student's t-test. RNAseq data was analyzed using DESeq2 [39].

An SmFTZ-F1 homolog is essential for parasite vitality
To investigate potential roles that nuclear receptors play in schistosome biology, we set out to examine their expression and function using both whole mount in situ hybridization (WISH) and RNA interference (RNAi). We cloned either full or partial length cDNAs for 17 of 21 receptors from adult S. mansoni cDNA. We were unable to clone the remaining four due to low expression in the adult stage [8]. Expression analyses revealed that a majority of the NRs were expressed in the reproductive systems of both the males and females (S1 Fig). VF1, a parasite-specific NR [40], was expressed in cells of the female vitellaria (a tissue responsible for making "yolk" cells) as well as what appeared to be primordial vitellaria of male worms (S1 Fig). Although many NRs were broadly expressed throughout the parasite tissues (e.g., Smftz-f1), the expression of some NRs was restricted to smaller sets of cells (e.g., SmNR4A4 and SmCOUP-TF1).
To explore the function of the schistosome NR complement, we performed RNAi for each NR and examined whether any of these treatments compromised parasite vitality in vitro ( Fig  1A). We found that RNAi-mediated depletion of the mRNA encoding the schistosome ftz-f1 homolog [12,22] resulted in a fully penetrant degenerative phenotype (Fig 1B and 1C). By day 9 (D9), male and female Smftz-f1(RNAi) parasites became unpaired and lost the ability to attach to the surface of the tissue culture dish, and by D20, movement in Smftz-f1(RNAi) parasites was virtually nonexistent in male worms (S1 Video). The mRNA of Smftz-f1 was found to be expressed throughout both sexes of the worm (Figs 1D and S2A). Similar phenotypes were observed with dsRNAs targeting two distinct regions of the Smftz-f1 gene, indicating these effects are specific to the reduction of Smftz-f1 level and not due to off-target effects (S2B- S2D  Fig).

RNAseq reveals possible direct targets of SmFTZ-F1
NRs are DNA-binding transcriptional regulators [41]. Therefore, we reasoned we could use transcriptional profiling to identify potential direct transcriptional targets and gain a deeper insight into the molecular changes responsible for the Smftz-f1(RNAi) phenotype. For these studies, we compared the transcriptional profile of control and Smftz-f1(RNAi) parasites at timepoints before ("early" D5 post-RNAi) and after ("late" D9 post-RNAi) we observed the degenerative changes in the Smftz-f1(RNAi) treatment group. We predicted that transcriptional changes that occur at the "early" timepoint following RNAi are most likely to represent direct targets of SmFTZ-F1, whereas changes at the "late" time point would represent both the direct and indirect effects of SmFTZ-F1 loss (Fig 2A). Since FTZ-F1 orthologs in other systems are transcriptional activators [34,42,43], we focused on genes down-regulated following RNAi. As predicted, on D5 following Smftz-f1(RNAi) treatment we noted that a relatively small number of genes (25) were down-regulated; this number increased to 263 genes by D9 of RNAi treatment ( Fig 2B) (log 2 fold-change -1.5, p < 0.01)(S2 Table). Importantly, 22 of the 25 genes down-regulated on D5 were also down on D9 (Fig 2B). qPCR validation studies of the 20 most downregulated of these 25 genes confirmed our observations from RNAseq for 19 out of 20 genes ( Fig 2C, S3 Table).

PLOS PATHOGENS
SmFTZ-F1 maintains esophageal gland function via transcriptional regulation of meg- 8.3 Previous studies using Electrophoretic Mobility Shift Assays (EMSA) showed that SmFTZ-F1 can bind to a human SF-1 response element [12]. To examine whether this sequence could be found in the regulatory regions of genes down-regulated at both D5 and D9, we searched 2kb upstream of the transcription start site of each gene for a related response element using FIMO [34,35]. This analysis revealed 64 putative SmFTZ-F1 response elements present upstream of 22 genes. We examined 30 of these putative response elements by EMSAs to determine which of these elements were bound by SmFTZ-F1 (S4 Table, S3 Fig). These studies revealed that SmFTZ-F1 bound efficiently to sequences upstream of seven genes ( Fig  2D), suggesting a potential consensus binding sequence for SmFTZ-F1 (Fig 2E). We examined these seven genes in more detail by performing WISH on control and Smftz-f1(RNAi)-treated parasites. Consistent with our RNAseq data ( Fig 2B) and qPCR studies (Fig 2C), each of the seven genes was markedly downregulated following RNAi treatment (Fig 3A). With the exception of Smp_331380, which was expressed specifically in the esophageal gland, these genes were broadly expressed throughout the worm, and their expression appeared to be uniformly reduced by Smftz-f1 knockdown. This observation is consistent with SmFTZ-F1 regulating gene expression across several cell types.

Smftz-f1 and meg-8.3 share similar RNAi phenotypes
We could envision two non-mutually exclusive scenarios for the phenotype observed following Smftz-f1 knockdown. In the first scenario, loss of Smftz-f1 disrupts the expression of multiple target genes and this broad transcriptional misregulation results in parasite degeneration and death. Alternatively, loss of Smftz-f1 leads to misregulation of individual target genes resulting in some or all of the observed phenotype. To distinguish between these models, we performed RNAi on each of the seven of the putative Smftz-f1 target genes. Consistent with the second scenario, we observed that RNAi of Smp_331380 led to detachment of parasites from the tissue culture dish beginning at D12 and a head curling phenotype identical to that observed following Smftz-f1(RNAi) (Fig 3B and 3C).
Based on BLAST homology, Smp_331380 appeared to be a member of the schistosome-specific MEG-8 protein family, sharing complete sequence identity with the previously described MEG-8.3 protein [44,45]. Thus, we will refer to Smp_331380 as meg-8.3 from this point forward. MEG proteins are small proteins of largely unknown function that are encoded by small exons (~6 to 36 bp) that were uncovered during the sequencing of the S. mansoni genome [46,47]. The meg-8 protein family includes four paralogs (meg-8. 1, 8.2, 8.3, and 8.4), whose homolog in Schistosoma japonicum MEG-8.2 appears to interact with host leukocytes as they pass by the worm's esophageal gland [48]. The meg-8.3 gene is predicted to encode an 11 kD protein with a predicted signal peptide (S4A Fig) generated from 10 exons [49], some of which are as small as 15 bp in length (S4B Fig). In principle, the observed loss of meg-8.3 expression in the esophageal gland following Smftz-f1 loss could be due to either misregulation of meg-8.3 expression or loss of the esophageal gland tissue entirely. To explore this, we queried scRNAseq data [23] and examined the expression of a panel of genes expressed in the esophageal gland following Smftz-f1(RNAi) treatment. In contrast to meg-8.3 expression that was rapidly depleted following Smftz-f1 knockdown (Fig 3A), the expression of six esophageal gland marker genes were not obviously different in Smftz-f1(RNAi) parasites (Fig 3D). This suggests that loss of Smftz-f1 appears to rapidly and specifically lead to the reduction of meg-8.3 expression in the esophageal gland.
We next evaluated the general morphology of the esophageal gland using the lectin PNA [50]. Under control conditions, the worms maintained a normal esophageal gland, however loss of either Smftz-f1 or meg-8.3 resulted in a loss of PNA staining in the esophageal gland

PLOS PATHOGENS
SmFTZ-F1 maintains esophageal gland function via transcriptional regulation of meg- 8.3 with little labeling observed by D18 of RNAi (Fig 3E). Simultaneously, we noted a significant reduction in the expression of the esophageal gland marker, meg-4 between D15 and D18 of meg-8. 3 RNAi treatment (S4C and S4D Fig). Taken together, these data are consistent with the model that SmFTZ-F1 regulates the expression of meg-8.3 in the esophageal gland, and that loss of meg-8.3 expression in the tissue results in loss of esophageal gland function that is at least partially responsible for the detachment and head curling observed in Smftz-f1(RNAi) parasites.
Given that the S. mansoni genome contains three other MEG-8 proteins with sequence identity to MEG-8.3, we wanted to rule out the possibility that our phenotype was due to offtarget effects. Therefore, we measured the levels of meg-8.3 and its closest relative meg-8.  (Figs 3E, S4C and S4D). Therefore, to examine the potential of RNAi off-target effects on meg-8.2 levels following meg-8. 3 RNAi, we examined the expression of meg-8.2 and an unrelated esophageal gland marker gene cystatin (Smp_328980) [23]. When compared to meg-8.3 levels, we observed modest decreases in the levels of both meg-8.2 and cystatin following meg-8.3(RNAi) (S4E Fig), suggesting the reduction in transcript levels is due to tissue dysfunction rather than off target RNAi effects. This observation, along with the fact meg-8.2 and meg-8.3 share limited sequence identity on the nucleotide level [51], indicates that meg-8.3 RNAi-treatment specifically targets meg-8.3 levels.

SmFTZ-F1 controls meg-8.3 expression
To determine if meg-8.3 is indeed a direct target of SmFTZ-F1 in the parasite, we generated an antibody against SmFTZ-F1 and performed chromatin immune precipitation experiments. Our SmFTZ-F1 antiserum recognizes an~110 kD protein in whole worm lysates (S5A Fig Given the specificity of the anti-SmFTZ-F1 antibody, we set out to determine if meg-8.3 is a direct target of SmFTZ-F1 in vivo by ChIP. For these studies, we amputated the heads of male worms to enrich for cells expressing meg-8.3, performed ChIP using either anti-SmFTZ-F1 or pre-immune serum, and then used quantitative PCR to evaluate relative SmFTZ-F1 binding to various sites in the genome (Fig 3F and 3G). As an initial step, we performed various controls evaluating SmFTZ-F1 binding at distal regions 5 and 10 kb upstream of meg-8.3 and at a region surrounding a SmFTZ-F1 consensus response element found upstream of foxA, a gene encoding a transcription factor that appears to be a master regulator of esophageal gland identity [28]; we did not note significant SmFTZ-F1 binding to any of these sites (S6A-S6D Fig). We next evaluated the consensus SmFTZ-F1 response element upstream of meg-8.3 that was bound by SmFTZ-F1 in vitro (Fig 2D). Evaluation of the sequence surrounding this consensus site revealed that it was repeated at several sites within the S. mansoni genome (S1 File), ruling staining. n > 10 worms, 3 biological replicates. (E) PNA labeling (green) of esophageal gland using RNAi treated worms at days 9, 12, 18. Maximum intensity projections are shown. n > 10 worms, 3 biological replicates (F) Cartoon of the ChIP-qPCR experiment (G) Schematic of the 2kb upstream region of the meg-8.3 promoter with the sequences and position of the response element and primer set used, length not proportional to the size. (H) ChIP-qPCR, shown as percentage of input DNA, for SmFTZ-F1 or preimmune serum antibodies at the putative promoter of meg-8. 3

PLOS PATHOGENS
SmFTZ-F1 maintains esophageal gland function via transcriptional regulation of meg- 8.3 out possibility of designing primers that could specifically amplify this region of the genome. However, since a large number of transcription factor binding sites are known to exist in repetitive elements [52][53][54], we proceeded with ChIP studies. Using qPCR primers that amplify the region surrounding the putative SmFTZ-F1 binding site upstream of meg-8.3, we noted a statistically-significant 10-fold increase when we performed ChIP using the anti-SmFTZ-F1 (Fig 3H). ChIP-qPCR using Smftz-f1 RNAi-treated worms showed that the enrichment of SmFTZ-F1 at the response element in the meg-8.3 promoter was abolished to levels similar to those observed using preimmune serum (S6E Fig). These data, along with our RNAseq studies (Fig 2B) and EMSA data (Fig 2D), strongly support the model that SmFTZ-F1 binds to a response element found in the meg-8.3 promoter.

MEG-8.3 is essential for the maintenance of head tissue
Our data show that SmFTZ-F1 maintains the expression of meg-8.3, and loss of meg-8.3 expression results in profound morphological changes in the male head. Given the specific expression of meg-8.3 in the esophageal gland, we predicted that this phenotype could be attributed to defects in esophageal gland function. The esophageal gland appears to be critical for schistosome feeding and the passage of materials (e.g., cells) into the intestine [55]. To explore this, we cultured Smftz-f1 and meg-8.3(RNAi) parasites in fluorescent dextran, which the worms ingest and eventually absorb into their intestine [23,56], and performed FISH for cathepsin B (ctsb) to assess gut structure [23]. Following 12 hours of culture with dextran, we noted that control parasites assimilated the dextran into their intestine (Fig 4A). In contrast, we noted varying rates of dextran uptake in parasites between 5 to 18 days of Smftz-f1 and meg-8.3(RNAi) treatment. At early time points, Smftz-f1 and meg-8.3(RNAi) parasites were indistinguishable from controls. However, as the experiment continued, Smftz-f1 and meg-8.3 knockdown parasites progressively lost the ability to accumulate dextran in their intestine ( Fig  4B and 4C).
We also noted that as Smftz-f1 and meg-8.3 knockdown parasites failed to accumulate dextran in their intestine, they began accumulating the label in their protonephridial (excretory) system (Figs 4A and S7). The protonephrida are a network of ciliated and non-ciliated tubules that collect materials from the worm's tissues and excrete them via a pore at the posterior of the worm [55]. Thus, we reasoned that the dextran may permeate the tissues of Smftz-f1 and meg-8.3 knockdown worms and subsequently be collected in the protonephridal system. One potential mechanism to explain the presence of dextran in the worms inner tissues is that loss of SmFTZ-F1/MEG-8.3 results in degenerative changes that result in an inability of the worms to maintain barrier function. To examine this, we used carmine red straining to examine tissue integrity focusing on the worm's head since this is where the meg-8.3 RNAi phenotype was localized. Consistent with Smftz-f1 and meg-8.3 being critical for tissue integrity in the worm head, we noted significant differences in head morphology between control and Smftz-f1 and meg-8.3 RNAi parasites. While controls maintained their head tissues and cellular architecture, Smftz-f1 (on D12) and meg-8.3 (on D18) RNAi treatment resulted in notable disruption in tissue morphology that manifested as "holes" in the heads of the parasites.

Conclusions
Microexon genes have been cataloged across schistosome species and their expression appears to be mainly (but not exclusively) associated with the esophageal gland [44,49,57]. Beyond the fact that most microexon genes are predicted to encode secreted or membrane-anchored proteins in the worms [44,49,57], their molecular functions remain largely elusive. Our data suggest that disruption of either Smftz-f1 or its transcriptional target meg-8.3 (Fig 3) results in

PLOS PATHOGENS
SmFTZ-F1 maintains esophageal gland function via transcriptional regulation of meg-8.3 Intermediate animals have normal gut structure and possess fluorescent dextran in the gut, but also contain dextran collecting in the protonephridial system in the head (arrows). Severe animals possess a relatively normal gut structure and very little, if any, dextran labeling in the gut. Instead, dextran labeling is found in the protonephridial system (see arrows). (C) Plots depicting the relative

PLOS PATHOGENS
SmFTZ-F1 maintains esophageal gland function via transcriptional regulation of meg- 8.3 tissue degeneration, which in turn prevents normal worm attachment (Fig 3B and 3C), feeding (Fig 4A, 4B and 4C), and barrier function (Fig 4D). Thus, control of meg-8.3 expression in the esophageal gland appear critical for the parasite.
Recent studies have highlighted the importance of the esophageal gland not only in blood feeding but also as a central player in the parasite's ability to evade host immunity [28,58]. Indeed, loss of the transcription factor foxA results in esophageal gland loss, blunting the ability of parasites to survive in immunocompetent, but not immunocompromised, hosts [28]. Curiously, foxA(RNAi) parasites appear phenotypically normal during extended in vitro culture. This is in contrast to what we observe when meg-8.3 is depleted from the esophageal gland by RNAi (Fig 3B). Since loss of foxA results in loss of all examined esophageal gland markers, and presumably meg-8.3 as well, it would appear that loss of meg-8.3 can be tolerated as long as it occurs in the context of the depletion of other esophageal gland-specific factors. We speculate that MEG-8.3 acts in an inhibitory fashion to blunt the activity of other esophageal gland-expressed factors, and when MEG-8.3's inhibitory activity is lost, these factors have destructive effects on the parasite's tissues. Thus, when the expression of meg-8.3 is lost simultaneously with other esophageal gland-specific factors (e.g., following foxA RNAi) these destructive effects are mitigated. Exploring the biochemical function of meg-8.3 and defining potential interacting partners will be essential for addressing this issue in the future.
Taken together, our data not only highlight the critical role for SmFTZ-F1 in controlling meg-8.3 expression but also demonstrate that perturbing the function of a single esophageal gland-specific factor can have deleterious effects on the parasite. Because many esophageal gland-specific factors appear to be parasite specific [44,47], targeting factors associated with this tissue could yield highly-selective therapeutics. Therefore, establishing a deeper understanding the esophageal gland could result in new classes of drugs to treat schistosomiasis.

PLOS PATHOGENS
SmFTZ-F1 maintains esophageal gland function via transcriptional regulation of meg-8.3 S1 Table. Oligonucleotide sequences used in this study. (XLSX) S2 Table. RNAseq analysis of transcriptional changes following Smftz-f1(RNAi) treatment. D5 and D9 tables represent entire datasets from Day 5 and Day 9 of RNAi, respectively. Subsequent tabs show subsets of data for the most down-regulated genes at D5 or D9 and the genes that were common between the D5 and D9 datasets. (XLSX) S3 Table. qPCR values of 20 most differentially down regulated genes following Smftz-f1 (RNAi) treatment. (XLSX) S4 Table. Response element sequences used in this study. Response elements that were tested as well as their relative binding affinity. Scores are-(no binding) to + (binding).