Nematodes, or roundworms, are one of the largest animal phyla and are found in all ecosystems: marine, fresh-water and soil (van den Hoogen et al., 2019). In addition, many nematode species are parasites of plants, animals, life stock or humans, resulting in their economic importance for agriculture, breeding and health (Lee et al., 2002). Most of these parasites are hard to culture in the laboratory, whereas some free-living species are among the most rapidly reproducing and most easily culturable species of all metazoans. One example is Caenorhabditis elegans, which was introduced as a potential model organism by Sydney Brenner in the 1960ies (Brenner, 1974). Building on a unique set of life history traits and a growing functional toolkit, the ‘C. elegans research community’ has grown rapidly to make fundamental discoveries in all areas of the life sciences. C. elegans can be grown on small agar plates in the laboratory, feeds on Escherichia coli and completes its life cycle in 3 days at 20 °C (Figure 1A). This species reproduces primarily by selfing of hermaphrodites, which produce around 200–300 sperm during larval development before switching to a female fate with oocyte production as adults. Consequently, the progenies of hermaphrodites are genetically identical and the resulting isogenic propagation represents a unique advantage among all animal model organisms. Original unbiased forward genetic screens have identified many mutants essential for animal survival, egg-laying, neurogenesis or life span (Wood and Researchers, 1988). With RNA interference and CRISPR/Cas9-based genome engineering being implemented, large scale reverse genetic screens have been performed aiming at the analysis of the knock-down or knock-out of all genes in the genome (Fire et al., 1998; Dickinson and Goldstein, 2016). Indeed, when the C. elegans genome was fully sequenced, the 100 Mb genome was shown to encode around 20,000 genes with several thousands of them being functionally characterized (Consortium, 1998). Together, these features made C. elegans one of the most important study systems in modern biology.

The related nematode Pristionchus pacificus shares many of these features with C. elegans and similarly builds on a short generation time (4 days, 20°C), self-fertilizing hermaphroditism, forward and reverse genetic tools and a fully sequenced genome (Sommer and Sternberg, 1996; Dieterich et al., 2008; Han et al., 2020). P. pacificus and C. elegans, like many nematodes, can undergo direct and indirect development (Figure 1A). Under favorable environmental conditions and the availability of food, they undergo direct development, rapidly proceeding through four juvenile (larval) stages to become adults. In contrast, in the absence of food and other unfavorable circumstances, they arrest in a long-lived and stress-resistant “dauer” stage, which is adapted to survive harsh environmental conditions (Figure 1A) (for review see (Sommer and Mayer, 2015)). Besides these conserved features, P. pacificus differs from C. elegans with regard to several morphological and organismal features that turned out to be under the control of sulfation-mediated processes. Specifically, while P. pacificus can be cultured on E. coli as single food source indefinitely, it is a potential predator of other nematodes (Figure 1B). It forms teeth-like denticles in its mouth that are unknown in C. elegans or most other free-living nematodes (Figure 1C) (Bento et al., 2010). These teeth come in two alternative mouth-forms, representing an example of developmental (phenotypic) plasticity (Sommer, 2020). Worms can either form a “eurystomatous” (Eu) morph with two teeth and a wide buccal cavity, or, alternatively, a “stenostomatous” (St) morph with a single tooth and a narrow opening (Figure 1C) (Ragsdale et al., 2013). While both morphs can feed on bacteria, only the Eu form is a potential predator with associated self-recognition systems preventing the killing of kin (Lightfoot et al., 2019). The role of sulfation enzymes, the sulfatase eud-1 and the sulfotransferase seud-1/sult-1, in the regulation of mouth-form plasticity were discovered through unbiased forward genetic screens and will be described below (Ragsdale et al., 2013; Bui et al., 2018; Namdeo et al., 2018).

Eukaryotes express cytosolic and membrane-bound sulfotransferases. Cytosolic sulfotransferases (termed SULTs) sulfate xenobiotics and small molecules like neurotransmitters and hormones. The membrane-bound sulfotransferases reside in the Golgi and mediate the post-translational sulfation of endogenous macromolecules (proteins, lipids and glycosaminoglycans) in the secretory pathway (Bojarova and Williams, 2008; Leung et al., 2016). Despite low sequence conservation, sulfotransferases possess a conserved protein structure and catalytic properties, which include the binding to the PAPS cofactor. However, sulfotransferases show broad substrate specificity and may sulfate an unlimited number of substrates (Mueller et al., 2015; Langford et al., 2017). The human genome contains 13 cytoplasmic and 39 membrane-bound sulfotransferases (Langford et al., 2017; Gunal et al., 2019). Based on published literature and Wormbase (version WS282), Table 1 lists the cytosolic and membrane-bound sulfotransferases currently identified in C. elegans (36), P. pacificus (18) and Strongyloides ratti (48), a common gastro-intestinal parasitic worm of the rat (Streit, 2008).

ssu-1 (suppressor of stomatin mutant uncoordination) is the only member of the cytosolic sulfotransferase gene family present in the genome of C. elegans (Carroll et al., 2006; Hattori et al., 2006; Mizuguchi et al., 2009). Orthologous proteins include the human SULT1B1, SULT2A1 and SULT2B1 enzymes (Wormbase curation). This sulfotransferase is expressed throughout development, particularly in embryos and adult stages, but shows increased levels in dauer larvae. Recombinant protein is able to sulfate simple phenol substrates, such as bisphenol A (Hattori et al., 2006). Expression of SSU-1 has also been detected in the amphid sensory neurons located in the head of the worm where it is hypothesized to modify endocrine signals (Carroll et al., 2006). A recent study observed that mutant ssu-1 animals failed to arrest development in response to osmotic stress. The authors suggest that in the sensory neurons SSU-1 sulfates a ligand of the nuclear hormone receptor NHR-1 to activate a transcriptional response to osmotic stress and induce developmental arrest (Burton et al., 2018).

Five paralogs of C. elegans SSU-1 are found in P. pacificus, termed SULT-1 to SULT-5 (Table 1) (Namdeo et al., 2018). CRISPR/Cas9-mediated knockout of these cytosolic sulfotransferases and a genetic screen for suppressors of an all-stenostomatous mutant of P. pacificus has identified a role for SULT-1 (PPA12547) in the regulation of mouth-form plasticity (see details below) (Bui et al., 2018; Namdeo et al., 2018). Additional SULTs can also be found in P. pacificus (4) and S. ratti (8). Their biological role remains unknown.

From prokaryotes to eukaryotes sulfatases are highly conserved in sequence, structure and mechanism of action (Hanson et al., 2004; Ghosh, 2007). They share a highly conserved N-terminal region containing two consensus sulfatase motifs. The first motif is characterized by the conserved sequence C/SxPxRxxxxTG (x is any amino acid) and is crucial to generate the post-translationally modified active-site aldehyde residue, the α-formylglycine (FGly; 2-amino-3-oxypropanic acid) (Knaust et al., 1998; Dierks et al., 1999). The second sulfatase signature motif is a 12-mer sequence GY/VxS/T-xxxGKxxH. The Lys and His residues are part of the active-site and required for catalysis (Waldow et al., 1999). The C-terminal region of sulfatases is the most diverse; however, it is proposed that this region provides substrate specificity (Hanson et al., 2004). Although a large number of eukaryotic sulfatases are active on small aromatic sulfates (aryl substrates) in vitro and thus classified as arylsulfatases, many sulfatases show strong substrate specificity, specific subcellular location and optimal activity under defined conditions (Hanson et al., 2004). Sulfatase genes encode proteins with a wide range of substrate specificity but little functional redundancy, and deficiency in a single sulfatase can lead to unique disorders and developmental defects in animals (Langford et al., 2017).

Eukaryotic sulfatases are targeted to the secretory pathway. In the endoplasmic reticulum (ER), the sulfatases are modified by glycosylation and FGly is generated (Dierks et al., 1997; Dierks et al., 1998a; Hanson et al., 2004). They can then remain in the ER, be secreted, or transported to other subcellular compartments, such as the Golgi complex and the lysosome (Table 2) (Hanson et al., 2004). The steroid sulfatase (STS, human arylsulfatase C) is integrated into the membrane of the ER or Golgi apparatus (Willemsen et al., 1988; Hernandez-Guzman et al., 2003) where it plays an important role in the regulation of endocrine responses and xenobiotic metabolism. STS is the main enzyme involved in steroid desulfation and regulates hormone levels by removing sulfate from precursors, such as estrone sulfate and dehydroepiandrosterone (DHEAS), and originating active steroid hormones (for review see (Mueller et al., 2015)). Lysosome-located sulfatases (human arylsulfatases A and B) participate in the catabolism of defective and unwanted sulfated glycolipids (sulfatides) and proteoglycans that are imported into this organelle by endocytosis. Lysosome storage disorders are therefore a consequence of lysosome-sulfatase deficiency (Langford et al., 2017). The secreted sulfatases, known as Sulfs, remove the 6-O-sulfate groups of the glucosamine units of HS regulating multiple developmental processes (Hanson et al., 2004; Bojarova and Williams, 2008; Langford et al., 2017).

Nematodes also contain multiple sulfatase genes: three in C. elegans and S. ratti, and five in P. pacificus (Table 2). Sequence-wise Wormbase indicates that SUL-1 proteins are orthologues of the secreted SULF-1 and SULF-2 sulfatases that act on HS. In contrast, SUL-2 proteins show similarity with the human arylsulfatases A, B that participate in the degradation of cerebroside-3-sulfate and keratan/dermatan sulfate or CS, respectively, the steroid sulfatase (STS) and N-acetylglucosamine-6-sulfatase (GALNS) (Ragsdale et al., 2013; Perez-Jimenez et al., 2021), another lysosome-resident protein involved in the catabolism of HS (Schlotawa et al., 2020). SUL-3 proteins are most similar to the lysosome sulfatase human arylsulfatase B that participates in the degradation of keratan/dermatan sulfate and CS, or the potentially secreted arylsulfatases I and J with currently uncharacterized substrates. The biological function, localization and substrates of the nematode sulfatases are largely unexplored. To date, efforts to understand desulfation in worms have been restricted to C. elegans SUL-2 and P. pacificus EUD-1 (SUL-2 paralog). The role of EUD-1 in the regulation of mouth-form and predation is discussed below.

C. elegans SUL-2, the worm steroid sulfatase (Perez-Jimenez et al., 2021), is expressed in sensory neurons of the head of the worm where it is proposed to regulate the levels of steroid hormones. Worms with loss-of-function mutation of sul-2 or treated with the steroid sulfatase (STS) inhibitor STX64 have higher levels of sulfate steroid hormones and increased longevity. The authors of the study hypothesize that sulfated steroid hormones produced in the gonads of the worm may act in response to environmental cues, such as nutrient availability, to alter neurotransmission and promote longevity. Furthermore, higher levels of sulfated hormones as a result of reduced SUL-2 activity, or treatment of worms with sulfated steroid hormones, reduced protein aggregation and proteotoxic stress in models of Parkinson and Huntington neurodegenerative disorders. These data indicate that inhibitors of STS activity and sulfated steroid hormones have great potential in the treatment of aging-related diseases (Perez-Jimenez et al., 2021). In the same study, genetic ablation of sul-1 and sul-3 had no effects on lifespan.

The post-translational modification that enhances sulfatase activity is catalyzed by an endoplasmic reticulum (ER)-resident protein, the FGE-generating enzyme (FGE, sulfatase modifying factor 1, SUMF1) (Dierks et al., 1998a; Cosma et al., 2003; Dierks et al., 2003; Appel and Bertozzi, 2015). The FGly modification is essential for sulfatase activity (Dierks et al., 1998b; Recksiek et al., 1998) and is able to greatly stimulate catalytic proficiency, which can reach a rate enhancement (k_cat/k_uncat) of 10²⁶ for alkylsulfates (most efficient enzymes known to date) (Edwards et al., 2012). In humans, lack of FGly modification in sulfatases due to mutations in FGE that alter its activity causes multiple sulfatase deficiency (Schmidt et al., 1995; Schlotawa et al., 2020). FGE orthologues are found in prokaryotes and eukaryotes. Yet, homology searches indicate that FGE homologs are not present in plants (which also lack sulfatases), nematodes and fungi, although these last two groups of organisms contain cysteine-type sulfatase genes (Landgrebe et al., 2003; Appel and Bertozzi, 2015). This observation is suggestive of a different FGly-generating system in nematodes and fungi with a yet elusive enzyme. Alternatively, as FGly modification has not been studied so far in these groups of organisms, it remains unknown if their sulfatases require this post-translational modification to be active.

The sulfatase-encoding gene eud-1 was the first gene identified to regulate mouth-form plasticity in P. pacificus (Ragsdale et al., 2013). In the P. pacificus wild type strain PS312, the majority of animals express the predatory Eu mouth-form. Therefore, genetic screens aimed at identifying eurystomatous form defective (eud) mutants that would only generate St animals. From a screen of less than 4,000 haploid genomes, 17 mutants with a Eud phenotype were isolated. Of these, four were dominant with heterozygous animals having a mutant all-St phenotype and all of them turned to be alleles of the same gene, eud-1. The cloning of eud-1 not only revealed this gene to encode a sulfatase but also that all four mutations reduce or eliminate gene function (Ragsdale et al., 2013). These reduction-of-function mutations and the associated dominant phenotype indicate this gene to be haploinsufficient and to act as a developmental switch. Indeed, eud-1 mutants can be rescued by the overexpression of a wild type copy of eud-1, whereas overexpression of the mutant version of eud-1 will not cause a Eu phenotype in wild type animals (Ragsdale et al., 2013). It should be noted that such dominant reduction-of-function mutants are extremely rare with only one example known in C. elegans after more than 50 years of forward genetic screening, whereas gain-of-function mutants with a dominant phenotype are quite common (Hodgkin, 2005).

Further characterization revealed that EUD-1 might act as a sulfatase. Ppa-EUD-1, like Cel-SUL-2, is most similar to the human arylsulfatases A, GALNS and STS (Table 2). Application of sulfate and phosphate ions in the worm diet, which are known inhibitors of arylsulfatases, mimicked the eud-1 phenotype (Ragsdale et al., 2013). As indicated above, the P. pacificus genome contains more sulfatase genes than the C. elegans genome and eud-1 is indeed one of the genes resulting from lineage-specific gene duplications. While the arylsulfatase Cel-sul-2 is a single copy gene on chromosome IV, there are three sul-2-related genes in P. pacificus (Table 2). The ancestral gene is also located on chromosome IV and is termed Ppa-sul-2.1. The two additional copies, eud-1 and Ppa-sul-2.2.1, are located on the X chromosome with an interesting head to head organization and a separation by 7 kb of potential regulatory sequence (Figure 2A). Ppa-sul-2.2.1 and Ppa-sul-2.1 were soon shown to play no role in mouth-form specification (Ragsdale and Ivers, 2016). However, the full complexity of the eud-1 locus was only revealed through several additional experiments. First, the conserved histone-acetyltransferase Ppa-lsy-12 is required for eud-1 expression suggesting that eud-1 expression and mouth-form regulation are subject to epigenetic control (Serobyan et al., 2016). Second, an antisense-eud-1 message exists at the locus that acts as a positive regulator of eud-1 (Serobyan et al., 2016). Finally, two more enzyme encoding genes are part of the eud-1 locus. These genes encode for N-acetylglucosaminidases (nag) and nag-1 and nag-2 are located adjacent to sul-2.2.1 and eud-1, respectively (Figure 2A). Inactivation of both genes results in the opposite phenotype of eud-1 mutants: all animals have the Eu mouth-form even in liquid culture conditions that induce the St morph in wild type animals (Sieriebriennikov et al., 2018). Interesting, nag-1, nag-2 and eud-1 are expressed in a small set of different sensory neurons suggesting that they play a key role in environmental perception (Figure 2B). Importantly, eud-1 is epistatic over nag-1 and nag-2 as a quadruple knockout of the complete multigene locus has an all-St phenotype similar to eud-1 single mutants (Sieriebriennikov et al., 2018). Thus, the sulfatase EUD-1 plays a crucial role in mouth-form regulation as the central developmental switch. First, it is involved in the sensing of the environment and second, it integrates different environmental inputs to guide final decision making of mouth-form. It is important to note that while these genetic experiments clearly show a key role of a sulfatase in regulating a plastic developmental decision, the exact biochemical function and the potential substrates remain currently unknown. It has been speculated based on the physical proximity of sulfatases and N-acetylglucosaminidases that they might act on the extracellular matrix of neurons. However, no experimental support of this hypothesis is currently available and future studies have to shed light on the actual substrates of EUD-1.

Further genetic studies have identified a complex gene regulatory network (GRN) to control mouth-form plasticity with eud-1 representing the key developmental switch (Figure 2C) (Sieriebriennikov et al., 2020). The identification of this GRN was in large parts based on the strong all-St phenotype of the eud-1 mutant itself, which allowed unbiased genetic screens for suppressors resulting in all-Eu animals.

First, three suppressor alleles with an all-Eu phenotype were shown to result from mutations in the nuclear hormone receptor Ppa-nhr-40 (Kieninger et al., 2016) and were later on shown to result from gain-of-function mutations (Sieriebriennikov et al., 2020). In contrast, the loss-of-function phenotype of Ppa-nhr-40 is all-St and thus, similar to the eud-1 mutant phenotype (Sieriebriennikov et al., 2020). Also, loss-of-function mutations in another nuclear hormone receptor, Ppa-nhr-1, have an all-St phenotype, but with additional morphological defects in teeth formation unknown from other mutants (Sieriebriennikov et al., 2020). These studies would be compatible with the EUD-1 protein regulating the sulfation state of potential ligands of nuclear hormone receptors. Note that while nematodes have undergone a substantial expansion of nuclear hormone receptors with more than 250 genes in P. pacificus and C. elegans, only one ligand has been identified: the steroid hormone dafachronic acid that regulates the nuclear hormone receptor DAF-12 in multiple nematode species (Motola et al., 2006; Ogawa et al., 2009). Thus, the ligands of nematode nuclear hormone receptors remain little understood and similarly, nothing is known about a potential role of sulfation in the regulation of such ligands.

Second, the same genetic screen for eud-1 suppressors resulted in the identification of a sulfotransferase, mutations in which also produced an all-Eu phenotype and thus were named seud-1 (suppressor of eud-1) (Bui et al., 2018). Transgenic overexpression of seud-1 revealed the sulfotransferase to be dosage-dependent, similar to eud-1 (Bui et al., 2018). When mutants of eud-1 and seud-1 were used to produce all types of homozygous and heterozygous combinations, a graduation of phenotypes was observed. For example, animals with one copy of the sulfotransferase seud-1 and two copies of the sulfatase eud-1 were Eu (Bui et al., 2018). Besides the work of Bui and co-workers, an independent study resulted in the identification of the same sulfotransferase. A pharmacological screen found bisphenol A to induce the St mouth phenotype in wild type animals (Namdeo et al., 2018). As already indicated above, in C. elegans the sulfotransferase ssu-1 sulfates bisphenol A (Hattori et al., 2006). Indeed, a systematic screen of the five sulfotransferases of P. pacificus that are most similar in sequence to Cel-ssu-1, identified a single gene, named sult-1, to control mouth-form plasticity (Namdeo et al., 2018). Note that these independent studies have named the same sulfotransferase gene as seud-1 and sult-1. Importantly, the expression of SEUD-1/SULT-1 (pharyngeal muscle cells) is distinct from EUD-1 (sensory neurons) and therefore, it is possible that two independent sulfation processes regulate mouth-form plasticity (Bui et al., 2018; Namdeo et al., 2018).

Completely independent insight pointing towards the role of sulfated molecules in the biology of P. pacificus nematodes came from studies in analytical chemistry. The predatory behavior of P. pacificus is thought to be of importance in the context of the ecology of this nematode. While P. pacificus is a soil nematode, it is most reliably found in association with adult scarab beetles, i.e. cock chafers, stag beetles and dung beetles. On the living beetle, nematodes are found in the dauer stage, the arrested and stress-resistant, alternative larval stage that can survive long periods of harmful conditions. It is important to note that several, unrelated nematode species can be found on beetles and also other insects, and many of them wait for their vector to die. Indeed, after the beetles’ death back in the soil, bacteria and fungi will grow on the carcass. These food signals will cause the exit of the nematodes from the dauer stage and they rapidly mature to feed and reproduce on this time-limited food source. Indeed, recent studies in P. pacificus have indicated the dynamics and succession of nematodes on beetle carcasses in the context of the microbiome of the insect cadaver (Meyer et al., 2017; Renahan et al., 2021; Renahan and Sommer, 2021). The predatory behavior of Eu animals of P. pacificus is thought to serve a dual role: on the one hand, it allows supplementation of its bacterial diet by preying on nematodes; on the other hand, it will result in the elimination of competitors for the limited bacterial food available. This phenomenon is known as intraguild predation and represents an important concept in ecology (Polis and Holt, 1992; Quach and Chalasani, 2020).

While there is no obvious evidence that P. pacificus and C. elegans share the same ecosystem, a study by Liu and co-workers indicated a defensive response of C. elegans when it is exposed to P. pacificus predators (Liu et al., 2018). Subsequent studies revealed the behavioral details of this predator-prey competition (Quach and Chalasani, 2020). Strikingly, the original defensive response of C. elegans larvae towards P. pacificus is caused by predator-secreted sulfolipids. Specifically, fractionation of the P. pacificus secretome and analytical chemistry identified several (w-1)-branched-chain sulfolipids to induce the escape behavior in C. elegans prey (Liu et al., 2018). These findings are consistent with previous studies that showed an avoidance behavior of C. elegans against sodium dodecyl sulfate (SDS) (Hilliard et al., 2004). Importantly, the production and secretion of branched chain fatty acids is specific to P. pacificus and likely its relatives (Liu et al., 2018). The presence of multiple secreted sulfolipids in P. pacificus might open the door to several independent functions in the organism. For example, it might well be that branched chain fatty acids can serve as ligands for nuclear hormone receptors although no functional studies are yet available to support this hypothesis. In conclusion, genetic and chemical studies provide strong support for the role of sulfation in the development and the behavior of this nematode.

The literature described in this review highlights the significance of sulfation and desulfation pathways during development of different nematodes. From neuronal organization, to the regulation of phenotypic traits and behavior, sulfatases and sulfotransferases appear to modify a wide range of substrates also in nematodes. Despite the work done so far, we still possess a limited understanding on the localization, substrates and regulation of the panoply of sulfation-associated enzymes present in worms. Many of the sulfotransferases and sulfatases remain uncharacterized; their substrates are unknown, we have no understanding of their biological significance or subcellular localization, and we lack defined knowledge on their pattern of expression. Moreover, specific sulfated molecules, such as sulfolipids, act as signaling molecules between animals (Quach and Chalasani, 2020). However, we still do not understand the molecular mechanisms that generate, interpret, and execute, or the universality of this communication pathway.

An exciting research area in sulfation relates to the regulation of the enzymatic activity by post-translational modifications, such as FGly and glycosylation. As mentioned above, nematodes lack a recognizable FGE. Future studies that aim at detecting this unique modification in the proteome of nematodes, the corresponding modifying enzyme and its mechanism of action have great potential to elucidate alternative systems that introduce the FGly modification in eukaryotic sulfatases.

The significance of sulfation pathways in parasitic nematodes, like S. ratti, has never been addressed. With the development of specific sulfatase inhibitors, it is therefore of great importance to study their impact on the infection and ability of the parasite to invade the host. A deeper understanding on the sulfation players and their functions in these pathogenic worms may lead to improved therapeutic strategies that aim at eliminating the pathogen from their natural host. We are therefore looking forward to use nematodes in the quest to obtain a comprehensive understanding of the mechanisms and roles that sulfation plays in organismal development.