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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section IVThe Nematode Surface

Nematode surfaces are targets for passive and active environmental assault, including attacks by the immune system of a host, the attachment of bacterial spores and fungal traps, and extremes of desiccation or salinity. The cuticle must therefore play a part in defense, and the biosynthesis and maintenance of the cuticle surface are of central importance (Bird and Bird 1991). For many drugs, it would appear that the hypodermal membrane is the diffusion-limiting structure (Ho et al. 1990; Geary et al. 1995). For attacking pathogens, cells, antibodies, or protein effectors, the perceived surface can be the surface coat, the epicuticle, or outer structural layers. We review here the nonstructural components of the cuticle; the structural components are described elsewhere (Kramer 1994b; see Kramer, this volume).

The cuticle is an acellular, dynamic, biochemical compartment, rather than a simple inert exoskeleton. The external plasma membrane of a nematode is that of the hypodermis (epidermis), but the cuticle is enveloped by the membrane-like epicuticle (Wright 1987). The cuticle is reconstructed at each molt, but pulse-chase labeling demonstrates continuous production and export of surface components, and transcripts for both collagen and noncollagenous proteins are found in intermolt and molting animals (Selkirk et al. 1989, 1990; Johnstone 1992; Johnstone et al. 1994; Gems et al. 1995).

A. The Surface Coat

The surface coat or glycocalyx lies external to the epicuticle (Blaxter et al. 1992; Spiegel and McClure 1995). It can vary widely in apparent thickness both between species and between life cycle stages in a species. Surface coats can be seen with the transmission electron microscope, either in negative stains or after staining with cationic ferritin or ruthenium red, and they have been demonstrated in most species examined (Bird and Bird 1991). The coat in C. elegans is~ approximately 5 nm thick and is seen in all stages (Zuckerman et al. 1979; Jansson et al. 1986). The surface coat is dynamic in that components appear to be synthesized continuously (Page and Maizels 1992), and coat material is readily shed on environmental or antibody insult. The surface coat of T. canis larvae is synthesized in pharyngeal and excretory glands and thus voided through the mouth and excretory pore. Transcuticular secretion of surface coat components also occurs in T. canis and Heterodera schachtii (Aumann et al. 1991; Endo and Wyss 1992).

The surface coats of nematodes are polyanionic (probably due to sulfate or phosphate groups) and contain carbohydrate and mucin-like proteins (Zuckerman et al. 1979; Himmelhoch and Zuckerman 1983; Jansson et al. 1986; Page et al. 1992). The composition of the C. elegans coat is not known in detail, but it probably includes the O-glycosylated target of the antibody M38 (Table 1) (Hemmer et al. 1991; Politz and Philipp 1992). Other candidates for components of the C. elegans coat are the products of the Exc mutants. These mutants have degenerate or pathological changes in the excretory cell ranging from swelling of the lumen into cysts (e.g., exc-1 and let-653 ) to the formation of multiple lumens (e.g., exc-3 ). The lumen of the excretory cell has a well-developed surface coat, and the let-653 gene product is a serine/threonine-rich protein with similarities to mammalian cell surface mucins (Jones and Baillie 1995). In infective larvae of the ascarid T. canis, the coat is made up of a small number of proteoglycans and mucin-like glycoproteins that share carbohydrate determinants (Maizels and Page 1990). The structure of this O-glycan has been solved and contains novel sugar linkages (Khoo et al. 1991). The major coat component (Mr 120,000) is a mucin-like protein containing 12 seven-amino-acid repeats rich in serine (Gems et al. 1995). An M. incognita adult-specific cDNA from the excretory gland, encoding a mucin-like protein, may be part of the surface coat (D. Bird and T.-J. Chen, unpubl.).

Table 1. Surface markers of C. elegans.

Table 1

Surface markers of C. elegans.

C. elegans wild-type cuticles bind few if any lectins (the male tail binds WGA and SBA at low levels, as does the vulval slit in hermaphrodites [McClure and Zuckerman 1982; Zuckerman and Kahane 1983; Link et al. 1988, 1992]). Attachment of the spores of some nematophagous fungi is confined to sensory openings and the vulva (Barron 1977; Jansson 1994). This absence of reactivity may reflect the relative success of a survival strategy whereby the information content of the surface is reduced, rendering it biochemically invisible to sensory or adhesive products of predators and parasites. Active shedding of the coat by animal parasites is thought to be an adaptive defense against immune attack (Blaxter et al. 1992). The same may be true for free-living species avoiding the adhesive traps and spores of fungi and bacteria. A dispensable coat may slough off attached molecules (and thus cells, spores, or traps) and act as a dispensable sink for enzymatic effector mechanisms. Shed material may then act as a decoy to divert attention from the corpus of the organism.

In contrast, the surfaces of plant-parasitic nematodes bind a range of lectins, often (depending on the species) in specific temporal and spatial patterns (Spiegel and McClure 1995). Carbohydrate-binding, lectin-like activity is expressed on the surface of M. javanica L2s (Spiegel et al. 1995). Many plant-parasitic nematodes avoid eliciting host defense or wound responses even during the invasion and migratory phases, or when resident in the host (see Fig. 5, left). It is possible that the surface coat mimics host self-identity, as antibodies raised to the M. incognita surface specifically cross-react with host phloem cells (Bird and Wilson 1994b) and the major surface coat glycan of T. canis resembles the host LewisX determinant (Khoo et al. 1991).

B. The Epicuticle

The epicuticle is an electron-dense layer at the exterior boundary of the cuticle visible in negatively stained sections of nematodes. It has the appearance of a trilaminate plasma membrane but is usually significantly wider (6−40 μm). In freeze-fracture studies of many secernentean species, the outer leaflet appears smooth, whereas the inner leaflet has sparse “intramembranous” particles (de Souza et al. 1993; Lee et al. 1993; Peixoto and de Souza 1994). The C. elegans dauer epicuticle differs by having particles in the outer leaflet (Peixoto and de Souza 1994). Transcuticular uptake of nutrients and drugs by Brugia and Ascaris suggests that the epicuticle may not be a homogeneous or complete lipid barrier (Howells 1983; Ho et al. 1990, 1992). In many enoplidan (adenophorean) nematode species, especially marine free-living ones, the cuticle and epicuticle contain microscopic pores (Bird and Bird 1991).

The epicuticle is the first new layer to be laid down during molting (Lee 1970; Wright and Hong 1989). Whether or how it is replenished during the intermolt periods is unknown. The epicuticle contains lipid. Surface labeling studies using IODOGEN reagent (which attaches radioiodine to unsaturated lipids) reveal that the epicuticular lipid is a distinct subset of the total lipid of the nematode and also that it varies with life cycle stage (Scott et al. 1988; Blaxter 1993a; M. Blaxter, unpubl.). Unlike mammalian cells, there appears to be no glycolipid on the surface of C. elegans (Blaxter 1993a), but glycolipids have been described from whole lipid extracts of C. elegans (Chitwood et al. 1995), Ascaris suum, Nippostrongylus brasiliensis (Dennis et al. 1995), and O. volvulus (Maloney and Semprevivo 1991).

Like more conventional membranes, the epicuticle will take up tagged lipid analogs from the surrounding medium in vitro (Kennedy et al. 1987), and a model of the organization of the epicuticle has been produced (Proudfoot et al. 1990, 1991). Adults of C. elegans and many parasitic species selectively take up 18-carbon aliphatic lipids but not analogs with shorter chain lengths. The headgroup of the C18 lipid must be anionic (Kennedy et al. 1987; Proudfoot et al. 1991). The mobility of these inserted lipids, measured by fluorescence recovery after photobleaching, is low in parasites compared to that seen in mammalian plasma membranes (Kennedy et al. 1987). In contrast, the nonpolar lipid probe NBD-cholesterol readily inserts into the adult parasite epicuticle and is laterally mobile (Proudfoot et al. 1991). Adult C. elegans show nearly unrestricted lateral mobility (Proudfoot et al. 1991), but dauer larvae (and infective larvae of many vertebrate parasites) are refractory to lipid probe insertion (Proudfoot et al. 1991). This may have a role in resistance to desiccation. One of the earliest biological markers of exit from the dauer stage in C. elegans is a change in surface lipophilicity. By 30 minutes after exposure to food, long before the molt to L4, the dauer surface starts to accept lipid probes (Proudfoot et al. 1991, 1993b). In parasites, a similar change is observed when arrested infective larvae encounter their definitive host (Proudfoot et al. 1991b). For filarial nematodes, the change is more rapid than that seen in C. elegans and can be triggered by exposure to host-like pH, carbon dioxide concentrations, or temperature (Proudfoot et al. 1991). There is evidence for the involvement of second-messenger-mediated signaling in the control of this event (Proudfoot et al. 1993a).

C. Noncollagenous Proteins at the Nematode Surface

Antisera from experimental and natural hosts and surface-directed labeling techniques have been used to identify noncollagenous “surface antigens” in many parasitic species. These proteins may have roles in nutrition, defense, or cuticle maintenance. The diversity of surface proteins of nematode parasites is low compared to that seen on mammalian cells and may reflect a necessity to avoid the immune response. In the best known system, Brugia malayi, several of these surface proteins have been cloned and shown to encode products with recognizable functions.

The major surface glycoprotein of B. malayi adults is an N-glycosylated glutathione peroxidase (GPX) homolog (Maizels et al. 1989; Cookson et al. 1992) which is made in the hypodermis and secreted through the cuticle (Selkirk et al. 1990). The filarial GPX is inactive against hydrogen peroxide but is active against lipid peroxides (Tang et al. 1995) and is thus well placed to protect the epicuticle from peroxidative disruption. Tissue-dwelling parasites are exposed to a chemical arsenal that can do severe damage to membrane lipids (Selkirk et al. 1993). A second protein on the filarial surface is a secreted superoxide dismutase that is presumed to eliminate superoxide generated by the lipoperoxidase (Tang et al. 1994; Ou et al. 1995a). The homolog of a third B. malayi surface protein was first cloned from the related cutaneous filarid O. volvulus (Lustigman et al. 1992) and is similar to cystatin proteinase inhibitors. It may be involved in cuticle maintenance or in abrogating the effects of proteases released by immune effector cells. An abundant complex of proteins from B. malayi, called the nematode polyprotein antigen (NPA) (McReynolds et al. 1993), has homologs in Ascaris suum, where it is the most abundant protein of the pseudocoelomic fluid and is not surface located (McGibbon et al. 1990; Spence et al. 1993), and in C. elegans, where the location is unknown (J. Moore and M. Blaxter, unpubl.). The NPAs are lipid carrier proteins (Kennedy et al. 1995) and are highly allergenic in Ascaris infections (McGibbon et al. 1990). They are made as large (>350 kD) polyproteins and then cleaved at tetrabasic protease sites to give 15-kD monomers (Poole et al. 1992; Paxton et al. 1993). In B. malayi, the processing is incomplete, resulting in a ladder of monomer, dimer, etc. (Tweedie et al. 1993). Homologs of all these filarial surface proteins have been isolated from C. elegans or identified in the genome or cDNA sequence (M. Blaxter, unpubl.).

In vertebrate parasitic species, the antigenicity and pattern of proteins present at the surface change during development. Surface properties can change at each molt or within one intermolt on timescales ranging from days to minutes (Maizels et al. 1983a,b; Philipp and Rumjanek 1984; Proudfoot et al. 1993a,b). These changes correspond to developmental events in the nematode and the generation of immune responses in the host and have been suggested to be part of an immune evasion mechanism (Philipp et al. 1980).

In C. elegans, the cuticles of different stages are morphologically distinct and contain distinct sets of collagens (Cox et al. 1981c, 1989; Politz and Edgar 1984) and cuticlins (Sebastiano et al. 1991). Surface labeling of C. elegans N2 adults with the nonpenetrating IODOGEN reagent reveals a simple pattern of surface proteins (Politz et al. 1990; Blaxter 1993a). Each stage has a distinct set of surface molecules (Table 1) revealed by antibody binding (Politz et al. 1987; Hemmer et al. 1991) and surface radioiodination profiles (Politz et al. 1987; Blaxter, 1993a; M. Blaxter, unpubl.). In particular, the dauer larva and the post-dauer L4 cuticle express the same surface proteins, but the post-dauer adult reverts to the normal pattern (Table 1) (M. Blaxter, unpubl.). The dauer-like surface phenotype of post-dauer L4s adds to data suggesting that this stage is not identical to directly developed L4 (Liu and Ambros 1989; Kramer 1994a). The finding that the free-living C. elegans also changes its complement of surface proteins (and antigenicity) suggests that modulation of the surface is a basic part of the nematode developmental program, albeit one that may have been exploited by successful parasites to evade the host. The C. elegans cuticle is an excellent model for understanding parasitic species (Politz and Philipp 1992). As most parasites are dioecious (and probably highly heterozygous) and brood sizes can be large, it is likely that significant antigenic polymorphism could be selected under immune pressure, but neither the existence of such polymorphism nor its importance has been assessed. The C. elegans locus srf-1 determines adult cuticular surface reactivity to an antibody raised against cuticle material; polymorphism at this locus has been detected in wild populations (Politz et al. 1987). Such variation may reflect selection by predators.

D. Genes Controlling Surface Identity in C. elegans

The Srf phenotype is defined by changed reactivity at the surface to antibody or lectin reagents in the absence of morphological disruption of the cuticle (Table 2). The srf loci can be divided into three groups. The first group, composed of srf-1 , srf-2 , srf-3 , and srf-5 mutants (Politz et al. 1987, 1990; Link et al. 1988, 1992), has only cuticle phenotypes that arise from the novel exposure of antibody epitopes or lectin-binding sites at the cuticle surface. The structure of the cuticle is otherwise intact, except in the srf-3 dauer larva which is abnormally SDS-sensitive. Surface coat or epicuticle lipid composition is changed (Blaxter 1993a), but there is no change in detergent- or 2-mercaptoethanol-soluble WGA-binding glycoproteins (M. Silverman et al., unpubl.). The srf-2 , srf-3 , and srf-5 L1 larvae do not bind the M38 antibody (S. Politz, pers. comm.). These srf mutations also abrogate expression of the radioactively labeled 6.5/12.5-kD species. The phenotype can be modeled as the loss of a masking or overlying layer which reveals moieties present but unavailable to extrinsic reagents in the wild-type cuticle.

Table 2. The phenotypes of srf mutants of C. elegans.

Table 2

The phenotypes of srf mutants of C. elegans.

The second group is composed of the pleiotropic srf loci, srf-4 , srf-8 , and srf-9 , that share an additional suite of defects in both internal and external structures. These include protruding vulvae and a low-penetrance multivulval phenotype (see Greenwald, this volume), male infertility, probably due to distorted copulatory bursae and abnormal spicules (see Emmons and Sternberg, this volume), defects in gonadal morphology, distortion of body shape, and uncoordinated movement (muscle cells are normal, but defects in neural cell projections were noted) (Link et al. 1992). These loci may be involved in secretion or specification of external matrix, both apically (cuticle) and basally (basement membranes). The internal defects would then arise from misrouting or misspecification of position, and the cuticle defects from misexpression of integral cuticle proteins (Link et al. 1992; see Kramer, this volume).

The third group of srf mutations affects the timing of surface antigen expression. Mutations in srf-6 result in the inappropriate expression at later larval stages of an antigen that is normally present only on L1 larvae. No other Srf-6 phenotype has been detected. Certain temperature sensitive dauer-constitutive (Daf-c) mutants are also in this class, revealing a link between dauer larva development and surface antigen switching. Heterochronic expression of the L1 antigen by these mutants is independent of dauer larva formation. Some genes affect both surface antigen switching and dauer larva formation (e.g., daf-1 and daf-4 ), whereas other gene activities (e.g., srf-6 and daf-12 ) are process-specific (see Riddle, this volume). Continued expression of the O-glycoprotein is probably due to failure of the switch from the L1 to the L2 cuticle type and similar controls may underlie both dauer- and cuticle-type switching (Hemmer et al. 1991; S. Politz, pers. comm.).

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK20022


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