Worm Breeder's Gazette 13(1): 82 (October 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

C. elegans as a parasite

Mark Blaxter

Imperial College, London SW72 BB
email mlb@bio.ic.ac.uk

Analogues of Parasite Surface Proteins on Dauer Worms

The dauer stage is specifically adapted in many ways to life in harsh environments: resistance to desiccation, reduced metabolism, altered behaviour. I am interested in the dauer cuticle surface because of its involvement in these adaptations, and because of the homology between the dauer stage and the infective stages of many parasitic nematodes. Surface molecules of such parasites may be determinants of infectivity or vaccine candidates: knowledge of the repertoire expressed by the freeliving C. elegans may indicate the sorts of structures and functions to be expected in parasites.

Adult C.elegans have a single heterodimeric surface-accessible protein (6.5 and 12 kDa subunits [M. Blaxter, JBC 268: 6600-6609]; hereinafter called surfin). Surfin is present on L2 through adult but is absent from the surface of L1 .The L3 stage is thus similar to the adult. Dauer (L3d) C. elegans have an additional 37 kDa molecule on their surface, which accounts for ~60% of the incorporated label in surface labeling experiments. Labeling is significantly enhanced at low pH suggesting that some charge barrier exists at the surface. The molecule is not disulphide-crosslinked, but does have a small mobility shift on reduction, so disulphide bonds may be important in its tertiary structure. It is also found on the surface of temperature-shift-induced dauers of daf-2 ( e1370 )worms (which have a normal pattern of surfin expression). The protein can be stripped off of the dauer surface without disruption of the worms by incubation in detergents (either SDS or TritonX-114).

Surfin was observed to behave as a hydrophobic protein, partitioning into the detergent phase in TritonX-114 phase separation experiments. The dauer 37 kDa shares this property, and this allows purification of the surface molecules from bulk Iysates of worms. While there are a large number of Coomassie and silver-staining detergent phase proteins (eg integral membrane proteins) visible on gels of this material, there are no stained bands corresponding to the surface labeled 37 kDa. Further work, and more worms, will be needed to obtain sufficient material for further analysis. What makes the surface molecules hydrophobic? They could have stretches of hydrophobic amino acids, and thus be true membrane proteins. Alternatively they could be anchored into the lipid of the cuticular surface by an acyl or glycosylphosphatidylinositol tail. As a first test of this possibility I have cleaved off any thio-ester liked acyl groups and re-phase separated: both the dauer 37 kDa and surfin change phase (ie become hydrophilic). This is good evidence for the hydrophobic interaction being mediated by a post-translationally added lipid anchor, and metabolic labeling experiments are in progress using candidate lipids.

Homologues of Parasite Surface Proteins: Glutathione Peroxidase

The major surface protein of the human-parasitic Iymphatic filaria is a 29 kDa glycoprotein with sequence similarity to mammalian glutathione peroxidases. An attractive role for this enzyme is defense against immune attack and downregulation of chemoattraction. Filarial nematodes, like C. elegans, lack detectable GPX activity when substrates such as H(2)0(2) or butyl hydroperoxide are used. The presence of the gene in the absence of activity in the filaria prompted me to probe the C. elegans genome by PCR for a homologue.

Using primers derived from conserved positions in 12 GPX sequences, a 350 bp PCR product was cloned and sequenced. It has 300 bp of coding sequence and a 52 bp intron. The intron is in the same position as one found in three species of filarial nematode. The predicted protein sequence confirms that it is GPX, and parsimony analysis places the C. elegans gene in a clade with the nematode sequences and distinct from the mammalian, plant and bacterial ones. So why no activity? Well, it could be that the substrate specificity of the nematode enzymes is different (and there is evidence for this in the filarial 29 kDa protein). More obviously, the segment I have sequenced (seven independent clones) has a stop codon (TGA) in place of an expected conserved tryptophan (TGG): this gene could be a pseudogene. I am attempting to confirm this by recloning new PCR products, and by mapping the fragment to polytene filters to obtain genomic lambda clones.