EXPERIMENTALPARASITOLOGY 74, 57-68 (1992)

Brugia

Patterns Surface-Associated maiayi:

of Expression Antigens

of

NEIL STOREY' AND MARIO PHILIPP*'~ New England Biolabs, Inc., Beverly,

Massachusetts

01915

STOREY, N., AND PHILIPP, M. 1992. Brugia malayi: Patterns of expression of surfaceassociated antigens. Experimental Parasitology, 74, 57-68. Patterns of expression of surface-associated antigens were analyzed in the iilarial nematode Brugia malayi immediately prior, and during development in the vertebrate host. Two surface-associated protein molecules, i.e., accessible to surface radioiodination and soluble in aqueous buffers, were investigated: M,s 29-30,000 and 16,000, both of which are antigenic in infected animals. The M, 2%30,000 glycoprotein is expressed in a surface-associated manner by adult worms and by fourth-stage larvae, but is not detectable in preparasitic third-stage larvae. The 16,000 component, which appears not to be glycosylated, is surface-associated in adult worms and fourth-stage larvae. In contrast to the 29-30,000 glycoprotein, the 16,000 protein is also expressed both by pre- and postparasitic third-stage larvae. However, it becomes smfaceassociated only after infection. Thus, immediately prior, and during development within the vertebrate host, B. malayi displays at least two different patterns of expression of surfaceassociated antigens: (i) de novo, initiated either immediately after infection (phase specific) or during genesis of the fourth-stage larva (stage specific); (ii) continuous, but with phasedependent surface exposure of previously cryptic antigens, during the transition from intermediate to definitive host. o IWZ Academic press, IIIC. INDEX DESCRIPTORS: Nematode, Brugia malayi; Cuticle; Epicuticle; Surface-antigen expression.

10 years a class of cuticular proteins and glycoproteins has been identified in many genera of parasitic (Philipp and Rumjaneck 1984; Selkirk et al. 1986) and free-living (Politz et al. 1990) nematodes. The defining attributes of the molecules of this class, termed hereafter “surface associated,” are accessibility to radiolabel from nonpenetrating labeling methods such as Iodogenmediated and lactoperoxidase-catalyzed iodination. Unlike the collagens, whose solubilization requires chaotropic and reducing agents, high temperatures, and denaturing detergents (McBride and Harrington 1967; Cox et al. 1981), surface-associated molecules can be solubilized from the cuticle by mild procedures involving homogenization in purely aqueous buffer solutions or in nondenaturing detergents. Indeed several of these easily solubilized proteins and glycoproteins have been shown to be actively released in vitro by living worms (Philipp et

INTRODUCTION

Organisms of the phylum Nematoda are bounded by an acellular cuticle. The best known role of this multilayered structure is that of an exoskeleton, but there is also evidence of its involvement in other vital functions such as uptake of nutrients (Howells 1987). In addition, cuticular antigens of nematode parasites have been shown to be either the source of induction or the targets of host-protective immune responses (reviewed by Philipp et al. 1988). Over the last i Current address: Department of Pharmacology,

BASF Bioresearch Corp., Cambridge MA 02139. ’ Current address: Department of Parasitology, Tulane Primate Research Center, Tulane University, Covington LA 70433. 3 To whom correspondence should be addressed at Department of Parasitology, Tulane Regional Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433. 57

0014-4894/92$3.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

58

STOREY AND PHILIPP

al. 1980; Philipp and Rumjaneck 1984; Kwan-Lim et al. 1989; Ibrahim et al. 1989).

Because of these properties, surfaceassociated components are consideredand in some cases have been proven to be -exposed at the surface of the cuticle (Philipp et al. 1981; Philipp and Davis 1986; Devaney 1987; Maizels et al. 1989). More importantly, given their solubility properties, they are in all likelihood nonstructural and thus could protagonize essential functional roles. This contention is supported by the observation that expression of molecules of this class often correlates temporally with parasite accession to a new host or host environment (Philipp and Rumjaneck 1984). For example, muscle larvae of the nematode Trichinella spiralis express surface proteins and glycoproteins that differ both structurally and antigenically from those of the intestinal adult worms and the migrating blood-dwelling newborn larvae. The putative functional importance of the surface-associated antigens of T. spiralis is further underscored by the fact that they are host protective (Silberstein and Despommier 1984; Grencis et al. 1986). Thus, their stage-specific expression pattern not only correlates with habitat changes but may also reflect an adaptation to survive during a primary infection the effects of a lethal immune response that is also stagespecific (Philipp et al. 1980, 1981; Bell et al. 1979; Wang and Bell 1987). We present here the results of an investigation of the expression patterns of two surface-associated proteins of Brugia malayi, M, 2%30,000 and 16,000. These molecules have been previously identified (Maizels et al. 1985; Alvarez e? al. 1989) and the biochemical properties of the M, 29-30,000 glycoprotein, have been studied (Maizels et al. 1989; Selkirk et al. 1990). In search of clues for the possible functional importance of these molecules, we were especially interested in changes in their expression occurring both at the onset of infection of, and during development in, the vertebrate host.

MATERIALS

AND METHODS

Parasites. Aedes aegypri mosquitoes infected with B. malayi were purchased from TRS Laboratories Inc.

(Athens, GA). Infective larvae were obtained by gently crushing mosquitoes in phosphate-buffered saline (PBS) containing penicillin (200 lU/ml), streptomycin (200 &ml) and fungizone (0.5 l&ml) (GIBCO, Grand Island, NY). Motile, undamaged, third-stage larvae were collected and injected into the peritoneal cavities of male jirds, Meriones unguiculatus, (Tumblebrook Farms, West Brookfields, MA). Postparasitic thirdstage larvae were recovered from jirds by peritoneal lavage on Days l-5 postinfection for the experiment described in Fig. 1A and on Day 3 postinfection for all other experiments. Fourth-stage larvae were recovered from jirds in the same manner on Day 10 postinfection. Adult worms were obtained from the peritoneal cavities of infected male jirds purchased from TRS Laboratories. Antisera. Antisera to infective larvae and adult worms were prepared as previously described (Arasu er al. 1987). Surface labeling and preparation of parasite extracts. Intact, motile worms were surface labeled with

lz51 by Iodogen-mediated iodination, as described by Philipp and Davis (1986). Detergent soluble parasite extracts were made either by homogenization at 4°C in PBS containing the detergent n-octyl-l3-D-glucopyranoside (n-OGP, 1.5%) (Calbiochem, San Diego, CA) and the protease inhibitors N-a-p-tosyl-L-lysine chloromethyl ketone-HCl (25 l&ml), N-tosyl+phenylalanine chloromethyl ketone (25 ugimi), EDTA (1 m&f), phenylmethylsulfonyl fluoride (1 n-&f), N-ethylmaleimide (5 r&f), all from Sigma Chemical Co. (Saint Louis, MO), or by suspending the parasites in sample buffer (see below) and incubating at 95°C for 15 min. The insoluble fractions were removed by a 14,OOOg centrifugation at 4°C for 15 min. Gel electrophoresis. Prior to analysis by onedimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), samples were denatured under reducing conditions by boiling for 2-3 min in SDS-PAGE sample buffer composed of Tris-HCl (0.05 M, pH 7.5), @mercaptoethanol (5%), SDS (2%) and bromphenol blue (0.00125%). Electrophoresis was performed on 15% acrylamide gels, using for reference molecular weight marker standards from Pharmacia (Piscataway, NJ) or BRL (Gaithersburg, MD). The latter, used only in Fig. lA, were prestained and recalibrated against unstained markers. Following fixation and drying, gels were autoradiographed on X-Omat film from Kodak (Rochester, NY) at -70°C in the presence of intensifying screens. Two-dimensional gel electrophoresis was performed as described (Philipp and Davis 1986) using the MiniProtean system from Bio-Rad (South Richmond, CA), with pH 5-8 ampholytes (Ampholine, Pharmacia-

B. malayi:

EXPRESSION PATTERNS OF SURFACE ANTIGENS

LKB) being employed for isoelectric focusing. Electrophoresis was carried out to equilibrium (approximately 5000 V-hr). The second-dimension separation was on 13.5% acrylamide SDS-polyacrylamide gels. Those gels with 3sSsamples were treated with the fluorography reagent Enlightning (New England Nuclear, Boston, MA) prior to fluorography on X-Omat film at - 70°C. Zmmunoprecipitation. Between 1 and 4 x lo5 cpm of labeled antigen (10 ~1) was mixed with 2% Triton X-100 in PBS (40 ~1) containing 10 pl of test antiserum or normal serum and incubated overnight at 4°C. A volume of 100 pl of a goat anti-rabbit immunoglobulin antiserum (Worthington, Malvern, PA) was then added and the mixture incubated for 2 hr at 37°C. Precipitates were washed five times by centrifugation using cold PBS. Metabolic labeling. Worms were incubated overnight at 25 or 37°C in methionine-deficient RPM1 1640 medium (Select-Amine Kit, GIBCO) supplemented with penicillin (200 IU/ml), streptomycin (200 &ml), Hepes (25 n&f), sodium bicarbonate (0.060/o),and 250 pCi L-[‘~S] methionine (SJ. 1015, Amersham, Arlington Heights, IL). Enzymatic

deglycosylation

of surface

associated

proteins. Extracts of iodinated worms were treated with the endoglycosidases N-Glycanase (peptide: N-glycosidase F) and 0-Glycanase (endo-d-Nacetylgalactosaminidase) from Genzyme, (Boston, MA). Prior to treatment with N-Glycanase, samples were treated for 2 min at 100°C with SDS (0.1%) and S-mercaptoethanol (0.1 M) to increase accessibility of the enzyme. The mixture was then incubated with N-Glycanase (10 Units/ml) in phosphate buffer (50 mM, pH 8.6) for 24 hr at 37°C. 0-Glycanase digestion was preceded by treatment with neuraminidase (1 Unit/ml) to remove sialic acid residues prior to incubation with enzyme (10 mUnits/ml) in phosphate buffer (50 mM, pH 6.0) for 24 hr at 37°C. RESULTS

Cuticular and Surface-Associated Proteins of Larval and Adult Stages of B. malayi before and after Infection of the Vertebrate Host

Mosquito-derived B. malayi third-stage larvae (L3) and postparasitic third-stage larvae (L3 ‘) recovered from jirds at l-day intervals after infection (up to Day 5) were radiolabeled with 125Iusing the Iodogen reagent, which has been shown to label only external layers of the cuticle of this parasite (Marshall and Howells 1985). The iodinated molecules were then extracted in SDS-PAGE sample buffer, sepa-

59

rated by electrophoresis, and visualized by autoradiography (Fig. 1A). The pattern of iodinated proteins changed considerably over these 5 days. Larvae isolated from mosquitoes (Day 0) had few clearly discernible iodinated molecules, but larvae taken from jirds 24 hr after infection had numerous and distinct species of surface molecules (more than 15 bands). Certain molecules of very high relative molecular mass were present from Day 0 through Day 5, notably a doublet in excess of 200,000. Thus, it appears that the components of the epicuticle, and perhaps also those of the external cortical layers of the larval cuticle, as determined by their accessibility to iodination, change drastically over 5 days following infection, and especially within the first 48 hr, with some components being lost and many more gained. It was noted, as reported previously by ourselves (Philipp et al. 1988) and by Selkirk et al. (1990), that the major surface-associated antigen of adult worms (M, 29-30,000) did not appear to be accessible to iodination up to and including 5 days postinfection (2-3 days before the third molt). In contrast, a prominent band of M, 15-15,500 in this particular gel, but of M, 16,000 in all others, was present after, but not prior to, 1 day postinfection. Three-day postinfection L3 + , lo-day postinfection fourth-stage larvae (L4), and mature male adult worms were then iodinated and their surface-associated components solubilized using the nondenaturing detergent n-OGP. The extract was analyzed by SDS-PAGE followed by autoradiography (Fig. IB). The predominant surfaceassociated molecule of the adult worms had an M, of 29,000 (Maizels et al. 1989) and was also expressed by the L4 stage, where it appeared to be a 30,000 precursor. In contrast, no such component was found on L3+, where an M, 16,000 species was the only major molecule (Philipp et al. 1988; Selkirk et al. 1990). The M, 16,000 component was present on the three stages examined (Fig. lB), yet not on preparasitic L3s.

60

STOREY AND PHILIPP

MW -218 I08 -78 -48

.26 19 15

FIG. 1. (A) Cuticular protein profile of iodinelabeled third-stage larvae of Brugia malayi recovered from mosquitoes (track 0) or from the peritoneal cavity of gerbils on Days l-5 postinfection (tracks l-5 respectively). Surface-labeled larvae were solubilized in SDS-PAGE sample buffer prior to electrophoresis on a 15% acrylamide gel. (B) Surface-associated protein profile of iodine-labeled Brugia malayi third-stage larvae recovered from the peritoneal cavity of gerbils 3 days after infection (track l), fourth-stage larvae (10 days after infection, track 2) and adult worms (track 3). Surface labeled worms were solubilized using the nondenaturing detergent n-octyl+-o-glucopyranoside and the soluble fractions analyzed by SDS-PAGE on a 15% a&amide gel.

Sample buffer extracts of these worms showed only a very diffuse dim band in this molecular weight region (Fig. lA, track 0). A minor band running at M, 20,000 was also

detectable on the surface of L4s and adult worms and it has been suggested that this molecule may be a breakdown product of the 29-30,000 molecule (Maizels et al. 1989). The M, 16,000 Surface-Associated Molecule is Biochemically Similar in All Three Postparasitic Stages Examined

To determine whether the M, 16,000 molecules from larval and adult stages had structural similarity, and to discern whether the band consisted of more than one molecular species, n-OGP detergent extracts of surface-labeled worms from pairs of different stages were coelectrophoresed in two dimensions on the same gel. 2-D gel resolution of mixtures of iodinated surface-associated proteins from L3 + and adult worms resulted in the presence of a single major spot in the M, 16,000 region, demonstrating that these stages share a component of identical relative molecular mass and a pZ of about 6.8 (Fig. 2). The same result was obtained when mixtures of extracts from L4s and adult worms were coelectrophoresed (not shown). An apparent microheterogeneity of the 16,000 spot, not visible on Fig. 2, was observed occasionally, mainly on material derived from L3+s. The M, 29-30,000 band usually did not focus within a pH gradient of 5-8, as it appears to have a basic pZ (Kwan-Lim et al. 1989). The M, 16,000 Molecule is Actively Synthesized by Adult Worms and by Preparasitic Third-Stage Larvae

From the results described so far it could be concluded tentatively that synthesis of the M, 16,000 surface-associated protein was initiated in the third larval stage, but in the phase following infection of the vertebrate host, and retained throughout the remaining stages of the life cycle. It was also conceivable, however, that this molecule was in fact synthesized before infection but remained cryptic, i.e., not accessible to

B. malayi: EXPRESSION PATTERNS OF SURFACE ANTIGENS

-KD 94 67 43 30 20

FIG. 2. 2-D gel analysis of a mixture of iodinated surface-associated proteins from postparasitic thirdstage larvae (L3+, Day 3) and adult worms of Brugia malayi. The arrow indicates the single spot corresponding to the M, 16,000 surface-associated proteins of both preparations. Electrophoresis in the first dimension was performed using pH 5-g ampholytes; separation in the second dimension was achieved on 13.5% acrylamide gels.

surface iodination until larvae were within the vertebrate host. In addition, its expression might have ceased after assembly of the adult worm cuticle, as happens with some of the cuticular collagens in nematodes such as Caenorhabditis elegans (Cox and Hirsh 1985). To determine whether the 16,000 surface antigen was being actively synthesized by the adult parasites, living adult male worms were metabolically labeled by culturing in -

the presence of [35S]methionine. Extracts of the labeled worms were made by homogenization in n-OGP and coelectrophoresed in two dimensions with a trace amount of the iodinated surface antigen preparation. Thus it was possible to distinguish between 35S- and 1251-labeledcomponents by comparing fluorographic and autoradiographic images of the same gel. The fluorographic image obtained within 2 days of exposure of the gel in the absence of intensifying screens is shown in Fig. 3a. It represents newly synthesized material containing the radioactive methionine. When the fluorographic image was obstructed with blackened X-ray film and the gel exposed for a longer time period in the presence of intensifying screens, a profile representing the iodinated components was obtained and is shown in Fig. 3b. By overlaying one image with the other it was found that the 16,000 surface-labeled component had an exact counterpart in the metabolically labeled preparation (arrows). The experiment was controlled by running in parallel a similar gel without the 1251tracer and showing that no fluorographic image could be obtained through the blackened film used to obstruct the 35Sfluorographic signal (not shown). In addition, no autoradiographic image was obtained after only 2 days of exposure of the gel with the combined isotopes but with

+

-

-MW 2;:

MW

4330-

4330-

20-

20-

+

6794-

14.4-

14.4 -

a.

61

b.

FIG. 3. 2-D gel analysis of a mixture of ‘251-labeledsurface-associated proteins and [“Slmethionine metabolically labeled proteins from Brugia malayi adult worms. Short-term exposure of the gel reveals

the fluorographic (35S)image (a), whereas long-term exposure with the fluorographic signal blackenedout reveals the autoradiographic (lz51) image (b). The arrowed spot common to both preparations corresponds to the M, 16,000 surface-associated protein. Electrophoresis in the first dimension was performed using pH 5-g ampholytes; separation in the second dimension was achieved on 13.5% acrylamide gels.

62

STOREY

AND

the fluorographic signal blocked and in the absence of intensifying screens. We concluded that the M, 16,000 molecule is actively synthesized by adult worms. However, the actual quantity of this molecule per adult worm at any point in time seemed to be very small indeed, for when several hundred micrograms of SDS-soluble adult worm protein material was coelectrophoresed in two dimensions with a trace amount of the iodinated surface-associated components and the resulting gel was stained, the iodinated 16,000 spot did not appear to have a Coomassie blue- or silverstained counterpart (not shown). To determine if L3s synthesized the M, 16,000 molecule a double isotope coelectrophoresis experiment as above was performed. Preparasitic third-stage larvae were metabolically labeled overnight with [35S]methionine at 25°C in order to avoid any initiation of expression that might be associated with the temperature change that acccmpanies the transition from insect to mammalian host. The labeled larvae were solubilized in SDS-PAGE sample buffer and coelectrophoresed in two dimensions with a trace amount of the iodinated surface antigen preparation (as above). +

a.

-

+

b.

PHILIPP

When the fluorographic image (Fig. 4a) was compared with the autoradiographic image (Fig. 4b) it was found that the iodinated M, 16,000 component had a metabolically labeled counterpart (arrows). In contrast, and consistent with the result obtained by SDS-PAGE analysis of sample buffer extracts of iodinated preparasitic L3s (Fig. IA, track 0), 2-D analysis of these same extracts showed no evidence of the M, 16,000 molecule (Fig. 4~). Thus, the latter is synthesized by the preparasitic third-stage larva but is not accessible to surface iodination, perhaps as a consequence of not being surface-associated during this phase of development. The M, 29-30,000 Surface-Associated Protein of LAS and Adult Worms Appears Not to Be Synthesized by L3s Iodination results indicated that the M, 2%30,000 molecule was available for surface iodination both on L4s and adult worms, but not on L3s of any developmental phase. To determine whether this was another case of cryptic expression we made use of rabbit antisera generated against living preparasitic L3s and whole adult worm tissues, respectively. Larvae used for rab-

-

+

C.

4. 2-D gel analysis of a mixture of [35S]methionine metabolically labelled proteins from preparasitic third-stage larvae (L3s) and ‘251-labelled surface-associated proteins from adult worms of Brugia malayi. Short-term exposure of the gel reveals the fluorographic (35S)image (a), whereas long-term exposure with the fluorographic signal blackened-out reveals the autoradiographic (“‘I) image (b). The arrowed spot common to both preparations corresponds to the M, 16,000 surface-associated protein, which appears not to be present in sample buffer extracts of surface-iodinated L3s (c). Electrophoresis in the first dimension was performed using pH 5-g ampholytes; separation in the second dimension was achieved on 13.5% acrylamide gels. FIG.

B.malayi:

EXPRESSION PATTERNS OF SURFACE ANTIGENS

bit immunization were left at 4°C overnight in PBS, a treatment they survived but which rendered them unable to molt to the L4 stage. Immunoprecipitation of iodinated surface-associated proteins from adult worms revealed that, while the antiadult worm antiserum was able to precipitate both the M, 29-30,000 and the 16,000 molecules, the anti-L3 antiserum reacted only with the latter component (Fig. 5). This result permits us to suggest that the preparasitic third-stage larva, and perhaps also the postparasitic L3, do not synthesize the M, 29-30,000 molecule.

63

malayi (Maizels et al. 1989) and the similar component of B. pahangi (Devaney 1988). Interestingly, in parasites of the genus Brugia and also in those of other genera, sugar

moieties are not available for lectin binding on intact parasites, and thus are probably not surface-exposed (Ortega-Pierres et al. 1984; Kaushal et al. 1984; Devaney 1988). However, since many surface-associated glycoproteins are released into the external milieu by viable parasites (Philipp et al. 1980; Maizels et al. 1984; Meghji and Maizels 1986; Kwan-Lim et al. 1989; Ibrahim et al. 1989), their sugar residues may nonetheless be the target of host responses affectThe M, 16,000 Antigen Does Not Appear ing their function. Therefore, we wished to to Be Glycosylated know whether the M, 16,000 molecule is glycosylated. This information could also Many surface-associated proteins of parbe useful to devise an appropriate strategy asitic nematodes are glycoproteins (Philipp for molecular cloning and expression of the and Rumjaneck 1984; Maizels et al. 1987), antigen. Extracts of labeled postparasitic including the M, 29-30,000 molecule of B. larvae and adult worms were treated with the enzymes N-Glycanase, which hydrolyzes asparagine-linked oligosaccharides from glycoproteins, and 0-Glycanase which hydrolyzes gal Bl-3galNAc core disaccharides linked to either serine or threonine residues. In neither case was there any reduction seen in the relative molecular mass of the M, 16,000 component (Fig. 6). This was also confirmed at the twodimensional level (not shown). However, as has been previously shown (Maizels et al. 1989), there was a marked shift in the M, of the 29-30,000 glycoprotein upon treatment with N-Glycanase. In addition, this molecule may also be 0-glycosylated, as suggested by the lower M, band in tract 7 of Fig. 6. We did not further investigate FIG. 5. Analysis of the immunogenicity of Brugia whether this band appeared as a consemalayi third-stage larvae and adult worms as measured by the induction of serum antibodies to the M, quence of partial 0-Glycanase digestion of 2%30,000 and 16,000 surface associated proteins. Io- the 29-30,000 molecule or of a radiolabeled molecule of similar Mr. Proteolytic activity dinated surface-associated proteins of adult worms were immunoprecipitated with sera from rabbits im- of broad specificity contaminating the enmunized with third-stage larvae (track 2) and with zyme preparation may be ruled out because adult worms (track 3), or from unimmunized animals of the invariance in the mobility of the (track 1). Immunoprecipitates were solubilized in SDS-PAGE sample buffer and electrophoresed on 16,000 molecule. Similarly, this invariance cannot be the consequence of inactivity of 15% acrylarnide gels.

64

STOREY

1234

5

6

7

AND

8

FIG. 6. Enzymatic deglycosylation of surfaceassociated proteins of Brugia malayi postparasitic third-stage larvae (tracks l-4) and adult worms (tracks 5-8). Surface-associated proteins of both stages were treated with the enzymes N-Glycanase (IS) or neuraminidase followed by O-Glycanase (3,7). Similar samples were incubated under the same conditions but in the absence of the corresponding enzymes (2,6; 4,8). Gel analysis was performed on a 15% acrylamide gel.

the endoglycosidases used, since they both effectively hydrolyzed the 2%30,000 component. Beta elimination with NaOH or periodate oxidation also had no effect on the electrophoretic mobility of the M, 16,000 band (not shown). DISCUSSION

Alterations in morphology and at the molecular level occurring upon accession of larvae to the definitive host have been documented in several species of filarids. Changes in morphology of filarial thirdstage larvae have been observed shortly after penetration of the vertebrate host. Perhaps the best documented change is the rounding of the anterior portion of the worms, occurring within 2 to 5 days following infection, in larvae of species as disparate as B. pahangi and Acanthocheilonema viteae (Schacher 1962; Tanner 1981). Five days after infection (2 to 3 days before the third molt), the cuticle of B. pahangi larvae

PHILIPP

becomes smooth over the entire length of the body (Schacher 1962). At the molecular level, changes take place that may be related to these alterations in morphology. A prominent M, 35,000 surface-associated antigen found on preparasitic L3s of Dirofilaria immitis (Davis and Philipp 1986) is released both in vitro and in vivo (Ibrahim et al. 1989)and appears to be lost within 24-48 hours after infection (T. B. Davis and M. Philipp, unpublished observation). Indeed, cast cuticles of D. immitis L3s no longer contain this 35,000 protein (Mok et al. 1988). Similarly, expression of a M, 23,000 major acidic protein of Onchocerca lienalis and Onchocerca volvulus ceases after transmission (Bianco et al. 1990). It is not known, however, whether this protein is surface associated. The shedding, and subsequent loss of a surface epitope identified through the binding of a monoclonal antibody (DIE,) also occurs in B. malayi larvae shortly after infection of the vertebrate host. This surface epitope is expressed only by late secondstage, and by preparasitic and early postparasitic third-stage, larvae of this parasite (Carlow et al. 1987a). It is shed in vitro from viable worms kept at 37°C at a rate so fast that its residence half-life on the worms’ surface is only 2.5 min (Philipp et al. 1988). Within 3 days of infection the D,E, antibody no longer binds to the larval surface, thus indicating the loss of the epitope (Carlow et al. 1987b). During this same time period B. malayi larvae also lose their ability to bind anti-C3 receptor antibodies (C. K. S. Carlow and M. Philipp, unpublished data). Simple stimuli, such as a rise in pH or in temperature from normal arthropod vector values (5.8-6.0, 26-27”(Z), to those of the vertebrate host (7.G7.4, 37°C) are also sufficient to induce, in vitro, surface modifications that enable the epicuticle of thirdstage larvae of B. pahangi and A. viteae to incorporate hydrophobic fluorescent lipid probes such as 5-N-octadecanoylamino-

B.malayi:

EXPRESSION PATTERNSOFSURFACE

fluorescein (L. Proudfoot and E. Devaney, personal communication). Taken together, these observations are consistent with a major change in the composition of the larval surface upon infection. Composition may be altered via a process of shedding of old components from the epicuticle, as these are gradually replaced by new ones. Alternatively, the detected phenomenon may be due to an entire epicuticular layer(s) breaking loose from the worms’ surface, thus exposing an interface of different composition. The latter mechanism is more consistent with the observed loss of surface molecules in large patches rather than in a uniform and gradual way (Carlow et al. 1987b). For either mechanism the effector could be a surfaceassociated protease activated by the temperature shift occurring upon transmission. and/or by a cofactor present only in the vertebrate host milieu. The results presented here are also consistent with these interpretations. Many more components were accessible to surface iodination on third-stage larvae within 5 days following infection than on preparasitic third-stage larvae. By Day 5 of infection more than 15 bands could be resolved on SDS-PAGE of sample buffer extracts of iodinated larvae, whereas fewer than 9 were present on similar gels of preparasitic L3s (Fig. 1A). Although it is possible that some of these molecules newly available to surface iodination were expressed de nova, synthesis of at least one of them, the Zt4, 16,000 surface-associated antigen, began prior to infection, and may have taken place also within the insect vector. We have not formally excluded, however, the possibility that removal of the larvae from the mosquitoes and exposure to culturemedium components in vitro at 25°C acted as the synthesis-initiation trigger. We were able to identify the M, 16,000 antigen on 2-D gels among metabolically labeled proteins of preparasitic L3s by using its welldefined iodinated version as a marker (Fig.

ANTIGENS

65

4). Identity of metabolically and surfacelabeled M, 16,000 molecules is therefore proven, to the extent that their M, and pZ are the same. No band or spot corresponding to the M, 16,000 region was discernible on one- or two-dimensional gels of sample buffer extracts of iodinated preparasitic L3s. The inaccessibility to Iodogenmediated iodination of the M, 16,000 protein synthesized by preparasitic L3s underscores one of the interpretations of our data, namely that an epicuticular layer is shed early after infection, thus rendering the M, 16,000 and perhaps other cuticular components available to surface-labeling. The possibility that the molecule was heavily glycosylated before infection and thus, although expressed as surface-associated, ran on 2-D gels with altered coordinates is unlikely since we (and also Maizels et al. 1989) were unable to find any level of glycosylation on the M, 16,000 component as obtained from adult worms (Fig. 6) and no major detectable alterations of its 2-D coordinates were observed during development from the L3+ to the adult stage. The M, 16,000antigen is thus expressed in a pattern that suggests it has functional importance within the vertebrate host, i.e., exposure as surface-associated immediately following infection, and persistence as such for the remaining portion of the life cycle. Expression of the M, 16,000 antigen by adult worms is also of practical importance, since cDNA libraries, made more easily from these worms’ mRNA than that of any of the much smaller larval stages, should contain cDNA inserts encoding the antigen. We were surprised, however, at the very small amounts of the M, 16,000 protein present in adult worm extracts, for no stained counterpart to the autoradiographic image of the radiolabeled molecule was detectable on 2-D gels of this material. As with many other surface-associated molecules, the M, 16,000 is probably released in vitro by living adult worms (Storey and Philipp, unpublished observations; Kwan-

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Lim et al. 1989). If this occurs at a rate comparable to that of synthesis, then this might explain the lack of a large antigen pool in the worms’ tissues. This paucity was disappointing, for we had hoped that synthesis of the antigen by the large adult worms would enable us to purify enough 16,000 protein to generate either monospecific antibodies or N-terminal amino acid sequence data for cloning purposes. Contrasting with the continual expression of the 16,000 molecule within the vertebrate host, expression of the M, 2930,000 surface-associated antigen appeared to begin at the fourth larval stage. The molecule was detectable neither in sample buffer extracts nor in n-OGP extracts of surface labeled pre- or postparasitic L3s (Figs. 1A and 1B). Furthermore, antisera raised against L3s that had been rendered unable to complete development to the fourth stage contained antibodies reacting with the 16,000 a,ltigen but not with the M, 29-30,000. This result strongly suggests that the M, 29-30,000 is not synthesized 5y the larvae before assembly of the L4 cuticle is initiated, for the molecule is indeed immunogenic under a variety of different circumstances. Antibodies to it (and to the 16,000 antigen) were found in serum from a rabbit immunized with adult worm material (Fig. 5), and similar antibodies are present in humans naturally infected with B. malayi (Maizels et cxl. 1985) and in mice given an intraperitoneal infection with L3s (our unpublished data); in mice the larvae molt to the L4 stage within a week of infection, but do not reach the adult stage (Carlow and Philipp 1987). Although we cannot formally rule out synthesis of the 29-30,000 antigen by L3s or early L3+s in quantities below immunogenic thresholds, our data indicate that expression of this surface-associated molecule is initiated by L4s or by late L3 + s during synthesis of the L4 cuticle. In this sense its pattern of expression is stagespecific. Our results also parallel those of Selkirk et al. (1990). These authors were

unable to immunoprecipitate the 29,000 antigen from extracts of surface- or metabolically labeled post-parasitic L3’s using an antiserum raised against gel-purified 29,000 protein. In contrast, Devaney and Jecock (1991), using a similarly prepared antiserum raised against the 29-30,000 antigen of B. puhungi, were able to immunoprecipitate this antigen from radiolabeled extracts of preparasitic B. puhungi L3s and also, but at much lower levels, from extracts of surface-labeled preparasitic L3s. The identity of this 29-30,000 component was compared and matched to that of the 29-30,000 molecule of adult worms both by 2-D gel electrophoresis and by peptide mapping. From these result it may be concluded that the pattern of expression of the 29-30,000 antigen in B. puhungi is not stage-specific, but similar to that of the 16,000 molecule in B. muluyi, i.e. expressed before infection, but not surface-associated or available to surface iodination. Our results have interesting implications concerning the modes whereby surface exposure of nematode antigens is initiated during development. Surface exposure may begin not only by synthesis initiation of a surface-associated antigen, but also by the spatial redistribution of proteins within the parasite. How are these processes controlled? Is synthesis initiation of the 29-30,000 glycoprotein regulated translationally or transcriptionally? What are the environmental triggers and the mechanisms that bring about the change in worm surface composition, or the putative shedding of a cuticular layer, at the onset of infection? These questions concern the very basis of nematode immuno- and developmental biology. Their answers may provide new approaches to the control of parasites of both medical and veterinary importance. ACKNOWLEDGMENTS The support of Dr. Donald G. Comb is gratefully acknowledged. We also thank our colleagues Drs. Clo-

B.malayi:

EXPRESSION PATTERNS OF SURFACE ANTIGENS

tilde K. S. Carlow, Samuel M. Politz, Carole J. PrainKeating, and Mr. Theodore B. Davis for their stimulating discussions and comments on this paper. Also acknowledged with thanks is the secretarial assistance of Ms. Sharon M. Nastasi, Department of Parasitology, Tulane Primate Research Center.

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