Conidial Wall Research Articles

Conidial morphology, host colonization, and development of shot hole of almond caused by Wilsonomyces carpophilus

Morphology and ultrastructure of shot hole disease of almond infected by conidia of Wilsonomyces carpophilus were examined using light, scanning, and transmission electron microscopy. The multicelled conidia of the fungus were thick-walled and darkly pigmented. The conidial wall was multilayered and mainly consisted of an electron-dense, outer-wall layer and an electron-translucent, inner-wall layer. Septa of conidia were also multilayered. An electron-translucent zone separated the electron-dense, septa-wall layers of adjacent cells, and this zone extended to the inside of the outer-wall layer of a conidium. Conidia lacked true septa (distoseptate) and germinated by rupturing the outer-wall layers. Germination hyphae penetrated indirectly through stomata or directly through the cuticle into leaf tissue from appressoria that were produced terminally or on lateral branches of germ tubes. An extracellular mucilaginous matrix was commonly observed around hyphae and germ tubes in contact with leaf surfaces. Within leaf tissue, hyphae ramified throughout intercellular spaces and degraded cell walls of epidermal cells apparently without cuticle degradation. Diseased host tissue was tan brown and collapsed; ultrastracturally, diseased leaf cells were reduced in size, nonvacuolated, and had disrupted chloroplasts. A healthy host tissue response, adjacent to an infection site on leaves of potted plants, was the formation of a wound periderm. Within 10–14 days after an infection period (16 h of wetness), the periderm became a lignified–suberized barrier at 15 °C or a suberized abscission layer at 22 °C based on the histological stains Safranin O, Sudan III, and Sudan Black. At 15 °C, no abscission occurred and meristematic cells remained isodiametric but their walls became suberized and lignified, whereas cells adjacent to diseased tissue became lignified. At 22 °C, abscission occurred as cells adjacent to diseased tissue became vacuolated, enlarged, and suberized. Subsequently, the epidermis ruptured and the enlarged cells separated along the middle lamella to form an abscission layer. Hyphal growth was limited to the boundaries of the walled-off diseased tissue. Under favorable environmental conditions, hyphae replaced host epidermal cells and aggregated above the palisade layer to form pulvinate sporodochia. Sporodochia consisted of hyphae, numerous sympodially developing conidiogenous cells, and conidia that ruptured the host cuticle. Key words: fungal morphology, fungal foliar diseases, wound periderm, host–parasite interactions.

Ultrastructure of Conidia and the Conidium Aging Process in the Plant Pathogenic Fungus Colletotrichum graminicola

The influence of age on conidial ultrastructure was investigated in Colletotrichum graminicola. The cytoplasmic volume of young conidia-those harvested from 5-7-d-old cultures-was dominated by lipid bodies. These conidia also were characterized by numerous small vacuoles with electron-dense contents. Other structures present included mitochondria, microbodies, ribosomes, elements of tubular ER, short strands of rough ER, multivesicular bodies, simple cisternal elements, Woronin bodies, microtubules, and large amounts of what appeared to be glycogen. Older conidia harvested from 30-35-d-old cultures exhibited a significantly different ultrastructure. They contained fewer lipid bodies, lacked the large glycogen deposits, were very highly vacuolate, and contained sheetlike strands of ER and extensive aggregations of tubular ER. Structures thought to be autophagic vacuoles also were present. The conidial wall appeared similar in conidia of both ages and consisted of a thicker inner electron-transparent layer ...

Ballistosporic Conidia in Basidiobolus ranarum: The Influence of Light and Nutrition on the Production of Conidia and Endospores (Sporangiospores)

The process of endospore cleavage (sporangiospore formation) within what were originally ballistosporic conidia was examined with light and electron microscopy in two isolates of Basidiobolus ranarum. A pathogenic isolate was obtained from skin ulcers on a frog (Davis isolate) and a nonpathogenic isolate was obtained from toad feces (Toad isolate). Electron microscopy revealed that endospore formation was not the result of successive bipartitioning or progressive cleavage as previously described. Instead, the protoplasm of the ballistosporic conidium was bisected by a wall, thus dividing the conidium into one or more sporangia within which endospores (sporangiospores) were delimited. The walls segregating the conidial cytoplasm into sporangia were continuous with the inner layer of the original conidial wall. As spores were demarcated, some cytoplasmic debris was left in the space between the conidial/sporangial walls and the spore walls. After a period of 16 hours of low light levels, turning on the laboratory lights stimulated the formation and discharge of conidia with a peak in conidial discharge 5–6 hours later.

Ballistosporic conidia in Basidiobolus ranarum: The influence of light and nutrition on the production of conidia and endospores (sporangiospores)

The process of endospore cleavage (sporan? giospore formation) within what were originally bal? listosporic conidia was examined with light and elec? tron microscopy in two isolates of Basidiobolus ranarum. A pathogenic isolate was obtained from skin ulcers on a frog (Davis isolate) and a nonpathogenic isolate was obtained from toad feces (Toad isolate). Electron mi? croscopy revealed that endospore formation was not the result of successive bipartitioning or progressive cleavage as previously described. Instead, the proto- plasm of the ballistosporic conidium was bisected by a wall, thus dividing the conidium into one or more sporangia within which endospores (sporangiospores) were delimited. The walls segregating the conidial cy? toplasm into sporangia were continuous with the inner layer of the original conidial wall. As spores were de- marcated, some cytoplasmic debris was left in the space between the conidial/sporangial walls and the spore walls. After a period of 16 hours of low light levels, turning on the laboratory lights stimulated the for? mation and discharge of conidia with a peak in conidial discharge 5-6 hours later.

An electron microscope study of conidiogenesis and wall formation of conidia of Aspergillus niger

An ultrastructural study of the wall formation of conidia of A. niger during conidiogenesis revealed that wall layer formation does not conform to classical concepts. An initial wall is formed which is eventually replaced by a second permanent wall. The initial wall forms enteroblastically. Prior to maturation the initial wall, which surrounded and delimited the immatur conidium, disintegrates. A new conidium wall consisting of an inner and outer layer is laid down by the conidium protoplast. The conidia are still covered by the surface coating. The phialide wall layers do not contribute to the formation of the wall layers of the mature conidium. Therefore the terms holoblastic and enteroblastic cannot be used in the usual context. The term endoblastic is proposed for describing this type of conidium wall formation and maturation.

Variation in conidial morphology among geographic isolates of Sphaeropsis sapinea

In the U.S.A., Sphaeropsis sapinea is believed to occur in at least two distinct forms (type A and B) that can be distinguished by their conidial wall ornamentation. In this study, mature conidia of 50 monoconidial isolates of S. sapinea from a worldwide collection were examined using SEM to determine whether these two forms occurred more widely. Twenty S. sapinea isolates consistently had conidia with smooth outer surfaces typical of type A. The remaining 30 isolates had typical pits or similar indentations occurring on between 6 and 38% of conidia examined. Pits, or indentations, either occurred uniformly on the outer surface or only on some parts of individual conidia. Authentic S. sapinea isolates, categorized as type A and type B in previous studies, were chosen for detailed examination of the effect of spore age, nutrition, and pigmentation on surface ornamentation. No distinct differences in conidial morphology between the two types were discernible between conidia of different ages, or those produced on media containing different carbon or nitrogen sources. The melanin inhibitor, tricyclazole, was used to compare melanin-deficient conidia with pigmented conidia. Although mature, melanin-deficient conidia were hyaline, this did not appear to influence the occurrence of pits on conidia. The results of these studies strongly suggest that conidial morphology of S. sapinea is a variable characteristic and, therefore, a poor taxonomic criterion.

Two bacteria causing farmer's lung: Fine structure ofThermoactinomyces vulgaris andSaccharopolyspora rectivirgula

The fine structure of Thermoactinomyces vulgaris and Saccharopolyspora rectivirgula is described by transmission electron microscopy. These two bacteria are the most common microbes causing farmer's lung. The fine structure of hyphae, germination of endospores and the details of conidial wall layers of T. vulgaris, as well as the fine structure of septate hypha and globose, polygonal conidia of S. rectivirgula are described. The conidial wall of T. vulgaris consisted of an inner multilayered spore coat, intermediate spore coat and outer spore coat. The findings are important for the investigations to find fragments of these bacteria in the lungs of exposed patients and experimental animals.

Light and Electron Microscopy of a New Species of Euryancale Producing Phallus-Shaped Conidia

Euryancale phallospora is described as a new species of the Zoopagales found in Japan. The conidium is phallus-shaped and composed of a bulbous basal portion tapering gradually towards the apex which is shaped like an arrowhead. Infection is initiated by the conidium, the apical portion of which lodges in the nematode's mouth. High voltage electron microscopy shows clearly that the mature conidium has a dome-shaped apex. The periphery of the apical dome has 14-15 electron-dense, bar-shaped appendages flaring like a skirt below the apex. Additional, almost median, appendages are present on the conidial wall. The apical dome is composed of an electron-dense hemispherical core covered with an extremely thick cell wall. The thick wall is coated with a layer of weakly staining material. A pouchy appendage connected to the outermost layer by a short and narrow stalk is clearly seen at the distal end of the conidium.

Interaction between Aspergillus fumigatus and basement membrane laminin: Binding and substrate degradation

Aspergillus fumigatus, the causative agent of human aspergillosis, binds to and degrades basement membrane laminin. Using immunoelectron microscopy, laminin binding appeared to be associated with the cell wall expansions of resting conidia, and progressively extended to the outer electron dense layer of the conidial wall during the germination process. Labeling of thin sections revealed numerous binding sites in the cytoplasm, whereas the inner cell wall and the plasma membrane were not labeled. Attachment of A fumigatus conidia on microtiter plates coated with laminin and its fragments P1 and E8 was also investigated. Conidia cells showed good adhesion to wells coated with laminin. As indicated by inhibition experiments, the interaction was specific and fragment P1 represented the major binding site on the laminin molecule. In addition, since A fumigatus produced an extracellular serine protease, we determined the susceptibility of laminin to this enzyme. We demonstrated that protease extract was capable to degrade laminin in solution as well as in tissue sections. The laminin cleavage products were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All the three chains were extensively degraded within 1 h. Treatment of the crude protease extract with the enzyme inhibitors, phenylmethylsulfonyl-fluoride and chymostatin, blocked the degradation of laminin, indicating a chymotrypsin-like serine protease activity. Immunofluorescence microscopy of cryostat sections of mouse and rat kidneys treated with the protease extract showed widespread loss of laminin epitopes from basement membranes. Enzyme treatment also removed immunoreactivity from lungs as observed after immunoperoxidase performed on paraffin sections. Binding and proteolytic degradation of laminin may together facilitate initial interaction of A fumigatus with the host tissues.

Specific binding of human fibrinogen fragment D to Aspergillus fumigatus conidia.

The interaction of purified human fibrinogen with Aspergillus fumigatus conidia was investigated by immunofluorescence and electron microscopy and binding assays with radiolabeled proteins. We described the localization of the binding sites on the A. fumigatus conidia and on the fibrinogen molecule and determined the binding characteristics. Immunofluorescence revealed that the fixation of purified fibrinogen was selectively associated with conidia and suggested a role for the D domains of the fibrinogen molecule. Binding assays performed with 125I-radiolabeled proteins confirmed that binding sites were located specifically in the D domains. No reaction could be detected with fragment E. The binding of 125I-fragment D to conidia was time dependent, saturable, and specific. Scatchard analysis of the data revealed an average of 1,200 binding sites per conidium, and an apparent dissociation constant (Kd) of 2.2 x 10(-9) M was estimated. Pretreatment of the cells with proteolytic enzymes or heat abolished binding, demonstrating the protein nature of the binding sites. Ultrastructural localization of the fungal receptors was determined by transmission electron microscopy. Labeling appeared to be associated with the outer electron-dense layer of the conidial wall and progressively decreased during the germination process. Labeling of thin sections with fragment D and an antifibrinogen immune serum revealed that binding sites also lay in the inner part of the wall and in vacuoles. These results indicate the presence at the conidial surface of specific receptors for fibrinogen which could act as mediators of conidial adherence to host tissues.

Interactions between conidia of Aspergillus fumigatus and human complement component C3.

Activation and deposition of C3 on Aspergillus fumigatus conidia have been previously demonstrated. This study investigated in further detail the interactions between complement component C3 and the conidia of A. fumigatus. Immunoblotting and 125I-C3 binding studies showed that C3 deposition was rapid (less than 15 min) and parallel to the formation of iC3b. Immunoblotting experiments identified a 54- to 58-kDa conidial protein which binds human complement component C3 and/or a C3 fragment(s). 125I labeling of the outer layer of the conidia demonstrated that this protein doublet was present on the surface of the spore. The further degradation of C3 to low-molecular-mass fragments (40, 37, and 30 kDa), in the absence of plasma, by intact living conidia and a preparation of the outer conidial wall layer indicated the ability of fungal components to cleave C3. These data suggest that interactions between conidia and C3 are not limited only to deposition via activation of the alternative complement pathway; they also include degradation of bound C3.

Ultrastructural aspects of conidiogenesis of Fusarium spp. in the section Liseola

Production of the first and all the successive microconidia in Fusarium spp. section Liseola is enteroblastic from the phialide. The conidium initials are pushed out through a sometimes thickened neck region of the phialide. The inner wall of the phialide produces the conidium wall in a wall building zone (ring zone) in the neck of the phialide. The inner wall layer of the phialide is continuous with the inner wall layer of the conidium from which the middle and outer wall layers of the conidium are formed. This is contrary to current concepts that the outer wall layer forms the inner wall layers. Since no outer wall remnants are deposited in the phialide opening it means that large numbers of successive microconidia are produced without proliferation of the phialide or plugging of the opening. The implications of this for the definition of enteroblastic conidiogenesis is discussed. Macroconidia are also produced enteroblastically in a mode corresponding to that of microconidia.

Coccidioides immitis fractions which are antigenic for immune T lymphocytes

The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity. We have generated a Coccidioides immitis antigen-specific murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays. The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components. The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay. Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive. In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C. immitis, which was constructed in lambda gt11 to identify clones that encoded T-cell-reactive fusion proteins (FPs). The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components. The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-cell response was determined. The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C. immitis. Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption. The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF. The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C. immitis. Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way. The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C. immitis.

Aspergillus nidulans wetA activates spore-specific gene expression.

The Aspergillus nidulans wetA gene is required for synthesis of cell wall layers that make asexual spores (conidia) impermeable. In wetA mutant strains, conidia take up water and autolyze rather than undergoing the final stages of maturation. wetA is activated during conidiogenesis by sequential expression of the brlA and abaA regulatory genes. To determine whether wetA regulates expression of other sporulation-specific genes, its coding region was fused to a nutritionally regulated promoter that permits gene activation in vegetative cells (hyphae) under conditions that suppress conidiation. Expression of wetA in hyphae inhibited growth and caused excessive branching. It did not lead to activation of brlA or abaA but did cause accumulation of transcripts from genes that are normally expressed specifically during the late stages of conidiation and whose mRNAs are stored in mature spores. Thus, wetA directly or indirectly regulates expression of some spore-specific genes. At least one gene (wA), whose mRNA does not occur in spores but rather accumulates in the sporogenous phialide cells, was activated by wetA, suggesting that wetA may have a regulatory function in these cells as well as in spores. We propose that wetA is responsible for activating a set of genes whose products make up the final two conidial wall layers or direct their assembly and through this activity is responsible for acquisition of spore dormancy.

Aspergillus nidulans wetA Activates Spore-Specific Gene Expression

The Aspergillus nidulans wetA gene is required for synthesis of cell wall layers that make asexual spores (conidia) impermeable. In wetA mutant strains, conidia take up water and autolyze rather than undergoing the final stages of maturation. wetA is activated during conidiogenesis by sequential expression of the brlA and abaA regulatory genes. To determine whether wetA regulates expression of other sporulation-specific genes, its coding region was fused to a nutritionally regulated promoter that permits gene activation in vegetative cells (hyphae) under conditions that suppress conidiation. Expression of wetA in hyphae inhibited growth and caused excessive branching. It did not lead to activation of brlA or abaA but did cause accumulation of transcripts from genes that are normally expressed specifically during the late stages of conidiation and whose mRNAs are stored in mature spores. Thus, wetA directly or indirectly regulates expression of some spore-specific genes. At least one gene (wA), whose mRNA does not occur in spores but rather accumulates in the sporogenous phialide cells, was activated by wetA, suggesting that wetA may have a regulatory function in these cells as well as in spores. We propose that wetA is responsible for activating a set of genes whose products make up the final two conidial wall layers or direct their assembly and through this activity is responsible for acquisition of spore dormancy.

Isolation and ultrastructural localization of a soluble protein from Ophiostoma ulmi

Two-dimensional polyacrylamide gel electrophoresis was used to identify and isolate a soluble polypeptide, the QP1 protein, which is characteristic of the vegetative hyphae of nonaggressive isolate Q412 of Ophiostoma ulmi. Individual QP1 spots were excised from 16 two-dimensional gels. Polypeptides were eluted from the gel spots by electroelution and lyophilized. The protein was injected into rabbits for the production of polyclonal antibodies. Antiserum specificity was tested by transferring polypeptides from a two-dimensional gel onto nitrocellulose and treating with QP1 serum. The resulting immunoblot contained a single spot that corresponded in shape and location to that of the QP1 polypeptide. Thin sections of fungal mycelia, from nonaggressive isolate Q412 and the aggressive isolate VA of O. ulmi, were treated with QP1 antibodies and protein A – gold. The gold label was localized in thin sections over conidial and hyphal cell walls of the nonaggressive isolate. The aggressive isolate was nonreactive. Mycelia from nonaggressive isolates Q412 and Q311 and aggressive isolates VA and CESS16K of O. ulmi were grown on solid medium, treated with QP1 antibodies, labelled with protein A – gold, and prepared for scanning electron microscopy. The gold-labelled QP1 polypeptide was detected on the leading edge of a small number of hyphae from nonaggressive isolates Q412 and Q311. Key words: immunogold labelling, Ophiostoma ulmi, soluble proteins.

Differentiation of Penicillium glabrum from Penicillium spinulosum and Other Closely Related Species: An Integrated Taxonomic Approach

Summary This first study by the Subcommission on Penicillium and Aspergillus Systematics is concerned with the status of the closely related species Penicillium glabrum, P. spinulosum, P. purpurescens, and P. montanense. Thirty five isolates tentatively identified as these species were studied as unknowns by the eight members of the Subcommission. Characters studied included traditional morphology and gross physiology plus secondary metabolite profiles, isoenzyme patterns and DNA restriction enzyme fragment length polymorphism. The various approaches provided consistent identifications for most isolates, indicating that the four species could be separated satisfactorily and should be maintained. Statistical models based on morphological and gross physiological data indicated that five characters were of value in distinguishing the species. These were conidial wall texture, colony diameters on Czapek yeast extract agar and 25% glycerol nitrate agar, phialide width and vesicle diameter. Practical aspects of differentiating these species are also discussed.

Conidium differentiation in Aspergillus nidulans wild-type and wet-white ( wetA) mutant strains

Conidium (asexual spore) differentiation in wild-type and the wet-white ( wetA) mutant of Aspergillus nidulans was compared in intact chains of successively older conidia. Carbohydrate cytochemistry helped define three stages (Stages I, II, and III) of wild-type conidium maturation on the basis of changes in the ultrastructure and composition of the conidium wall. Conidia of the wetA6 mutant strain formed normally but failed to mature during Stages II and III. Specifically, the inner wall layer of wetA6 conidia did not condense during Stage II and two wall layers that stained for carbohydrates did not form during the transition to Stage III. Concomitantly, wetA6 conidia formed large cytoplasmic vacuoles and underwent lysis. The wetA gene appears to have a conidium-specific function for the modification of the conidium wall during Stages II and III. These modifications of the conidium wall are essential for the stability of mature, dormant conidia.

Ultrastructural localization of β-1,4-glucan-containing molecules in the cell walls of some fungi: a comparative study between spore and mycelium

An exoglucanase, purified from a cellulase produced by the fungus Trichoderma harzianum was complexed to colloidal gold and used for localizing β-1,4-glucan-containing molecules in the cell walls of some fungi. With the exception of Aspergillus niger, β-1,4-glucan-rich molecules were found to be associated with conidial walls of Trichoderma viride, Fusarium oxysporum f.sp. radicis-lycopersici, Verticillium albo-atrum, Penicillium thomii, and Ophiostoma ulmi. The abolition of wall labeling following previous cellulase digestion suggested that the compunds detected by the exoglucanase–gold complex were likely of cellulosic nature. Differences in cell wall composition between conidia and mycelium were reflected by the absence of β-1,4-glucan-containing molecules in the vegetative walls of most fungi tested. This raises the question as to what extent the chemical composition of spore walls should be considered as an important criterion in the taxonomy and phylogeny of fungi. The disappearance of these molecules upon conidial germination (with the exception of O. ulmi) suggests that lytic enzymes are produced to cause wall breakdown. The presence of molecules with β-1,4-linkages in conidia probably contribute to reinforcement of the wall architecture. Key words: fungi, spores, β-1,4-glucan, gold cytochemistry.

Isolation of tube precipitin antibody-reactive fractions of Coccidioides immitis

Patients presenting with primary coccidioidal infection have been shown by earlier investigators to produce immunoglobulin M (IgM) precipitin antibodies to lysates of mycelial and spherule phases of Coccidioides immitis. This humoral response has been detected by tube precipitin (TP) and immunodiffusion (ID)-TP assays of patient sera, which are valuable aids in early diagnosis of coccidioidomycosis. Several reports of antigenic fractions which show reactivity with patient TP antibody have been published. However, confusion persists with respect to the nature of the specific serologically reactive macromolecule(s). In this study we isolated two TP antibody-reactive antigens (TP-Ags) from an alkali-soluble, water-soluble fraction of the inner conidial wall and a culture filtrate plus toluene lysate of the mycelial phase of C. immitis. The crude antigens were first separated by concanavalin A (ConA) chromatography. The TP-Ags were identified in ID-TP assays as 120- and 110-kilodalton (kDa) fractions which were electroeluted from reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of the ConA-bound conidial wall extract and ConA-bound culture filtrate plus lysate preparation, respectively. Following electroelution, the 120-kDa fraction was subjected to gel filtration chromatography which yielded a major 240-kDa and minor 120-kDa component. The apparent dimer may be a product of disulfide bond formation resulting from reassociation of the reduced, monomeric components (120 kDa). The latter was suggested by the presence of cysteine in the isolated fraction. The electroeluted 110-kDa fraction was subjected to ion-exchange chromatography. The DEAE-isolated, TP antibody-reactive fraction was identified as antigen 2 in the coccidioidin-anti-coccidioidin reference system. Homogeneity of the TP-Ags was demonstrated in silver-stained sodium dodecyl sulfate-polyacrylamide gels of the respective chromatographically isolated fractions. The two purified TP-Ags showed reactivity in the TP and ID-TP assays and were capable of binding patient IgM but comparatively little IgG antibody, as determined by an enzyme-linked immunosorbent assay. It appears that the diagnostic TP reaction between sera from patients with coccidioidomycosis and the ID reference antigens examined in this study is a composite of IgM binding to both a 120-kDa and a 110-kDa antigen.