Eur. J. Epidemiol.0392-2990
Vol. 8, No. 3
May 1992,p, 452-459
CYTOLOGICAL I M M U N O D E T E C T I O N OF YEAST G L Y C O P R O T E I N SECRETION J.C. CAILLIEZ.1, D. POULA1N*, D.W.R. MACKENZIE** and L. POLONELLI*** *Unitk 42 I N S E R M - D o m a i n e du C E R T I A - 369 rue J u l e s G u e s d e 59650 Villeneuve d ' A s c q - France. **Mycological R e f e r e n c e L a b o r a t o r y - C e n t r a l P u b l i c H e a l t h L a b o r a t o r y - 61 C o l i n d a l e A v e n u e L o n d o n N W 9 5 H T - U.K. ***Istituto di M i c r o b i o l o g i a - Universitdt Degli S t u d i di P a r m a - Via A . G r a m s c i 14 43100 P a r m a - Italia.
Keywords:Yeast - Glycoprotein secretion - Monoclonal antibodies - Immunodetection Expression of antigenic epitopes shared by secreted yeast glycoproteins was studied using specific immunological probes. Application of cytological and ultrastructural methods of immunodetection, employing monoclonal antibodies, permitted us to localize these glycoproteins in the cytoplasm, through the cell wall and at the yeast cell surface. Importance of glycosylationsecretion relationships were evaluated in the secretion process of these molecules. The cell wall crossing and the cell surface distribution of antigenic glycoproteins was described in immunoelectron microscopy and immunofluorescence. Some preferential secretion "ways" were suspected through the yeast cell wall leading to an heterogenous distribution of cell surface glycoproteins destined to be excreted into the medium. Antigenic variability of cell wall glycoproteins expression was discussed in relation with the glycloprotein secretion.
Yeast protein glycosylation
Glycoproteins secreted in yeasts are molecules of great interest because of their fundamental properties but also for their significant involvement in metabolism. The application of immunological methods have permitted the exploration of yeast biology by studying the biosynthesis, cytoplasmic transport and excretion modalities of metabolic products. This report reviews the role of glycosylation in the yeast secretion pathway and the methods to immunodetect secreted glycoproteins at the ultrastructural level by using specific monoclonal antibodies. 1 Corresponding author.
Primarily studied in Saccharomyces cerevisiae, Nglycosylation represents 95% of the whole yeast protein glycosylation (56). The process has been studied from the peptidic synthesis in the endoplasmic reticulum to the secretion of mature molecules into the medium (1, 17, 35, 48, 51). The neosynthesized peptide is translocated to the lumen of the endoplasmic reticulum, where an internal oligomannosaccharidic chain (core) is set up attached to an asparagine (asparagine-X-threonine/serine sites) by the intervention of a lipidic carrier (dolichol) (30, 31). A modification of this oligosaccharidic core by successive glucosidase and mannosidase activities leads to the elaboration and elongation of an external
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polymannosaccharidic chain (outerchain), conferring the final form to the mature glycoprotein (21). An Oglycosylation process provides some proteins of an oligosaccharidic structure composed of 1 to 4 mannose units linked to a serine or a threonine. In yeast cells, however, the O-glycosylation steps do not occur in the Golgi apparatus as in all other eukaryotic cells, since the first mannose is added to the protein in the Golgi lumen (51).
Glyeosylation-secretion relationships To evaluate the importance of glycosylation in yeasts, several studies have concentrated on protein precursors of well characterized glycoproteins by using tunicamycin (15, 26-29, 35, 50, 62), endo-13-Nacetylglucosaminidase (58), thermosensible secretory mutants (sec) (17, 18) and mutants possessing enzymatic defects characterised by incomplete glycosylation (mnn) (2). Glycosylation has been shown to play a significant role in the cytoplasmic transport of metabolic products and their secretion into the medium. In S. cerevisiae, the period of time required for the secretion of invertase is longer when tunicamycin is present (17, 18). In Cryptococcus albidus, the mature 48 kDa xylanase is secreted 2.5 times more rapidly than the corresponding 40 kDa deglycosylated enzyme produced in the presence of the antibiotic (37). Glycosylation can also be involved in the elaboration and preservation of the enzymatic activity of some glycoproteins secreted by yeasts. In C. albicans, the inhibiting effects of tunicamycin on Nglycosylation is responsible for biological disruptions, particularly in the synthesis of the outer fibrillar cell coat which normally mediates the adherence of yeast to epithelial cells and inert surfaces (47, 60).
Immunocytological probes In the past few years, a great number of monoclonal antibodies (MAbs) have been prepared against antigenic determinants of different yeast species (3-5, 11, 13, 19, 23, 25, 33, 34, 40, 41, 46, 54, 55, 57). Some have been shown to be specific for cell wall glycoproteins which can be secreted into the medium (16). Because of their high specificities compared to polyclonal antibodies, MAbs minimize the risk of cross-reactivity between epitopes shared by different glycoproteins. MAbs can be more properly and conveniently used as biological probes characterized by higher specificity and affinity. The high rate of MAbs specificity has increased our knowledge of the secretion modalities of yeast glycoproteins (38, 39). Fundamental studies have been performed with MAbs reacting with yeast glycoproteins involved in the pathogenesis of fungal infections (8, 44) or characterized by definite biological and biochemical properties, such as enzymes, toxins, adhesins, cellular receptors or sexual pheromones (33, 41). 453
In this report, the cytological study of glycoprotein secretion is discussed according to two different yeast models. The first concerns C. albicans glycoproteins which are secreted in vitro into the culture medium by blastoconidia and germ tubes and detected in vivo in the sera of patients affected by systemic candidosis (43). Polysaccharidic moieties of these glycoproteins are recognized by a monoclonal immunoglobulin M (MAb 5B2). The second model is represented by a Pichia anomala killer toxin which is characterized by a wide spectrum of antimicrobial activity (41). Different MAbs have been produced against the crude toxic extract of this killer strain. One of these (MAb KT4) was shown to be an immunoglobulin G producing a sharp precipitin band in the double immunodiffusion procedure in the reaction with the crude toxic extract and neutralising in vitro the killer activity of the secreted yeast killer toxin (41). MAbs 5B2 and KT4 have been used as immunological markers with different microscopic procedures to visualize the secretion of their corresponding glycoproteins. MAb KT4 was proven to be specific for the protein part of the killer toxin, whereas MAb 5B2 was directed to the polysaccharidic fraction of the complementary glycoprotein. Immunocytological localization of the superficial antigenic determinants is commonly visualized in yeasts by immunofluorescence (IFA) and immunogold silver staining (IGSS) assays whilst ultrastructural detection is performed by immunoelectron microscopy (IEM).
Cell surface distribution of antigenic glycoproteins All secreted glycoproteins accumulate in the cell wall before being released into the culture medium. Cell wall labeling observed by IFA and IGSS confirms that MAbs may react with the complementary epitopes of the yeast glycoproteins before their secretion. Direct fluorescence assays have shown that yeast populations originating from a single cell give heterogeneous reactions with polyclonal antibodies. Later, a similar heterogeneity of surface labeling was described with agglutinating MAbs produced against C. albicans antigenic determinants (3, 5). Two different heterogeneities of labeling should be considered. The first is clearly independent from the concentration of the MAb used for immunodetection and shows that some cellular elements are better recognized by MAbs. A difference of intensity in labeling could be Observed on daughter-cells, young buds or germ tubes in comparison with their mothercells or mature blastoconidia (Fig. 1). The second type of heterogeneity in labeling is directly linked to the concentration of MAb. In this form, a homogeneous labeling, which attests to the presence of the antigenic glycoproteins in the whole periphery of the cell, can become heterogeneous simply by increasing the dilution of the MAb used to perform the immunodetection. It appears that the distribution of the epitope reacting with MAb is not homogeneous at
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Figure 1. - Indirect immunofluorescence assay of Candida albicans V W . 32 germ tubes reacting with MAb 5B2 ascitic fluid. A homogenous labeling was observed at the surface of the yeast mother-cells, whereas the apex of the germ tubes were weakly labeled. Bar = 10 tam.
the yeast cell surface. Some cell wall areas may present a greater concentration of antigenic glycoproteins which may be due to either a more intense secretion or to the thickness of the cell wall, as sometimes is observed in the septum or the bud and birth scars. This heterogeneous immunolabeling is only detectable with optimal concentration of MAb which permits a weak to a high fluorescent labeling to be distinguished. The heterogeneous distribution of the epitope might be masked in the case of strong fluorescence due to higher MAb concentration Reasonably, this heterogeneous labeling could be due to a differential secretion of yeast glycoproteins across some, as yet undefined, cell wall areas. In immunodetection, antigenic determinants never become completely saturated with antibodies. Unoccupied antigenic sites, therefore, may exist which are probably due to a rapid and heterogeneous secretion of newly synthesized glycoproteins through the cell wall. By decreasing the MAb concentration, a "punctuated" fluorescence can be observed at the surface of yeast cells often appearing like dashes or punctuation marks (Figs. 2 and 3). A linear labeling, which can be detected on a great number of cells, corresponds in most cases to their bud and birth scars. In this area, the greater thickness of the cell wall seems to be principally responsible for the great quantity of MAbs which are fixed because, to date, no work has demonstrated a preferential secretion of yeast glycoproteins in bud and birth scars. To confirm that this "punctuated" labeling was not due to an artefact caused by mechanical changing in the shape of yeast cells when dried on glass slides, the same IFA must be carried out in batches with cells kept live in buffered solution. However, in these experimental conditions, a remarkable amount "of yeast cells do not fluoresce because of immuno-agglutination.
Figure 2. - Immunogold silver staining of C. tropicalis cells reacting with G40 gold-conjugated MAb 5B2 (particles of 40 nm). The gold labeling was visualized by a silver enhancement. A "punctuated" labeling was observed on the surface of yeast cells (large arrows). This particular labeling could also be observed on the germination element, showing a significant fLxation of MAb 5B2 (free arrow). Bar = 0.5 lain.
Figure 3. - Indirect immunofluorescence assay of Pichia anomala UCSC 25F killer cells reacting with MAb KT4. A heterogenous fluorescence was observed at the yeast cell surface. Cells were covered by a punctuated labeling related to some undefined cell wall areas. Bar = 10 tam.
Presumably, such cells are not accessible to the FITC-conjugated antibodies. "Punctuated" and "patched" labelings suggest the existence of preferential cell surface areas of secretion, which can represent the last step of an organized secretion pathway for yeast glycoproteins.
Immunoelectron microscopy procedures
Immunoelectron microscopy (IEM) is a suitable assay to visualize antigenic determinants of yeast
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glycoproteins at the ultrastructural level. In order to be used as specific probes for IEM studies, antibodies are linked to electron-dense complexes like colloidal gold particles. Yeast cells may be embedded in hydrophilic resins such as Lowicryl, London resin white or Metacrylate resins as well as frozen in liquid nitrogen. Ultrathin sections are then prepared and collected on carbonated and membraned nickel grids. Sections are first incubated with a monoclonal or monospecific polyclonal antibody reacting with secreted glycoproteins and, in a second step, with a gold conjugated antibody directed against the class and species of the first antibody. Alternatively, MAbs can be linked to gold colloidal particles and used in direct immunodetection. Such immunolabelings involve a single incubation in purified MAbs which are directly linked to colloidal gold particles. The decrease in sensitivity of the reaction is compensated by a greater specificity and a lower level of background labeling (24, 52). Direct immunodetection does not present any problem of interpretation of the labeling because all gold particles which are fixed on the yeast section effectively reveal epitopes reacting with the complementary MAb. With indirect immunodetection, the utilisation of purified MAbs is not required. Reactions can be carried out in two (Mab + goldconjugated antibodies) or three (MAb + biotinylated antibodies + gold-conjugated streptavidin) steps and permit the use of a panel of different commercial antibodies and other ligands conjugated with gold particles which can be produced in a variety of sizes generally ranging from 5 to 40 nm. Hence, tracers coupled to different sized colloidal gold particles can be discerned visually in IEM studies and permit performing double- or triple-labeling experiments. By a process of amplification, the sensitivity of immunodetection increases with the number of steps involved, but this in turn has the disadvantage of producing greater background labeling. This nonspecific reaction is particularly common working with C. albicans because a number of laboratory animals used to produce commercial conjugated antibodies are natural carriers of the opportunistic yeast. Therefore, it might be necessary to previously adsorb the conjugated antiserum with the C. albicans cells used in the immunodetection assay.
Cytoplasmic transport and cell wall crossing The two main functions of the cell wall in yeasts are to maintain the cell shape and to constitute a dynamic barrier between the fungus and its environment. This latter function is of particular importance for the study of the secretion of glycoproteins, molecules which may actively interact with other microbial or mammalian cells. The complexity of the yeast cell architecture emphasizes the difficulties inherent to the immunodetection of antigens localized in the cell wall and cytoplasm (14). The cell wall is a rigid multi-layered structure (44),
composed of polysaccharides and proteins, which is largely responsible for the immune response. It is also often involved in interactions between the fungal cell and its environment. In pathogenic yeast species, surface glycoproteins exert an important role in hostparasite relationships (49). In C. albicans, particularly, the cell wall is surrounded by an adhesive fibrillar coat whose superficial layers, constituted by mannoproteins, are responsible for the yeast adherence to the host epithelial cells and actively promote colonization (47, 59, 60, 61). Indirect IEM assays have been performed on ultrathin sections of yeast cells fbxed and embedded in hydrophilic resins or frozen in liquid nitrogen and immunodetections have been performed with MAbs for evaluating the cytoplasmic and cell wall distributions of glycoproteins destined to be secreted. A cytoplasmic labeling is always obtained and this shows that MAbs are able to recognize their complementary epitopes on molecules, which can be due to either intracellular form or to precursors of the mature glycoproteins. Immunolocalization of a yeast killer toxin has been reported in the cytoplasm of P.anomala isolates using MAb KT4 (Fig. 4) (manuscript submitted for publication). Biochemical studies are currently in progress to characterize the nature of the cytoplasmic molecules which carry the reacting epitopes (protoxin or mature non-excreted killer toxin).
Figure 4. - Ultrastructural immunodetection with MAb KT4 recognized by G15 gold-conjugated goat anti-mouse immunoglobulin G (particles of 15 nm) on ultra-thin sections of P. anomala UCSC 25F killer cells embedded in Araldite. The labeling mainly occurred in the cytoplasm and into the vacuole (V). Only a few colloidal gold particle were observed on the cell wall (arrows). Bar = 0.5 lam.
The number of gold particles observed throughout the cell wall layers must, in practice, be sufficient to permit an accurate description of the glycoprotein secretion. In C. albicans, some preferential secretion "ways", characterized by linear
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distribution of the epitopes throughout the cell wall, have been described in IEM (44) (Fig. 5). An agglutination of gold particles, directly linked to the MAb 5B2, has been described like a "patched" labeling. If the linear immunolabeling observed by IEM in the cell wall of C. albicans corresponds to preferential secretion "ways", the "punctuated" and "patched" labeling described in IFA and IGSS respectively could be associated with the emergence of the glycoproteins before their secretion into the medium (9, 44). Under our experimental conditions in IFA, the P. anomala killer toxin epitopes reacting with MAb KT4 appeared also to be preferentially expressed through defined regions of the yeast cell wall even if no distinct secretion "ways" were detected by using the IEM assay (Fig. 6) (manuscript in preparation). Unexpected labeling can be obtained in IEM assays according to the nature of fixation procedures which may alter the epitope conformation and affect its accessibility to the MAb. The ability of viable and glutaraldehyde-fixed yeast cells of C. albicans to bind concanavalin A and monospecific antibodies has been examined in IEM (36). The findings showed that glutaraldehyde-fixed yeast cells bound in a minor amount the lectin (210/0) and the antiserum (19°/0). Recently, the glutaraldehyde f~xation procedure has also been involved in the dissociation of some C. albicans antigenic determinants recognized by specific monoclonal antibodies (32). These experiments, however, were unable to show whether the difference in surface labeling was qualitative or quantitative. In P. anomala, the absence of MAb KT4 fixation in the cell wall of yeast killer cells embedded in Araldite resin might be referred to the high concentration of glutaraldehyde used in the fixative solution.
Figure 5. - Ultrastmctural immunodetection with MAb KT4 reacting with biotinylated goat anti-mouse immunoglobulin G and G15 gold-conjugated streptavidin (particles of 15 nm) on ultra-thin sections of P. anomala UCSC 25F cells embedded in Lowicryl K4M resin. The labeling mainly occurred in the cell wall (CW). The labeling of the cell wall outer-layers was not homogenous at the whole cell surface (arrows). Bar = 0.5 lam.
Figure 6. - Ultrastructural direct immunodetection with G40 gold-conjugated MAb 5B2 (particles of 40 nm) on an ultra-thin section of C albicans VW.32 blastospore. A linear distribution of colloidal gold particles might be observed through the cell wall (arrows). Bar = 0.5 pm.
Glutaraldehyde is known to react primarily with the Eamino groups of lysin in the protein and, possibly, with the glucosamine of the fungal chitin (22). Thus, the tertiary structure of the epitope may be altered by the formation of artificial linkages between the protein amine residues of the killer toxin (manuscript submitted for publication).
Antigenic variability The variability in the antigenic expression of (secreted) glycoproteins has been described in the C. albicans cell wall by many authors who have emphasized the importance of studying comparatively in the IEM assay different isolates and culture conditions (3-7, 10, 12, 20, 23, 55). The eventual absence of MAb fixation on the yeast cell surface does not necessary mean that the reactive glycoproteins are not synthesized. A cytoplasmic or a cell wall innerlayer labeling may be detectable by IEM assay, even although the cell surface is totally unreactive by IFA (7, 8). In C. albicans, a variable antigenic expression of surface glycoproteins has been reported using polyclonal (42, 53) and monoclonal antibodies (3, 5). The transitory reactivity of some cell wall glycoproteins, described in IEM with MAbs, has been due to the discontinuous excretion of these molecules from the periplasmic space to the yeast cell wall outer layers (9, 44). Recent cytochemical and immunological studies using labeled lectins, MAbs and enzymatic substrates, have suggested that glycoproteins may be subject to some rearrangements which could explain the irregular appearance of the distribution of these molecules at the yeast surface (50, 61). Possible mechanisms for cell wall reorganization during the cell cycle could be attributed to (i) a masking of some
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surface glycoproteins by other synthesized components, (ii) an enzymatic degradation of the cell wall surface glycoproteins resulting in their liberation into the medium, and (iii) the capping of these cell wall components in some part of the yeast cell surface and their eventual redistribution over some other cellular elements. It may be noted, however, that the movement o f yeast glycoproteins in the cell wall has, to date, never been reported. Many authors have reported that the in vivo or in vitro antigenic expression at the surface of C. albicans is a straindependent dynamic process which is influenced by environmental and nutritional factors (3-7, 12, 20, 42, 55). Yeast glycoprotein secretion, therefore, should be studied in a variety of strains and media over time to avoid missing immunodetections (7).
Ballou L., Alvarado E., Tsai P., Dell A. and Ballou CE. (1989): Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class : support
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Regardless of their potential values for diagnostic (45, 57), taxonomic (34, 40) or epidemiological (5, 13) purposes, no definitive and complete study with MAbs produced against yeast glycoproteins can be achieved without cytological analysis of the distribution o f the antigenic determinants with which they react. The knowledge of the expression and mode of biosynthesis of glycoprotein antigenic determinants under different culture conditions (in relation to the cell cycle) among yeast species and strains is essential to understand the physiology of secretion, a continuous process more than a sequence of distinct events. MAbs proved to be very effective tools for visualizing the presence o f (pro)glycoproteins in the cytoplasm of yeast cells and the emergence of the mature glycoproteins before their secretion into the culture medium. The relationship between a dynamic secretion process and static glycoprotein immunolocalization remains, however, difficult to be establish and requires critical interpretation. Acknowledgments
This study was supported by the research program of the European Economic Community "Cooperative European Research on Candida albicans Cellular Biology and Pathogenicity" (contract number SCI 0361 EDB), the National Research Council (CNR) Target Projects "Biotechnology and Bioinstrumentation", "Prevention and Control of Disease Factors" (contract number 91.00121.41 / 115.15897) and by the Ministero della Sanith, Istituto Superiore di Sanit/t Progetto A.I.D.S. (1990), Roma, Italy. The authors are grateful to Mr. Gilbert Lepage and Ms. Annie Benigand, Unit6 42 1NSERM, Villeneuve d'Ascq, for their technical assistance. REFERENCES
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