Fungal fimbriae

EXPERIMENTAL

MYCOLOGY

10,

19-27 (1986)

Fungal Fimbriae V. Protein A-Gold

A. SVIKEV,

lmmunocytochemical Lableling of the Fimbriae of Ustilago violacea

R. SMITH,

R. B. GARDINER,

I.

M.

RACKI,

AND

A.

W.

DAY

Departmenr of Plant Sciences, University of Western Ontario, London. Ontario, Cancida Accepted for publication August 20. 1985 SVIRCEV. A., SMITH, R., GARDINER, R. B.. RACKI, I. M.. AND DAY, A. W. 1985. Fungal fimbriae, V. Protein A-gold immunocytochemical labeling of the fimbriae of Ustilago l,io/acea. .&per-imental kJyco/ogy 9, 19-27. Protein A-gold immunocytochemical labeling of long protein appendages tfimbriae) on the surfaces of cells of Ustilago violacea is described. The specificity of the technique is confirmed by a series of serological and morphological controls. The technique allows specific identification and localization of fimbrial antigens in electron microscopic studies of a variety of fungi in both pathogenic and saprophytic situations. c 1986 Academic Press. Inc. INDEX DESCRIPTORS: fimbriae; protein A-gold labeling; immunocytochemistry; Ustiiago; Rhdotorula.

INTRODUCTION

Ouchterlony tests involving cells or fimbriae of the other species (Cardiner et uJ., 1982; Gardiner and Day, 1985). Surface fibrils of similar diameter, length, and moiecular weight were later detected on a large number of other smut species and in many of the related beterobasidiomyceto~s yeasts (Gardiner et al., 1981). S brils of similar diameter (6 10 nm) have recently been detected on the su ces of many ascomycetous yeasts (Ga ner et al., 1982) and on several ~larne~t~~s fuungi (manuscript in preparation). In parallel serological experiments involving agglutination tests or Ouchterlony tests. antigens responding to AU, AR, or both antisera were detected on most of the fungi mentioned above (Gardiner ef al., 1981, 1982: Gardiner and Day, 1985). These observations suggest that a family of immunolo~ica~~~ related proteins with a molecular weight of about 74,000 Da is widespread in fungi, appearing as 7-nm surface fibrils of varying lengths. The electron microscopic observations and serological tests described above rep

The cells of the smut fungus Ustilago violncea produce long surface appendages very similar to bacterial pili or fimbriae (Poon and Day, 1974). These fungal fimbriae are clearly revealed by shadowing (Fig. 1) or by negative staining (Fig. 2), appearing as flexuous fibrils with a diameter of 7 nm and lengths of up to 30 km (Poon and Day, 1974, 1975). The fimbriae are composed of a polymerized protein with subunits of 74,000 Da; no carbohydrate moiety has been detected (Poon and Day, 1975; Day and Cummins, 1981; Gardiner and Day, 1985). An antiserum, termed antiserum AU, has been raised against the 74,000 Da protein from cells of U. violacea strain aI (Gardiner et al., 1981), and a second antiserum (AR) has been raised against the 74,000 Da protein from similar surface fibrils on the basidiomycetous yeast, ~ho~oto~~~ia rubva (Gardiner et al., 1982). These antisera agglutinate fimbriated, but not defimbriated, cells of the corresponding species but show only slight cross-reactivity in agglutination tests and 19

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SVIRCEV

ET AL.

GOLD LABELING

resent two separate observations and therefore do not establish that the observed fibriIs contain the antigens detected. To establish this point directly, it was necessary to employ an immunocytochemical test. This paper reports the application of the protein A-gold immunocytochemical staining technique (Roth et al., 1978) to show the presence and specific location of fimbrial antigens on the surface of cells of U. violncea. This technique is applied here to the detection of fimbriae on whole cells, but it has the further advantage that it can also be used to detect and locate fimbriae in thin sections of cells. This was not previously possible with sections because of the small size and electron transparency of these fibrils. This work forms the basis for investigations in this laboratory into (a) the presence of fimbrial antigens on other fungi, and (b) the location and possible functions of fimbriae in tissues of animals or plants infected by fungi. MATERIALS

AND METHODS

Stock cultures. Stock cultures of wildtype strains of U. violacea (ATCC Nos. 22000 and 22001) were maintained and prepared for shadow casting or negative staining in 1% ammonium molybdate by the methods described by Poon and Day (1975). Defimbriation of cells was carried out either by high-speed agitation (Poon and Day, 1975) or by heating a ceil suspension to 50°C for 15 min (Gardiner and Day, 1985). In either method, the defimbriated cells

OF FIMBRIAE

2

were centrifuged and the pellet was washe in distilled water, then used immediately or kept for short periods at 4°C in the presence of 100 bg/ml cycloheximide to prevent fimbrial regeneration (Poon and Day, 1975). Stocks of R. vubra (Strain 80-4, Gardiner et al., 1982) were also maintained and treated in the same way. Preparation of protein A-gold complex. The reader is referred to the excellent review of protein A-gold immunocy~och~mistry by Bendayan (1984) for full details of the procedures used. Colloidal gold particles of 15-nm average size were folrmed by reducing c~lor~a~ric acid (HAuCl, * 3H,O, Baker) with sodium citrate (Pi-ens, 1973). Four milliliters of 1% aqueous sodium citrate was added to Ii0 ml of a boiling solution of 0.01% chloroauric acid and boiled for a furt until a wine red color was achieved dayan, 1984). The colloidal gold was at 4°C in the dark, conditions under which it remained stable for several months. To prepare the protein A-gold complex, 10 ml of the colloidal suspension was brought to pH 6.9 using 0. added to 0.3 mg of protein ical Co.) diss,olved in 0.2 ml plex was then centrifuge at 47,SOOg to remove exce tein A. The supernatant containin protein A was carefully aspirate carded. The dark red protein A-go plex in the pellet was resu of 0.01 M phosphate-bu 7.4, and this stock solution was stored at

FIG. 1. A well-fimbriated cell of U. vio/ucea shadowed with tungsten oxide showing numerous intertwined fimbriae arising from the cell surface. ( x 66,800) FIG. 2. A cell similar to that in Fig. 1, negatively stained with 1% ammonium molybdate showing fimbriae arising from the cell surface (bottom left) as well as loose fimbrial fragments. The individual fimbriae are constant in diameter at 7 nm. (X 66,800) FIG. 3. A fimbriated cell of U. violacea following treatment with antifimbrial antiserum (AU). Individual fimbriae appear decorated with antibody molecules, raising the overall diameter of the timbriae to about 3.5 nm. The antibody molecules coat the fimbriae at high density (minimum spacing 15-20 nm)-shown particularly clearly at the arrowed site-suggesting that antigenic sites are distributed at intervals at least as small as this. ( x 66,800)

22

SVIRCEV

4°C. The complex remained stable under these conditions for 6-8 weeks. AntiJimbvial antisera. The antisera used in this study were those described by Gardiner et al. (1982) prepared against (1) the fimbriae from a, cells of U. violacea (= antiserum AU), and (2) the fimbriae from strain 80-4 of R. rubra (= antiserum AR). Dr. B. G. Atkinson (Department of Zoology, University of Western Ontario, London, Ontario) kindly provided a sample of mouse anti-M-line antiserum. The controls used are described under Results. Preimmune serum was obtained from the rabbits prior to injection of fimbrial protein. Protein A-gold staining. Washed fimbriated or defimbriated cells were placed on Formvar-coated 400-mesh grids (Poon and Day, 1975). The grids were floated cell side down on drops of undiluted antiserum for 15-30 min at room temperature, then gently but thoroughly washed with distilled water. Two methods of washing were used: (a) transfer of the grids cell side down through a series of water droplets, and (b) exposure to a stream of distilled water squirted through a fine pipet. Experience suggested that the former technique was preferable, as there appeared to be some redistribution of gold particles when the squirting became too vigorous (see Bendayan, 1984). Following washing, the grids were floated cell side down on drops of the protein A-gold complex stock solution for 15 min at room temperature. The grids were washed thoroughly again with distilled water for 5 min. When required, shadow casting with tungsten trioxide (Poon and Day, 1975) or negative staining with 1% ammonium molybdate (Gardiner and Day, 1985) was done at this stage; the former after the grids had dried, the latter before they had dried. Gold labeling of isolated fimbriae was carried out using similar procedures, following deposition of samples of purified fimbrial protein (Gardiner and Day, 1985) onto Formvar-coated 400-mesh grids.

ET AL. RESULTS

Decoration of Fimbriae of U. violacea with Antibodies Fimbriated cells were treated with AU, negatively stained, and examined in the electron microscope to establish that antibodies in the AU antiserum bind directly to fimbriae and to determine the frequency of the antigenic sites along the fibril. Antibodies were detected on the fimbriae as particles coating the fibril and increasing its diameter from 7 to about 35 nm (Figs. 2, 3). The diameter of the decorated fimbriae in Fig. 3 is consistent with the known diameter of immunoglobulin G molecules of about 1.5 nm. Antibodies appeared to be packed without intervening spaces along the fimbriae, suggesting that the protein subunits are also closely packed together, with consequent high density of antigenic sites. These observations provide direct confirmation of other evidence (Gardiner and Day, 1985) that antibodies present in antiserum AU bind to fimbriae. Gold Labeling of Fimbviated Cells of U. violacea Heavily fimbriated cells of the a, strain of U. violacea were placed on grids and negatively stained, either shadowed, treated with AU + protein A-gold (pAg),’ or treated with AU + pAg and then either shadowed or negatively stained. The shadowed and negatively stained treatments were used to establish that the cells were indeed heavily fimbriated. As described previously (Poon and Day, 1974, 197.5) the fimbriae frequently associated and dissociated in loose cable-like structures and often crossed over each other (Fig. I). Those cells treated with antiserum AU then pAg were surrounded by a “cloud” of gold particles frequently extending 5-20 pm around the periphery of the cell (Figs. 4,5>. The gold was normally present as single ’ Abbreviation

used: pAg, protein A-gold.

GOLD

LABELING

particles, but in some cases aggregate particles were observed. These aggregates form during storage of the pAg complex and most were removed by low-speed centrifugation just prior to use of the pAg (Bendayan, 1984). The individual gold particles frequently formed straight or curvilinear lines, presumably following the track of individual fimbriae (Fig. 4). Individual particles in such lines were rarely closer than 20 nm apart and generally were 30-40 nm apart. This result is consistent with the size and nature of the pAg complex: in some negatively stained preparations the protein A was visible as a disk-shaped globule of about 3O-nm diameter with a gold particle of approximately 15-nm diameter at its center. As the frequency of antigenic sites is not a limiting factor, individual gold particles should bind to the fimbriae at minimum intervals of about 15-20 nm. Background levels of gold labeling were very low and attributable to detached fimbriae since they decreased as the number of washings increased (see Figs. 4, 5). In an effort to display the underlying fimbriae in such ‘“clouds” of gold around cells, some grids were shadowed after the pAg treatment. Unfortunately, while the gold labeling remained good, the fimbriae were niot clearly displayed. Nevertheless, occasional ridges overlain with gold particles were observed (Figs. 6, 7). This difficulty in displaying fimbriae by shadowing after AU treatment is attributed to material in the antiserum which dries down as surface deposits on the grid, burying the fimbriae and thus preventing their detection by shadowing. This interpretation is supported by observations of cells on other grids that were treated with antiserum AU only before shadowing. A rough background deposition was seen ah over the Formvar surface and the fimbriae were detectable only as ill-defined ridges. Further refinement of the technique involving purified antiserum preparations and treatment of the cells with AU prior to placing them on the grid are

OF

FIMBRIAE

23

being attempted in order to better shadowed fimbriae with simulta~e~ labeling. More satisfactory results were Qb~a~~e~ when gold-labeled fimbriae from a cell suspension, or from a preparation of purifie fimbrial protein (Gardlner and Day, %985)? were subsequently negatively stained (Figs. 8- 11). A major problem encountered in t procedure was that the negative stain tended to wash off the particles of gold. With care, however, fimbriae with antibodies and with reasonably dense pAg particles were observed. Even in these preparations, however, some Ioss of gold particles had probably occurred as judged by comparisons with control preparations lacking the negative stain treatment. Attached or detached fimbriae from a cell preparation (Figs. 8, 9) remanned as Bong fibrils and were not agglutinated together i clumps by the antibodies as much as was the purified fimbrial protein preparation (Figs. 10, 11). In both cases the highly specific labeling of the fimbriae by the protein A-gold complex was apparent. Controls

A variety of controls, including the series recommended by Bendayan (~9~4~, was used to establish the specificity of labeling. These controls were: (i) omission of the treatment with antiserum AU to detect nonspecific adsor~t~~~ of the pAg to the material; of preimmune serum or use of unrelated antisera such as mouse anti-M line antiserum to detect nonspecific binding of immunog~obulin molecules tcst material; (iii) absorption of AU with an excess of fimbrial antigen for 12 h at 4°C to verify the specificity of the antibody-antigen reaction; (iv) incubation with AU followed by pAg preabsorbed with an excess of either AU or preimmune serum;

24

GOLD LABELING

(v) incubation with AU followed by a 30minute incubation with protein A (0.2 mgi ml) and then by a 30-minute incubation with pAg to verify the specificity of the imrnunoglobulin-protein A interaction.

OF FIMBRIAE

25

pAg complex provides a highiy specific label for the 74,000-Da protein present in fimbriae. DISCUSSION

Specific staining of fimbriae with protein In all of these controls, gold labeling was A-gold depends on (a) the specificity ofthe absent or relatively sparse. antibody-antigen reaction, and (b) the In addition to these immunological conspecificity of the antibody-protein A reactrols, controls involving defimbriated cells tion. Several observations attest to the of e/. violacen and the cells and antifimbrial specificity of antiserum AU for fimbrial antiserum of R. mbra were also used. Deprotein. Thus, it has been shown elsew fimbriated cells were very lightly Iabeled on (Gardiner and Day, 1985) that: or very near the cell surface, but no clouds of particles in the extramural region were (i) isolated fimbrial protein (74,~~O seen. Fimbriated cells of R. JXL!Y~ had large that was used to inject the rabbits poiy-, clouds of gold particles in the extramural merizes under suitable conditions to form region after exposure to antiserum AR and fibrils of a similar diameter and fle pAg, but not after treatment with AU and as attached fimbriae (Gardiner an pAg. Similarly, very few gold particles be- 1985); came bound to the walls or extramural area (ii) all conditions, including genetic when cells of U. vioiacea were exposed to events, temperature conditions, as well as AR and pAg. This result was expected, as chemical, mechanical, and thermal defimthe antigens and antisera from these two briations, which remove visible fimbriation species do not cross-react in agglutination from cells of e/. vioiacea result in no agglutests (Gardiner et al., 1982), Ouchterlony tination following AU treatment; tests (Gardiner and Day, 198.51, or immu(iii) isolated and purified fimbriaf nofluorescence tests (unpublished data), reattaches to defimbriated ceils a and it serves as a useful confirmation of the stores both the appearance of ~~~~~ia~~~~ specificity of the gold labeling technique. and the ability to be agglutinated by AU; We conclude from these results that the (iv) positive precipitin lines form in combination of antifimbrial antiserum and Ouchterlony tests of AU against either isoFIG. 4. A well-fimbriated cell of U. violace~~ treated with AU antiserum and stained with protein A-gold complex, showing the distribution of fimbrial antigens in a “cloud” around the cell. Note ii) the absence of background labeling. (ii) the clear orientation. in several cases, of gold particules in straight or curved lines (arrows), an orientation interpreted as the labeiing pattern of individual fimbriae, and (iii) the spacing of gold particles at approximately regular intervals (minimum 20 nm), except for tight clusters that are due to clumping of gold prior to treatment. This spacing follows from the physical dimensions of the IgG molecules and the protein A-gold complex, as well as the frequency of antigenic sites (see text). The fimbriae themselves are not visible because they are electron transparent. ( x 17,8QO) FIG. 5. A cell similar to that in Fig. 4, but labeled with fewer and less extensive go!d particles which are restricted mostly to the cell wall and immediate extramural layer. The cell was from a population less fimbriated than that in Fig. 4. (x 13,500) FIGS. 6. 7. Cells of U. violucea treated with AU antiserum and protein A-gold complex and shadowed with tungsten oxide, showing labeled ridges presumed to be fimbriae. Treatment with the crude antiserum used deposited a rough background on the grids and, to a large extent, buried individual fimbriae (7 nm), making them difficult to detect compared to those on cells that were shadowed without prior antiserum treatment (Fig. 1). The ridges in the examples shown may represent a cable of intertwined fimbriae rather than individual fibrils. (Fig. 5, X 40.000; Fig. 7, X 44,50(3)

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SVIRCEV

ET AL.

GOLD LABELING

Hated fimbriae or fimbriated defimbriated cells.

OF FIMBRIAE ACKNOWLEDGMENT

cells, but not

The visualization of binding of antibodies to fimbriae (Fig. 3) provides direct confirmation of this specificity. That the protein A-antiserum interaction is also highly specific is demonstrated by the immunological controls and by the direct visualization of gold-labeled fimbriae (Figs. 8- 11). Protein A-gold immunocytochemical labeling thus provides a highly specific method for locating the fimbriae of U. violacea. However, the method can also be applied to detect and locate fimbriae in other fungi which have surface antigens responding to antifimbrial antisera (Gardiner et al., 1981, 1982; Gardiner and Day, 1985; and manuscript in preparation). Protein Agold labeling following treatment with AU or AR should therefore be a powerful tool to (i) detect fimbrial antigens in a variety of fungi, (ii) show whether or not these antigens are located on the 7-nm surface fibrils that appear to correspond to fimbriae on these species (Gardiner et al., 1981, 1982; Gardiner and Day, 1985), (iii) identify sites of synthesis of fimbrial proteins within the fungal cell, and (iv) identify fimbriae produced by pathogenic fungi in their host tissues to determine to what extent they penetrate host tissues (Day et al., 1984). The results obtained should provide valuable insights into the distribution and possible functions of fimbriae in fungi.

The work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (Grant A0290). REFEREMCES M. 1984. Protein A gold electron microscopic immunocytochemistry: Methods, apphcations and limitations. J. Electron Miscrosc. Tech. 1:243-270. DAY, A. W., AND CUMMINS, I. E. 1981. The genetics and cellular biology of sexual development in Usti/ago violacea. In Sexual Interactions in Eukat-yotic Microbes (D. M. Q’Day and P. A. Horgen, Eds,). pp. 379-402. Academic Press, New York. DAY, A. W., SVIRCEV. A. M., SMITH, R., APEDGAT(DINER. R. B. 1984. Fungal fimbriae and host infection. Phyto,patha/ogy 74: 833 (Abstract). FRENS, G. 1973. Controlled nucleation for the reguiation of the particle size in monodisperse gold soiutions. Nalure Ph?;s. Sci. 241: N-22. GARDINER, R. B., CANTON, M., AND DAY, A. W. 1981. Fimbrial variation in smuts and heterobasidiomycete fungi. Bar. Gaz. 442: 147-150. GARDINER. R. B., AND DAY; A. W. 8985. Fungai Embriae. IV. Composition and properties of fimbriae from Ustilago vioiacea. Exp. Mycol. 9: 334-350. GARDINER. R. B., PODGOR~U. C., AND DAY, A. W. 1982. Serological studies on the fimbriae of yeasts and yeastlike species, Bot. Gaz. 143: 534-541. POON, N. H., AND DAY, A. W. 19i4. Fimbriae in the fungus Vstiiago violacea. Nature i&cmdorr) 250: 648-649. POON. N. II., AND DAY, A. W. 1975. Fungal fnmbriae. 1. Structure. origin and synthesis. Canad. J. Microbiol. 21: 531-546. ROTH. .T., BENDAYAN. M.. AND ORCI, L. 1978. Ultra-. structural localization of intracellular antigens by the use of protein A-gold complex. J. Hisrocirem. Cytocllem. 28: 55-57. BENDAYAN,

FIGS. 8- 11. Note the antibody molecules decorating the fimbriae. raising their diameter to approximately 35 nm (cf. Figs. 2, 3). and the gold labeling of these antibody molecules at the sides of the fimbriae. Some gold has been lost during the negative stain treatment as judged by comparisons with similar preparations lacking negative stain. The small number of gold particles that are not associated with visible fimbriae may be due to (i) random background attachments, (ii) dissociated subunits of fimbrial protein, and/or (iii) fimbrial fragments that have not been outlined well by the negative stain. (X 66,800) FIGS. 8, 9. Detached fimbriae from a cell suspension of U. vioiacea treated with AtJ antiserum and protein A-gold complex, then negatively stained with 1% ammonium molybdate. FIGS. 10, 11. A purified. polymerized fimbrial protein preparation treated with antiserum AIJ and protein A-gold complex, then negatively stained with 1% ammonium molybdate.