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Immunolocalization of a proteinase produced by the sap-staining fungus, Ophiostome piceae Hoffert, Cyrla 1995

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IMMUNOLOCALIZATION OF A PROTEINASE PRODUCED BY THE SAP-STAINING FUNGUS, OPHIOSTOMA  PICEAE  by CYRLA HOFFERT B.Sc, McGill University, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Faculty of Forestry, Department of Wood Science)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  June 1995 © Cyrla Hoffert, 1995  In presenting  this  thesis in  degree at the University of  partial  fulfilment of  the  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted department  or  by  his  or  her  representatives.  It  is  by the head of  understood  that  copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  The University of British Columbia Vancouver, Canada  DE-6  (2/88)  ABSTRACT  Ophiostoma piceae, the most frequently isolated sap-staining fungus in Canada, produces an extracellular subtilisin-like, serine proteinase. This enzyme is presumed to serve an important nutritive function for the fungus as it grows in wood where nitrogen is found primarily in the form of protein. To further characterize this proteinase, an attempt was made to detect and localize it in O. piceae cells. The fungus was grown in a minimal, liquid medium supplemented with either protein or inorganic nitrogen as nitrogen sources. The approaches taken to localize the proteinase in the cells included a) detection of cell-associated proteolytic activity and b) immunological detection of this proteinase in both whole cells and cell fractions. Proteolytic activity was assessed using both the general substrate azocoll and a subtilase specific substrate, succinyl(Ala)2-Pro-Phe-p-nitroanilide. For immunological detection, polyclonal antibodies were prepared to localize the proteinase in O. piceae by immunoblotting methods and immunogold labelling with transmission electron microscopy. Polyclonal antibodies were first raised against proteinase K, an enzyme that is similar to the O. piceae proteinase, and then against the O. piceae extracellular proteinase purified by hydrophobic interaction chromatography. Characterization of these antibodies indicated that they recognized the O. piceae proteinase. In comparison to extracellular proteolytic activity, little activity was detected in washed cells and homogenized cells of O. piceae grown in soya milk medium. An even lower amount of activity was detected in cultures supplemented with inorganic nitrogen. By immunodetection, the enzyme was found to be associated with the O. piceae cells from both inorganic nitrogen cultures and protein provided cultures. Gold labelling was observed primarily in the cell wall and the extracellular sheath. The results suggested that the proteinase can be detected in the O. piceae cells whether or not they are secreting active proteinase. This was the first time the extracellular proteinase produced by O. piceae was studied with respect to the cells.  ii  TABLE  OF CONTENTS Page  Abstract  ii  Table of Contents  iii  List of Abbreviations  v  List of Tables  vi  List of Figures  vii  List of Plates  ix  Acknowledgements  x  1.0 Introduction  1  1.1 Sapstain control and prevention  2  1.2 Sapstain fungi in Canada: their dispersal and their growth in wood  3  1.3 Nutrition of sapstain fungi  4  1.4 Proteolytic activity of fungi  5  1.5 Characterization of the major extracellular proteinase produced by O. piceae  7  1.6 Immunogold labelling technique for studies of wood colonizing fungi 1.7 Objective  8 10  2.0 Materials and Methods 2.1 Fungal strain 2.2 Culture conditions and biomass determination  11 11 11  2.3 Cell homogenization 2.3.1 Harvesting and washing 2.3.2 Cell disruption 2.3.3 Protein extraction and protein determination of fractions  12 12 12 13  2.4 Detection of proteolytic activity 2.4.1 Proteolytic activity - azocoll substrate 2.4.2 Proteolytic activity - succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate 2.4.3 Proteolytic activity - zymogram  14 14 14 15  2.5 Production and purification of polyclonal antibodies 2.5.1 Anti-proteinase K serum 2.5.2 Anti-O. piceae proteinase serum 2.5.3 Purification of polyclonal antibodies  15 15 16 17  iii  Page 2.6 Protein gel electrophoresis  17  2.7 Immunoblotting 2.7.1 Dot blot preparation 2.7.2 Western blotting 2.7.3 Chemiluminescent detection of proteins  19 19 20 20  2.8 L i g h t microscopy and immunogold silver enhanced staining (IGSS)  21  2.9 Tissue preparation for transmission electron microscopy and immunolabelling  22  2.10 Post-embedment immunogold labelling  23  3.0 Results  24  3.1 G r o w t h and proteolytic activity in l i q u i d cultures of O. piceae  24  3.2 Production of purified polyclonal antibodies  30  3.3 Characterization of the purified polyclonal antibodies  31  3.3.1 Electrophoretic analysis of proteins used for antibody characterization 3.3.2 Characterization of the anti-proteinase K IgG 3.3.3 Characterization of the anti-O. piceae proteinase IgG 3.4 Localization of the proteinase of O. piceae using the p K I g G and the p O p I g G 3.4.1 C e l l disruption and protein extraction 3.4.2 Immunoblot detection of O. piceae proteinase using the purified antibodies 3.4.3 Immunogold labelling - Microscopic detection of O. piceae proteinase using the purified antibodies  31 36 41 44 44 49 54  3.4.3.1 Immunogold labelling of the proteinase i n O. piceae cells 62 3.4.3.2 Immunogold labelling of the proteinase in O. piceae cell w a l l fragments 63  4.0 Discussion  85  4.1 G r o w t h and proteolytic activity of O. piceae in l i q u i d culture 4.2 Production and characterization of antibodies for proteinase localization 4.2.1 A n t i - O . piceae proteinase monoclonal antibodies 4.2.2 Anti-proteinase K polyclonal antibodies 4.2.3 A n t i - O . piceae proteinase polyclonal antibodies 4.3 Detection of the extracellular proteinase i n O. piceae cells b y immunoblotting 4.4 Proteinase localization by i m m u n o g o l d labelling and microscopy  85 89 89 90 92 94 96  5.0 Conclusion  102  6.0 Literature cited  105  i v  LIST OF ABBREVIATIONS  BCA- bicinchoninic acid BSA- bovine serum albumin ELISA- enzyme-linked immunosorbent assay FPLC- fast protein liquid chromatography HIC- hydrophobic interaction chromatography IEF- isoelectric focussing IGSS- immunogold silver staining MAC- membrane affinity chromatography method 1A- 2% glutaraldehyde fixation followed by embedment in LR White resin method 4B- 2.5 % glutaraldehyde and 2.0% para-formaldehyde fixation, 1% OsC>4 post-fixation, embedment in Epon resin. OSO4- osmium tetroxide PAGE- polyacrylamide gel electrophoresis PBS- phosphate buffered saline pKIgG- purified anti-proteinase K IgG PMSF- phenyl methyl sul fo ny 1 f luo ri d e pOpIgG- purified anti-O. piceae proteinase IgG PVDF- poly-vinylidene fluoride SDS- sodium dodecyl sulphate TEM- transmission electron microscopy  v  LIST OF TABLES  Page Table 1: Proteolytic activity in liquid cultures of O. piceae, determined with the azocoll substrate.  28  Table 2: Proteolytic activity in liquid cultures of O. piceae, determined with the peptide substrate, succinyl-(Ala)2-Pro-Phe-p-nitroanilide.  29  Table 3: Summary of results of TEM processing trials.  56  vi  LIST OF FIGURES Page  Figure 1: Biomass and proteolytic activity of O. piceae grown in organic (a) and inorganic nitrogen (b) supplemented minimal medium. 25 Figure 2: Total proteolytic activity in fractions from cultures of O. piceae that were provided with organic nitrogen or inorganic nitrogen. 27 Figure 3: Silver stained SDS-PAGE Phast gel (8-25% gradient) showing O. piceae proteinase and proteinase K. 33 Figure 4: Silver stained IEF gel (pi 3-9) of O. piceae proteinase... Figure 5: Silver stained SDS-PAGE Phast gel (8-25%) of commercial preparations of various proteins that were used for screening the purified polyclonal antibodies for cross-reactivity.  34  35  Figure 6: Immunodot blots of various proteins probed with the pKIgG (A) or buffer (B) in the primary incubation. 38 Figure 7: Western blots of proteins shown in Fig. 3 that were probed with the pKIgG. Figure 8: Native-PAGE separation (Bio-Rad Mini-Protean II) of the semi-purified proteinase (lane 1) and proteinase purified by HIC (lane 2).  39  40  Figure 9: Immunodot blot of various proteins probed with the purified rabbit pre-immune serum IgG fraction (A) or the pOpIgG (B). 42 Figure 10: Western blot of various proteins probed with the pOpIgG (A) and the pre-immune serum IgG fraction (B). Figure 11: Phase contrast photomicrographs of stationary phase O. piceae cells from soya milk culture before (A) and after (B) homogenization.  43  46  Figure 12: SDS-PAGE (8-25%) and silver staining of extracts and intracellular fractions from stationary phase cultures provided with organic nitrogen (O) or inorganic nitrogen (I). 47 Figure 13: Silver stained SDS-PAGE (8-25%) gels of extracellular protein from soya milk culture (A) and inorganic nitrogen culture (B).  vii  48  Figure 14: Dot blot of fractions from organic nitrogen supplemented cultures probed with the pKIgG diluted 1 /500 (A) or the pOpIgG (B) diluted 1 /18 000. Figure 15: Dot blot of fractions from inorganic nitrogen supplemented cultures probed with the pKIgG diluted 1 /500 (A) or the pOpIgG (B) diluted 1/18 000.  50  51  Figure 16: Western blot of extract from cell wall fragments and intracellular filtrates from stationary phase cultures of O. piceae grown in medium containing organic nitrogen (O) or inorganic nitrogen (I). 53 Figure 17: Dark field photomicrograph of O. piceae cells labelled using the pKIgG for IGSS.  viii  55  LIST OF PLATES Page  Plate 1: O. piceae cells fixed with 2.0% glutaraldehyde (method 1) and embedded in LR White (A) or Epon (B) resin.  58  Plate 2: O. piceae cells fixed with 2.5% glutaraldehyde plus 2.0% paraformaldehyde (method 2) and embedded in LR White (A) or Epon (B) resin.  59  Plate 3: O. piceae cells fixed with 2.0% glutaraldehyde, post-fixed with 1.0% OsC>4 (method 3) and embedded in LR White (A) or Epon (B) resin.  60  Plate 4: O. piceae cells fixed with 2.5% glutaraldehyde plus 2.0% paraformaldehyde, post-fixed with 1.0% OSO4 (method 4) and embedded in LR White (A) or Epon (B) resin.  61  Plates 5 through 8: Transmission electron micrographs of O. piceae cells processed by method 4B and immunogold labelled with the pKIgG.  66-69  Plates 9 through 12: Transmission electron micrographs of O. piceae cells processed by method 4B and immunogold labelled with the pOpIgG.  71-74  Plate 13: Immunogold labelling of O. piceae cells, grown in minimal medium supplemented with ammonium nitrate, and processed for TEM by method 1 A. 76 Plate 14: Immunogold labelling of method B processed O. piceae cells that were taken from exponential phase culture supplemented with ammonium nitrate. 77 Plates 15 and 16: Negative controls for gold labelling of cells from soya milk provided culture (Plate 15) and inorganic nitrogen provided culture (Plate 16) processed by method 4B.  78-79  Plates 17 through 20: Transmission electron micrographs illustrating gold labelling of O. piceae cell fragments.  81-84  ix  ACKNOWLEDGEMENTS  I thank my supervisor, Dr. Colette Breuil, who's concern, enthusiasm and generosity "sans faille" enabled me to complete this work and provided me with valuable experience. You are a source of infinite energy. And thank you to my co-supervisor, Dr. David Brown, for his helpful advice and guidance. I extend my deepest gratitude to Linda Abraham, Srabani Banerjee and Serge Gharibian for their generous help, advice and support throughout my studies. Your input was invaluable. I also want to thank my electron microscopy teachers, Beatrice Valentine and Dr. David Brown, for helping me and sharing their knowledge and experience with me. I feel very fortunate to have been one of your students. And, thanks to Forintek Canada Corp. for allowing me to use their equipment and facilities, and to Maria Chan for her help while I worked there. Thank you friends and family for your care and understanding. And, Andrew, thank you for your support, patience and understanding, and for always being there without question (Agape). The purified antibodies that are described in this thesis, and were essential to completing this work, were produced by Michael Chester, Xiumei Feng and Cecilia Stocker. The ammonium sulphate precipitate of culture filtrate proteins described in this thesis was provided by Claudia Yagodnik, and the HIC purified proteinase was provided by Linda Abraham. This research was financially supported by a strategic grant from the Natural Sciences and Engineering Research Council of Canada.  1.0 INTRODUCTION  Wood has long been appreciated for its clarity and natural beauty. Consumers have always paid a premium for wood that is clear and free of visible defects. The importance of blemish-free lumber is evidenced by the millions of dollars in product quality claims sustained yearly by Canada's lumber industry as a result of sapstain. Sapstain, also known as bluestain, usually refers to the blue, grey or black discoloration of sapwood resulting from its colonization by fungi with darkly pigmented hyphae. Though these fungi cause little structural damage to wood, the aesthetic defect they produce reduces the value and marketability of wood (Wilcox 1973; Zabel and Morrell 1992). Therefore, bluestain is a serious problem for wood products industries worldwide. There are approximately 200 fungal species known to stain sapwood (Seifert and Grylls 1991). These include non-decaying fungi that, in general, are either superficial (surface moulds) or penetrating (sap-stainers) colonizers of wood (Findlay 1959; Seifert and Grylls 1991). The surface moulds, such as Aspergillus spp. and Trichoderma spp., produce masses of pigmented spores which can be easily planed or brushed off of wood (Zabel and Morrell 1992; Seifert 1993). These fungi are not as economically important as the sap-stainers which penetrate the wood deeply and discolour it with their darkly pigmented hyphae or by their production of pigments that diffuse into the wood tissue (Findlay 1959; Phillips and Burdeken 1982). These fungi, which have been isolated from most economically important softwood species, including those of Abies, Pinus, Picea, Larix, Pseudotsuga and Tsuga genera, are primarily responsible for the monetary losses sustained by the Canadian lumber industry due to wood discoloration (Seifert and Grylls 1991; Seifert 1993). Consequently, millions of dollars in Canada alone are invested annually in sapstain prevention (Smith 1991).  1  1.1 Sapstain control and prevention In a d d i t i o n to the i m p l e m e n t a t i o n of preventative l o g g i n g practices ( P h i l l i p s a n d B u r d e k e n 1982), there are generally t w o methods that have been e m p l o y e d for sapstain prevention: k i l n d r y i n g and chemical treatment. K i l n d r y i n g reduces the w o o d moisture content to b e l o w 19% o n average, w h i c h creates a n environment unsuitable for fungal g r o w t h .  This  treatment can be very effective as long as the w o o d is kept dry. But there are both economic a n d practical problems associated w i t h this approach.  K i l n d r y i n g is expensive, particularly for  w o o d s w i t h inherently high moisture contents (Byrne a n d Smith 1987; S m i t h 1991).  It is not  w o r t h the expense for offshore shipments, since freight charges are based on volume, not weight. M o r e o v e r , w i t h no foolproof and universally accepted w a y to prevent rewetting of the w o o d d u r i n g transport overseas, k i l n d r y i n g could be futile.  In addition to these limitations, certain  customers prefer green lumber for specific end uses, and large dimension lumber tends to be more susceptible to d r y i n g defects (Seifert 1993). C h e m i c a l protection is the traditional approach, h a v i n g been e m p l o y e d since the early 1900's (Zabel a n d M o r r e l l 1992).  C h e m i c a l l y treating w o o d w i t h protectants, such as  chlorophenates, has been s h o w n to be very effective (Zabel a n d M o r r e l l 1992; Seifert 1993). W h e n employed i n conjunction w i t h good d r y i n g practices, lumber can be protected throughout all stages of shipment. H o w e v e r , a significant problem w i t h this method of sapstain prevention is the non-specificity of the chemicals used. Chlorinated phenols, w h i c h are considered to be the most effective for p r e v e n t i n g sapstain, are toxic to other life forms.  Furthermore, many  formulations contain dioxins w h i c h persist i n the environment (Smith 1991; Seifert 1993; Byrne and Smith 1987). N o t surprisingly, these chemicals have been opposed b y the environmentally concerned a n d restrictions have already been legislated o n them. In recent years, chemicals that are less damaging to the environment have been developed and utilized at some mills (Byrne and Smith 1987). H o w e v e r , not only are these formulations less effective than the chlorophenols at preventing the development of sapstain, these chemicals are generally more expensive, some are hazardous to the m i l l worker and some are highly corrosive to equipment (Byrne and Smith 1987;  2  Zabel and Morrell 1992; Seifert 1993). Furthermore, nine of the products that are presently in use in Canada, have only received temporary registration (Byrne and Smith 1987; Smith 1991). Therefore, there is a need for new treatments which specifically target the offending organisms without causing problems in implementation and in the environment. This might be achieved by gaining a better understanding of the fungi that cause sapstain. For this, fundamental research on the physiology and growth requirements of sapstain fungi is necessary.  1.2 Sapstain fungi in Canada: their dispersal and their growth in wood The prevalence and distribution of sapstain fungi in Canada was recently assessed by Keith Seifert and Bryan T. Grylls (1991). They isolated various staining fungi from a range of economically important wood species and grouped them into four main categories: Ophiostomatales, dematiaceous moulds, green moulds, and black yeasts. The Ophiostomatales category includes most of the sap-staining fungi and is comprises a number of Ascomycetes including species of Ceratocystis and/or Ophiostoma genera and their anamorphs (Class Deuteromycete), Sporothrix, Graphium and Leptographium. The Ophiostomatales cause sapstain by their production of darkly pigmented fruiting bodies, spores and hyphae (Seifert and Grylls 1991). Ophiostoma species produce ascospores that are borne in long necked perithecia, while the asexual forms produce conidia from synnemata (Alexopolous and Mims 1979; Seifert and Grylls 1991). Spores from both ascogenous and conidial fruiting bodies are released non-violently within a mucilagenous matrix to be dispersed by air currents or water films and droplets (Alexopolous and Mims 1979, Seifert and Grylls 1991). These routes allow the spores to be introduced to the wood through exposed wood rays or ruptured tracheids (Zabel and Morrell 1992; Phillips and Burdeken 1982).  Alternatively, the sticky  masses of spores can be dispersed by adhering to bark beetles and/or phoretic mites which carry the spores beyond the otherwise unpenetrable bark (Subramian 1983; Hudson 1986). Wood is therefore most susceptible to infection by sapstain fungi during seasoning, transport and storage of lumber before the wood is dried (Zabel and Morrell 1992; Seifert 1993).  3  When environmental conditions in wood are favourable for fungal growth (e.g., appropriate nutrient, moisture and free oxygen contents), spores can germinate into mycelia which colonize the sapwood.  Hyphal growth is primarily radial, through the sapwood ray  parenchyma, though growth can also be axial through longitudinal tracheids in softwoods or through vessels in hardwoods (Ballard et al. 1984). In general, the heartwood is more resistant to growth of staining fungi, since it lacks easily metabolized nutrients and contains toxic extractives (Findlay 1959; Subramian 1983). Sapwood colonization by staining fungi can be quite rapid. Under ideal conditions they can grow as much as 0.5 mm  tangentially, 1 mm  in the radial plane  and 5 mm longitudinally in a 24 hour period (Zabel and Morrell 1992). Hyphal growth into adjacent cells is believed to be mechanically mediated (Findlay 1959; Subramian 1983; Ballard et al. 1984). The hypha forms an appresorium like structure with a penetration peg ("Transpressorium") which allows the fungus to enter adjoining parenchyma cells by penetrating pits or by creating tiny bore holes in the wood cell wall (Findlay 1959; Ballard et al. 1984; Zink and Fengel 1989; Zabel and Morrell 1992). Colonization of tracheids, in softwoods, is similarly mediated by hyphae which push through or break down the torus of bordered pits (Ballard et al. 1984). There has been no evidence of enzymatic dissolution of the wood cell walls as is observed with wood decay fungi (Findlay 1959; Subramian 1983; Ballard et al. 1984).  1.3 Nutrition of sapstain fungi The proliferation of sapstain fungi in wood parenchyma cells is indicative of the nutrients required by these organisms. Unlike decay fungi, sapstain fungi do not obtain their nutrients from the structural components of wood (Seifert 1993); most sapstain fungi lack the enzyme activity associated with this type of degradation (Liese 1970). It is thought that they obtain their nutrients from the stored macromolecules found in wood (Wilcox 1973). The sapwood parenchyma cells therefore, serve as an ideal environment for fungal growth, since they contain a large amount of storage compounds, including proteins, soluble sugars (e.g., starch) and lipids (e.g.,  4  glycerides)which sapstain fungi can metabolize (Findlay 1959; Kramer and Kozlowski 1960; Subramian 1983; Gao et al. 1994). To obtain carbon and nitrogen from the parenchyma cell nutrient stores, sapstain fungi require appropriate metabolic activity. Nitrogen, for example, which comprises 0.1%, or less, of the dry weight of most woods (Cowling and Merrill 1966), is found predominantly in the form of protein in wood, often stored in vacuoles (Langheinrich and Tischner 1987; Sauter and Wellencamp 1988). So, not only is there a small amount of nitrogen in wood, but the majority of it is in a form that fungi cannot readily assimilate. Therefore, a proteolytic enzyme system, which would serve to break down these proteins into smaller assimilable nitrogenous compounds, is likely essential for the fungus to acquire the nitrogen it requires for growth.  1.4 Proteolytic activity of fungi Fungi from all of the major taxa produce intracellular and extracellular proteolytic enzymes (Cohen 1980) that serve various functions to promote the growth and survival of the fungus. For example, proteases are important for nutrition (Lilly et al. 1991; Burton et al. 1993; Zhu et al. 1994; Venable et al. 1995), inactivation or activation of enzymes (Deshpande 1992; Choi et al. 1994), morphogenesis, including, branching and hyphal extension (Bartnicki-Garcia 1968; Mahadevan and Mahadkar 1970; Lilly et al. 1991; Deshpande 1992) and pathogenesis (St. Leger et al. 1987; Reichard et al. 1990; Frosco et al. 1992; Kolattukudy et al. 1993; Markaryan et al. 1994). Many sap-staining fungi have been shown to produce proteolytic enzymes during active growth that are presumed to serve a nutritive function by providing the fungus with the nitrogen it requires in an assimilable form (Breuil and Huang 1994; Banerjee et al. 1995a; Banerjee et al. 1995b; Breuil et al. 1995). Ophiostoma piceae (Munch) H. and P. Syd., the most frequently isolated sapstain fungus in Canada (Seifert and Grylls 1991), produces a variety of proteolytic enzymes. These include cell associated carboxy- and amino-peptidases (Banerjee et al. 1995a,b; Breuil et al. 1995) and extracellular proteases (Abraham et al. 1993). The activity of these enzymes has been detected  5  when O. piceae is grown in liquid medium, as well as when the fungus is grown in blocks of lodgepole pine wood (Breuil and Huang 1994; Banerjee et al. 1995a,b; Breuil et al. 1995; Abraham and Breuil 1993). These enzymes are believed to play an important role in the physiology of O. piceae in wood. Extracellular proteolytic activity in liquid cultures of O. piceae appears to be partially dependent on the type of nitrogen source present in the growth medium. When O. piceae is grown in a minimal liquid medium that is supplemented with protein, a large amount of proteolytic activity is detected in the culture supernatant (Abraham et al. 1993). In fact, one major extracellular proteinase constitutes the majority of extracellular protein released by the fungus in the culture supernatant (Abraham and Breuil 1995b). Proteolytic activity is also detected in the culture supernatant of O. piceae liquid cultures that are depleted of or starved for nitrogen (Abraham et al. 1993). However, when O. piceae is provided with easily assimilable forms of nitrogen, such as ammonium or amino acids, proteolytic activity is barely detectable in the culture supernatant (Abraham et al. 1993). Therefore, extracellular proteolytic activity is only evident when assimilable forms of nitrogen are not available. These findings support the assumption that proteases are required by O. piceae when it grows in wood where nitrogen is mainly organic, as protein. In addition, proteolytic activity detected in wood that was inoculated with O. piceae demonstrates a similar pattern of inhibition to that found for liquid culture (Abraham and Breuil 1995b). This suggests that the proteases produced by O. piceae under both growth conditions are the same. Therefore, an understanding of the proteases produced in liquid culture would help in determining how to specifically inhibit these key enzymes in wood and thereby, the growth of O. piceae on wood.  6  1.5 Characterization of the major extracellular proteinase produced by O. piceae  The major extracellular proteinase that is secreted by O. piceae in protein-supplemented liquid medium has been isolated, purified and characterized (Abraham and Breuil 1995b). The molecular weight of this enzyme is 33 kDa and it has an isoelectric point of 5.6. Preliminary studies indicate that this enzyme is a serine proteinase because it is completely inhibited by phenylmethanesulphonyl fluoride (PMSF). Its activity is also somewhat inhibited by the chelating agent, ethylenediaminetetraacetic acid (EDTA) which indicates that it has a requirement for metal ions (e.g. Ca 2 ) for stability. +  This proteinase was finally classified as a class II subtilisin, since it possesses various properties in common with other proteinases of this category. These properties include a sensitivity to autolysis, similar patterns of inhibition, and a broad cleavage specificity with a slight preference for aromatic residues in the PI position (Siezen et al. 1991; Abraham and Breuil 1995a,b). In addition, enzymes from this group, commonly referred to as subtilases, show a high degree of sequence homology and considerable conservation of their secondary structure (Siezen et al. 1991). Since the N-terminus sequence of O. piceae proteinase was similar to those of known class II subtilases, including proteinase K, its classification was confirmed (Abraham and Breuil 1995b). Proteinase K, one of the most extensively characterized members of this group of proteinases, is a commercially available enzyme that is isolated from the soil ascomycete Tritirachium album Limber (Ebeling 1974). Like proteinase K, the O. piceae proteinase is optimally active at neutral to alkaline pH at 37°C with a total loss in activity at pH below 3 which is possibly due to changes in secondary structure (Bajorath et al. 1988; Abraham and Breuil 1995a). However, in contrast to proteinase K and other subtilases, the O. piceae proteinase is extremely unstable at high temperatures due to autolysis (Abraham and Breuil 1995a). So far, the major extracellular proteinase has been characterized by biochemical analysis of the enzyme that was purified from liquid culture supernatant (Abraham and Breuil 1995a,b). Localizing this proteinase in O. piceae will further its characterization. One way to localize it is by immunological detection methods. 7  1.6 Immunogold labelling technique for studies of wood colonizing fungi Immunological probes can be very sensitive and highly specific for a particular antigen. Antibodies are commonly used for the specific detection of cellular components or metabolic products of organisms including wood-inhabiting fungi. The enzyme-linked immunosorbent assay (ELISA), for example, has been shown to be effective for the detection of soft rot caused by Phialophora sp. (Daniel and Nilsson 1991) as well as for the early detection of sapstain fungi in wood (Breuil et al. 1988). Polyclonal and monoclonal antibodies that were raised against O. piceae cell wall proteins could potentially serve as tools for early diagnosis of sapstain in wood as well as for the specific detection of O. piceae in wood among other wood inhabiting fungi (Breuil et al. 1988; Banerjee et al. 1994). Antibodies have also been used for detecting or localizing fungal cells or components by light and electron microscopy. Immunogold labelling is a relatively new microscopic technique, particularly in its application to studying wood-colonizing fungi. Briefly, immunogold labelling involves the use of colloidal-gold-conjugated-antibodies as affinity probes to specifically label an antigen for visualization by microscopy (VandenBosch 1991). Many researchers have used this method for the detection or localization of fungal metabolic products or cellular components in fungi that are grown in wood or artificial media. For example, monoclonal antibodies raised against a toxic glycopeptide produced by Ophiostoma ulmi, the "Dutch elm disease" pathogen, were used to localize this toxin in infected seedlings by immunogold labelling. The results permitted an evaluation of the distribution of the toxin in host tissue (Benhamou et al. 1985). In another study, Benhamou et al. (1986) localized fimbrial proteins in O. ulmi by using an antiserum raised against fimbriae of the basidiomycete fungus Rhodotorula rubra. The results not only suggested the importance of fimbrial protein-host interactions for the establishment of disease, but, technically, it showed that antibodies raised against antigens of one organism could crossreact with and localize similar antigens in another organism.  8  In addition, immunogold labelling has been used for localizing fungal wood-degrading enzymes including those involved in lignin degradation (Blanchette et al. 1989; Daniel et al. 1989, 1991 and 1992; Lackner et al.; Nicole et al. 1992) or cellulose degradation (Sprey 1988; Blanchette et al. 1989; Gallagher and Evans 1990, Green III et al. 1992) in liquid cultures and/or in situ. These studies have contributed to an improved understanding of the growth and/or pathogenesis of wood colonizing fungi by providing valuable information on their nutrition, the interaction of different enzymes and metabolites, the function of extracellular fungal sheaths, in addition to the degradative effects these enzymes can have on wood. Similarly, immunolocalization of the proteinase produced by O. piceae will add to our knowledge and understanding of its role in the physiology of this sapstain fungus.  Fungal  proteolytic activity has been given relatively little attention in comparison to ligninolytic and cellulolytic activity in wood biodegradation studies. In fact, to my knowledge, proteases of wood colonizing fungi have not been localized by immunogold labelling although, proteases of entomopathogenic fungi (St. Leger et al. 1987; Goettel et al. 1989) and human pathogens (Homma et al. 1992; Moutaouakil et al. 1993; Markaryon et al. 1994) have been studied and immunolocalized.  9  1.7 Objective  As previously mentioned, most of the studies of O. picture's major extracellular proteinase have so far been focused on the proteinase, isolated from the supernatant of liquid cultures. Knowledge of where the enzyme is located during growth of O. piceae could help in the design and development of specific inhibitors or alternative control strategies. In previous work with O. piceae, polyclonal antibodies and monoclonal antibodies that were raised against cell wall proteins from this fungus, were specific for this fungus and were utilized in ELISA, immunoblotting and immunogold labelling of liquid culture and wood grown O. piceae (Luck et al. 1990; Banerjee et al. 1994). The same techniques could also be used to detect and localize the major proteinase in both liquid culture of O. piceae and in sapwood infected by O. piceae. This would confirm that the proteinase produced by O. piceae in liquid culture is the same as that produced during its colonization of wood. It could also provide information that could help in targeting this enzyme to inhibit O. piceae growth in wood. The objective of this work was to localize the 33 kDa extracellular serine proteinase that is produced and secreted by O. piceae when it is grown in liquid medium supplemented with protein. The main method used to achieve this objective was immunological detection because of its potential specificity and sensitivity. Presented first is an assessment of growth and proteolytic activity in liquid cultures of O. piceae, supplemented with inorganic nitrogen or protein as a nitrogen source. This is followed by a description of the production and characterization of polyclonal antibodies that were required to immunolocalize this proteinase in O. piceae cells. Finally, the utilization of these antibodies in immunodetection methods, including immunoblotting and immunogold labelling for transmission electron microscopy, is illustrated. The data provide a preliminary assessment of the activity and localization of this proteinase in O. piceae cells.  10  2.0 M A T E R I A L S A N D M E T H O D S  2.1 Fungal strain A culture of Ophiostoma piceae 387N (Munch) H. and P. Syd. (O. piceae) was obtained from the Forintek Canada Corp. (Quebec, QC, Canada) culture collection. This strain had originally been isolated from softwood chips at the MacLaren Mill in Mason, Quebec. The fungus was grown on 2% malt extract agar and then stored as 3 mm cores in 10% glycerol at -80°C.  2.2 Culture conditions and biomass determination O. piceae was grown in a semi-synthetic medium (Media B) composed of 0.4 g CaCl2,1.0 g KH2PO4, 0.8 g Na2HP04, MgS04-7H20, 3.0 g KH-phthalate, 20.0 g soluble starch, 1 mL of 1000 times concentrated micronutrient solution (Vogel 1956) and 1 mL of filter sterilized vitamin solution (Montenecourt and.Eveleigh 1977) per litre. The medium was supplemented either with 1.6 g/L N H 4 N O 3 (inorganic nitrogen medium) or 200 mL/L of diluted, filter sterilized unsweetened soya milk (Sunrise Markets, Vancouver, B.C., Canada) containing 2.1% protein, 0.93% fat and 0.6% carbohydrate (organic nitrogen medium). The inocula (0.01 mg/mL) were pregrown in medium containing either inorganic nitrogen or organic nitrogen for 3-4 days from the stored 3 mm cores of O. piceae . Cultures were grown in darkness on a rotary shaker (250 rpm) at 23°C for 10 days. Fungal growth was monitored daily by determining the dry weight of the fungus. Two mL of culture were filtered through a pre-weighed glass microfibre filter paper (Whatman), dried in a microwave (4 minutes at high power) and then allowed to cool to room temperature in a desiccator before being weighed.  11  2.3 Cell homogenization  2.3.1 Harvesting and washing  Liquid cultures of O. piceae grown in both organic and inorganic nitrogen containing media, were harvested at late exponential growth phase i.e.; near the end of the active growth phase, and in stationary growth phase, and were washed as follows to remove any loosely bound extracellular components. Whole cultures were transferred into 50 mL poly-allomer centrifuge tubes and spun down for 20 minutes at 9690 g in a Sorvall RC24 centrifuge. The culture supernatant was recovered and the cells were washed in an equal volume of sterilized PBS.  Cells were  centrifuged at 9690 g for 10 minutes and then washed similarly two more times with a final spin at 27 000 g to form a compacted pellet. The resulting washed pellet was removed with a sterilized spatula and transferred into a disposable, sterile 50 mL polypropylene tube. The washed cells were spun for 5 minutes at 3500 rpm in an International Equipment Corp. (IEC) benchtop centrifuge.  2.3.2 Cell disruption  The washed pellet was kept on ice throughout the following homogenization/disruption steps*. Initially, a small volume (approximately 3 mL) of homogenization buffer (0.1 M TrisHC1, 10 mM CaCl2-2H20, pH 8.0) was added to the 0.5 g (equivalent of dry weight) pellet. The suspension was made homogeneous before being transferred into a 75 mL Duran bottle with a glass rod or, for more compacted pellets, an Omni 2000 probe homogenizer (Diamed Lab Supplies, Missisauga, Ont, Canada). Chilled homogenization buffer and washed glass beads (425-600 nm; Sigma Chemical Co., St. Louis, Mo., U.S.A.) were added to the bottle to achieve a final, approximate 1:1:1 volume ratio of cells:buffer:beads. The cells were then disrupted in a Braun homogenizer (MSK Braun, Germany) for 10 and 20 seconds intervals and cooled on ice for at least 30 seconds between shakings. Cell disruption was monitored by phase contrast microscopy and continued until at least 80% of cells in a given field appeared to be damaged or lysed. Once the 1 In this text, the terms "homogenization" and "disruption", and variations, are used interchangeably to describe the breaking open and fragmenting of fungal cells. 12  cells were sufficiently disrupted, the homogenate was vacuum filtered through nylon stocking into a side arm flask in order to remove the glass beads. The homogenate was then transferred into a 15 mL corex centrifuge tube. The flask was rinsed with 1 mL of homogenization buffer which was then added to the corex tube. The homogenate was then forced through a 22G1 syringe (Becton Dickinson and Co., Lincoln Park, N.J., U.S.A.) 5 times and then centrifuged at 10 800 g for 30 minutes. The filtrate was removed (intracellular fraction) and an approximately equal volume of homogenization buffer was added to resuspend and wash the remaining cell fragments. After centrifuging for 25 minutes at 12 000 g, this first wash was recovered (wash) and the cell fragments were similarly washed two more times to obtain a final pellet of washed cell fragments. Samples of the unwashed and washed cell wall fragments were taken for electron microscopy processing as described in section 2.9. Volumes and wet weights were monitored throughout the experiment.  2.3.3 Protein extraction and protein determination of different fractions  Protein was extracted from the various pellets and the whole cell homogenate by boiling samples in 1% SDS in 0.1 M Tris-HCl, pH 8.0 buffer for 10 minutes. Samples were prepared at a concentration of 0.2 mg (wet weight)/mL extraction buffer. Supernatants were recovered by centrifuging the boiled suspensions for 10 minutes at room temperature at 13 000 rpm in an Eppendorf micro-centrifuge. Protein was determined for all homogenization fractions by the bicinchoninic acid (BCA) method using a kit purchased from Sigma. Appropriate dilutions were applied in 20 uL volumes per well on a 96 well Falcon Probind microplate (Becton Dickinson). BCA reagent was added at 200|j.L/well with a multi-pipettor. The plate was sealed and incubated for 30 minutes at 50°C, then cooled on ice for 15 minutes. Absorbances were measured at 562 nm on a Thermomax microplate reader (Molecular Devices Corp., Menlo Park, Calif., U.S.A.). Standard curves were prepared using a stock solution of 1 mg/mL bovine serum albumin (BSA).  /  13  2.4 Detection of proteolytic activity  2.4.3 Proteolytic activity - azocoll substrate  Total proteolytic activity in liquid culture samples of O. piceae was detected and quantified spectrophotometrically using the substrate azocoll (<50 mesh, Calbiochem, La Jolle, Calif., U.S.A.) in a colorimetric assay modified from Chavira et al. (1984) and as described in Abraham et al. (1993). In 18 mm diameter test tubes, azocoll was prepared in 0.1 M Tris-HCl buffer, pH 8.0 on the basis of 4 mg/mL of azocoll per tube. Assays were conducted at 37°C in a shaking water bath (320 rpm) for 20 or 40 minutes for organic nitrogen culture samples and 15 to 24 hours for inorganic nitrogen culture samples. The reactions were stopped with 50 uL of 50% (w/v) trichloroacetic acid (TCA) per tube. The contents of each tube were poured into micro-centrifuge tubes and centrifuged for 10 minutes at 13 000 rpm. Triplicate aliquots of 200 uL were transferred to a 96 well Falcon Probind microplate and the absorbances were measured at 525 nm (A525) using a Thermomax microplate reader. Activity (U) was defined as the amount of enzyme required to give an A$25 increase of 0.1 OD units per mL per minute (organic nitrogen culture) or per mL per hour (inorganic nitrogen culture) of reaction time. Samples were assayed on the basis of volume. Pellets were assayed for activity by preparing a suspension of pre-weighed sample in a volume of 0.1 M Tris-HCl, 20 mM C a C l 2 - H 2 0 buffer, pH 8.0, to give a 0.2 mg (wet weight) per mL concentration. A blank or negative control of substrate only, was included with each assay.  2.4.2 Proteolytic activity - succinyl-Ala-Ala-Pro-Phe-p-nitroanilide  The  substrate  peptide substrate, Succinyl-(Ala)2-Pro-Phe-p-nitroanilide was used for a  qualitative determination of proteolytic activity in cells of both inorganic nitrogen and organic nitrogen cultures. This substrate was prepared in 0.1 M Tris-HCl, pH 8.0 to a final concentration of 1 mM. Samples of 10 uL from supernatant, whole cell wash and cell wall material from both soya milk supplemented and inorganic nitrogen supplemented cultures, were added to micro-centrifuge  14  tubes containing 200 uL substrate. Reactions were carried out at room temperature over a period of 7 days. Activity was noted as a change in colour relative to the control conditions. Controls included, buffer only (negative) and ammonium sulphate precipitate of O. piceae supernatant proteins which included the proteinase (positive).  2.4.3 Proteolytic activity - zymogram  Native-PAGE gels (Section 2.6) were used for contact print zymograms for the detection of proteolytic activity. Proteolytic bands were detected using unprocessed X-Omat RP-XRP-1 X-ray film (Kodak, Rochester, N.Y., U.S.A.) as described in Abraham et al. (1993). The X-ray film with its gelatin coated surface, was incubated in contact with the gel for 4 -20 minutes at 37°C in a moisture chamber. After incubation, the film was carefully removed from the gel surface and rinsed under a stream of cool water. Proteolytic activity was represented by zones of clearing which indicated gelatin hydrolysis i.e., the presence of proteolytic activity  2.5 Production and purification of polyclonal antibodies  Polyclonal antibodies used for the immunological detection of the O. piceae proteinase were generated against a) proteinase K from Tritirachium album and against b) the extracellular proteinase of O. piceae.  2.5.1 Anti-proteinase K serum  Two female New Zealand white rabbits were sub-cutaneously immunized at multiple sites. Each rabbit received 1.3 mg of proteinase K (Sigma) dissolved in 0.5 mL of phosphate buffered saline, pH 7.2 (PBS) and mixed with an equal volume of Freund's complete adjuvant. This initial immunization was followed by 5 intra-muscular boosts that were 12-14 days apart which consisted of two 0.5 mL injections of 1.3 mg/mL proteinase K in Freund's incomplete adjuvant. Test bleeds were taken at the second, fourth and fifth boosts while the final bleeds  15  were taken 11 weeks after the initial immunizations. Direct ELISA was performed at each bleed to monitor antibody production and reactivity.  2.5.2 Anti-O. piceae proteinase serum  For the preparation of antibodies against the proteinase of O. piceae, this enzyme was purified from soya milk culture supernatant in two main steps. First, total protein was precipitated from the culture supernatant with ammonium sulphate at 90% saturation. The precipitate was resuspended in 100 mM Tris-HCl, pH 8.0 (Breuil and Huang 1994). This semi-pure preparation of the proteinase was further purified by hydrophobic interaction chromatography (HIC) in a Pharmacia (Pharmacia, Uppsala, Sweden) fast protein liquid chromatography (FPLC) system using a Phenyl-Superose HR5/5 column from Pharmacia (Abraham and Breuil 1995b). The column was equilibrated with 100 mM Tris-HCl, 1.7 M ammonium sulphate, pH 8.0 and protein was eluted by applying a decreasing linear salt gradient from 1.7 to 0 M ammonium sulphate in the buffer. The peak corresponding to the proteinase was recovered and treated with 1 mM phenylmethylsulfonyl fluoride (PMSF) to prevent autolysis of the proteinase. The excess PMSF was removed by washing with PBS, pH 8.0. The proteinase was concentrated in an Amicon (Amicon, Beverly, Mass., U.S.A.) Centricon-10 with a molecular weight cut-off (MWCO) of 10 000. The total amount of proteinase that was recovered from each purification run varied from 1.6 mg to 2.4 mg as determined by BCA assay. Two female New Zealand white rabbits were immunized sub-cutaneously at multiple sites. Each rabbit received 1 mL of the PMSF-inactivated O. piceae proteinase prepared in PBS and mixed with an equal volume of Freund's complete adjuvant. Four subsequent boosts, each 2 to 4 weeks apart, consisted of multiple site intra-muscular injections with the O. piceae proteinase in PBS mixed with an equal volume of Freund's incomplete adjuvant for a total of 1 mL. Test bleeds were taken 2 weeks following each boost while the final bleeds were taken 17 weeks after the initial immunization.  Direct ELISA was performed at each bleed to assess antibody titre and  reactivity.  16  2.5.3 Purification of polyclonal antibodies  The polyclonal and pre-immune sera were "purified" by isolating the IgG class of antibodies using Amicon Protein A membrane affinity chromatography (MAC) discs (Amicon). The methods described in Malakian et al. (1993) and the manufacturer's recommendations, were followed as described here. Twenty MAC discs, stacked in a MAC disc holder, were pre-washed with 60 mL PBS, then 60 mL 0.15 M glycine, pH 2.3 and finally, 100 mL of equilibration buffer (200 mM Tris-HCl, pH 8.0). Buffers were added to the MAC unit at a flow rate of 55 mL/minute using a peristaltic pump (Cole-Parmer Instrument Co., Chicago, 111., U.S.A.). The A28O of the equilibration buffer was considered as "baseline". Serum was diluted in sample preparation buffer (3 M NaCl and 1.5 M glycine, pH 8.9) at a ratio of 1/5. The serum was slowly injected into the MAC unit with a syringe and was recirculated 11 times. The unit was then washed with equilibration buffer until the ^280 returned to baseline. Antibodies were then eluted from the membranes with approximately 30 mL of 0.15 M glycine, pH 2.3. Fractions of 1 mL were collected in test tubes containing 87.5 uL 1 M Tris-HCl, pH 8.0, in order to rapidly bring the pH of the fraction to neutral. Following A28O readings of fractions, those having the highest absorbances were combined. The purified anti-proteinase K antibodies (pKIgG) were stored as 500 uL aliquots at -80°C. Anti- O. piceae proteinase purified antibodies (pOpIgG) and pre-immune serum IgGs were concentrated at 4°C using Amicon's centricon-10 concentrators, (MWCO 10 000). Protein determinations (see section 2.3.3) were performed to estimate the concentration of recovered IgG.  2.6 Protein gel electrophoresis. Gel electrophoresis was carried out to analyze proteins in various O. piceae derived samples as well as in commercially prepared samples. Two electrophoresis systems were used: the Bio-Rad Mini-Protean II apparatus (Bio-Rad Laboratories, Richmond, Calif., U.S.A.) and the Pharmacia LKB PhastSystem (Pharmacia).  17  Proteins were separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) and native-PAGE in 12% gels (Laemmli 1970) using the Mini-Protean II apparatus. SDS-PAGE sample buffer containing p-mercaptoethanol was prepared fresh prior to use (Laemmli 1970). Protein samples for native-PAGE were not boiled and SDS was omitted from sample buffer, gels and running buffer. To allow for resolution of low molecular weight proteins, the SDS-PAGE method of Shagger et al. (1980) was followed for gel preparation and buffer preparation. Separation by SDS-PAGE and native-PAGE were carried out at 110 V at 200 V, respectively until the bromophenol blue marker reached the bottom of the gel. With the PhastSystem, protein samples were analyzed by SDS-PAGE (8-25% gradient), native-PAGE (8-25% gradient) and/or IEF gel electrophoresis (pi range 3-9). Running conditions were as follows: 30 minutes at a constant voltage of 250 V for SDS-PAGE, 30 minutes at a constant voltage of 400 V for native-PAGE and for IEF, proteins were separated on pre-focussed (5 minutes at 1000 V and 2.0 mA) gels at a constant current of 2.0 mA for a total of 431 Vh. Pre-cast gels and the appropriate buffer strips were purchased from Pharmacia. Samples for SDS-PAGE were prepared in sample buffer as recommended by Pharmacia with the fresh addition of (3mercaptoethanol. Samples for native-PAGE and IEF were prepared in Nanopure water (Barnstead/Thermolyne, Dubuque, la., U.S.A.). Following electrophoresis, proteins were either electro-blotted onto protein binding membranes (see section 2.7.2), or stained with coomassie blue {0.1% R-250 coomassie blue (BioRad), 40% methanol, 10% acetic acid}. Alternatively, Phast gels were silver stained in the Phast system, according to the manufacturer's recommendations. SDS-PAGE low molecular weight standards were purchased from Bio-Rad and Pharmacia. IEF standards used were purchased from Pharmacia. The amount of protein loaded depended on the sample and the use of the gel. Commercially purchased enzymes (see section 2.7.1) were prepared in 20 mM Tris-HCl, pH, 8.0. PMSF was sometimes added to O. piceae proteinase samples and proteinase K for SDS-PAGE separation, to prevent enzyme autolysis upon sample boiling.  18  2.7 Immunoblotting  2.7.1 Dot blot preparation  A 0.2 um pore size nitrocellulose membrane (Bio-Rad) was prewetted in PBS, lightly blotted dry on Whatman filter paper, and then placed into a Bio-Dot apparatus (Bio-Rad). Protein samples, diluted in PBS were dotted in 20 uL or 10 uL volumes at various concentrations. Mild suction was applied intermittently for 1 to 2 minutes to accelerate the drying of the membrane. The membrane was left to dry overnight at room temperature and was removed from the apparatus the following morning for immunodetection. The samples of dotted O. piceae derived proteins included: a) pellet extracts (unwashed whole cells, whole cells, homogenized cells and cell wall fragments) and other fractions obtained from cell disruption, b) culture supernatants, c) ammonium sulphate precipitate of culture supernatant proteins from soya milk supplemented liquid cultures, and d) extracellular proteinase purified by preparative HIC (Abraham and Breuil 1995b) from culture supernatant of soya milk supplemented cultures. Other samples included various commercial preparations of fungal and bacterial enzymes: a) proteinase K from Tritirachium album (Sigma, P-6556 ) prepared in 20 mM Tris-HCl, 100 mM CaCl2"H20, pH 8.0, b) lysing enzymes from Trichoderma harziamim (Sigma, P-2265), c) protease Type X (thermolysin), a metallo-protease from Bacillus thermoproteolyticus (Sigma, P-1512), d) chitinase from Streptomyces  griseus (Sigma C-1525), and e) a-amylase from  Bacillus  amyloliquifaciens (Boehringer and Mannheim, 161764, GmbH, Germany). These were prepared in 20 mM Tris-HCl buffer, pH 8.0.  19  2.7.2 Western blotting  For Western blotting, protein samples derived from O. piceae cultures as well as commercially bought enzymes were subjected to gel electrophoresis as described in section 2.6. Separated proteins were transferred to either nitrocellulose or poly-vinylidene fluoride (PVDF) membrane by one of two systems: a) PhastSystem (semi-dry transfer) or, b) in a Bio-Rad TransBlot apparatus. For both systems, membranes cut to the size of the gel, were pre-equilibrated in chilled transfer buffer (10 mM CAPS buffer, pH 11 with 20% methanol). A backing membrane of nitrocellulose was included in the transfer sandwich to bind protein that may have passed through the primary membrane. The Phast transfer was performed at a constant current of 25 mA for 25-35 minutes with the cooling bed set at 23°C. The Bio-Rad transfer was performed at a constant voltage of 45V for 3 hours in a transfer buffer filled chamber fitted with a cooling coil. Following transfer, gels were stained with coomassie blue and membranes were stained with 0.2% Ponceau red to determine the success of the transfer. PVDF membrane used was 0.1 um pore size Immobilon P^Q (Millipore, Bedford, Mass., U.S.A.). Nitrocellulose membrane used was as described in section 2.7.1.  2.7.3 Chemiluminescent detection of proteins  Nitrocellulose and PVDF blots were labelled with the purified polyclonal antibodies by the following procedure. The membrane was first rinsed in PBS and then incubated in a blocking buffer of 5% skim milk in PBS. After three 10 minute washes with 0.2% skim milk in PBS, the blot was left to incubate for 90 minutes in either the pKIgG or the pOpIgG diluted in 0.2% skim milk in PBS. The blot was then thoroughly washed for 50 minutes with the same buffer, and then incubated for 60 minutes with goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase, diluted 1/2000 in 0.2% skim milk in PBS. Finally, the blot was washed for 5 minutes in 0.2% skim milk PBS and then for an additional 30 minutes in PBS. All incubations and washes were performed at room temperature with gentle rocking. Labelling was detected using Amersham's enhanced chemiluminescence (ECL) reagents following manufacturer's instructions  20  (Amersham International pic, U.K.). A light safe casssette and Hyperfilm X-ray film (Amersham) were used for the detection of blue light emission resulting from the oxidation of luminol by horseradish peroxidase in the presence of peroxide. Controls included incubation with pre-immune serum IgGs or buffer in the primary incubation.  2.8 Light microscopy and immunogold silver enhanced staining (IGSS)  O. piceae cells were fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 (Na-cacodylate) for 2 hours and then washed twice for 5 minutes in Na-cacodylate.  Cells were then permeabilized with 0.5% Triton X-100 in Na-  cacodylate for 20 minutes, washed twice with Na-cacodylate buffer for 5 minutes each and after two more washes in PBS, the sample was incubated in 0.05 M glycine in PBS to quench free aldehyde groups. Samples were incubated for 30 minutes in blocking buffer (0.8% bovine serum albumin (BSA), 0.1% gelatin and 5% normal goat serum (NGS) with 2 mM sodium azide in PBS, pH 7.4} and then washed for 10 minutes in PBS. Cells were pelleted and resuspended in the same buffer then divided into small aliquots which were then pelleted and resuspended in incubation buffer {0.8% BSA, 0.1% gelatin and 1% NGS with 2 mM sodium azide in PBS, pH 7.4} containing the pKIgG at various dilutions. After a one hour incubation, samples were washed three times 10 minutes in washing buffer (0.8% BSA, 0.1% gelatin, with 2 mM sodium azide in PBS, pH 7.4} and then left for one hour in secondary antibody (biotinylated donkey anti-rabbit IgG; Amersham) diluted 1/200 in incubation buffer. After washing in washing buffer, samples were incubated for 45 minutes with streptavidin linked 1 nm gold (Janssen Auroprobe One; Amersham) diluted 1/50 in incubation buffer. Following extensive washing in washing buffer and then PBS, samples were post-fixed for 10 minutes in 2% glutaraldehyde in PBS. Once rinsed in Nanopure water, gold particles were silver enhanced for approximately 15 minutes using the Janssen IntensEM kit (Amersham). Cell suspensions were mounted onto glass slides with 50% glycerol and viewed by dark-field illumination with a Zeiss Universal light microscope. All incubations were carried out at room temperature with gentle rocking.  21  2.9 Tissue preparation for transmission electron microscopy and immunolabelling  O. piceae samples from liquid cultures (Section 2.1) were processed for transmission electron microscopy (TEM) at room temperature as follows: 500 uL aliquots of culture or cell wall fragments were pelleted, the supernatants removed and the cells resuspended in fixative consisting of either a) 2.0% glutaraldehyde or, b) 2.5% glutaraldehyde and 2.0% paraformaldehyde in Na-cacodylate buffer. Cells were fixed at room temperature for two hours with gentle rocking, washed in Na-cacodylate buffer (for 10 minutes twice) and then incubated in 0.05 M glycine for 30 minutes (to quench aldehyde groups). After washing twice with Nacacodylate, some samples were stored overnight at 4°C. Others were post-fixed with 1% OSO4 in Na-cacodylate for 1 hour and then washed 2 times for 15 minutes each, in the same buffer, prior to overnight storage at 4°C. The following day, cells were resuspended in PBS to form a dense, milky suspension and samples were drawn into hematocrit tubes by capillary action. Cells were pelleted for 90 minutes at high speed in an IEC benchtop centrifuge fitted with a hematocrit head. Pellets (approximately 1mm X 3mm) were then removed and dehydrated in a graded ethanol series for either LR White, acrylic resin (Sigma) embedment or, Epon resin embedment. For LR White embedment (method A), samples were dehydrated as follows: 1 X 20%, 1 X 40%, 1 X 50%, 2 X 70% and finally, 2 X 90% ethanol at 10 minute intervals. These pellets were gradually infiltrated with LR White: 90% ethanol over a period of 3 days (1:3, 1:2, 1:1, 2:1, 3:1 ). Finally, after 2 changes (10-15 hours each) of 100% LR White, samples were embedded in gelatin capsules that were subsequently filled with resin. Blocks were polymerized at 50°C for 20-24 hours. For Epon embedment (method B), samples were dehydrated as follows: 1 X 20%, 1 X 40%, 1 X 50%, 1 X 70%, 2 X 80%, 2 X 90% and finally, 3 X 100% ethanol at 10 minute intervals. This was followed by 2 changes of propylene oxide (PO) for 15 minutes each. Pellets were gradually infiltrated over 2 days with Epon resin (Luft,1961; 51.12 g eponate 12,16.76 g DDSA (dodecenylsuccinic anhydride), 32.12 g NMA (nadic methyl anhydride), and 2.0 mL of DMP-30 (2,4,6-rri(dimethylaminomethyl)phenol) for every 100 g resin) which was initially mixed with  22  PO (2:1,1:1,1:2 of PO:Epon) and then finally as 100% Epon overnight. Samples were embedded in Epon filled BEEM capsules at 60°C for a minimum of 23 hours. Resin components were purchased from Pelco, Ca., U.S.A. Cell fragments (section 2.3.2) were similarly processed but were fixed and embedded as follows:  a) 2.0% glutaraldehyde in LR White, and b) 2.5% glutaraldehyde plus 2.0%  paraformaldehyde with 1% OsC>4 post-fixation and embedment in Epon.  2.10 Post-embedment immunogold labelling Silver/gold (60-70 nm) sections were cut with a diamond knife on a Leitz Reichert Ultracut microtome and were collected on 2% collodian coated 300 mesh nickel grids. Grids carrying sections of liquid cultured O. piceae were rinsed with PBS for approximately 5 minutes and then blocked with 1% BSA in PBS (blocking buffer) for 30 minutes. The grids were then washed with PBS for 6 to 8 minutes and then probed for 1 hour with either the pKIgG or the pOpIgG diluted in blocking buffer. After washing with PBS, the sections were labelled for 45 minutes with 10 nm gold conjugated to goat anti-rabbit IgG (Sigma) diluted 1/100 in blocking buffer. Grids were rinsed with PBS and then with Nanopure water. The sections were stained in the dark with 2% uranyl acetate for 10 minutes, rinsed in Nanopure water and finally, air dried. Sections were viewed and photographed with a Zeiss EM-10C transmission electron microscope at 60 or 80 KV. Controls included primary incubation with normal rabbit IgG (Caltag Laboratories, So. San francisco, Calif., U.S.A.) or, incubation buffer only. An additional control for the pOpIgG was rabbit pre-immune serum IgGs similarly diluted. All solutions and buffers were filtered with Nalgene filters (0.2 urn) prior to use. Washes and incubations were carried out by flotation of grids on 20 uL drops.  23  3.0 RESULTS  3.1 Growth and proteolytic activity in liquid cultures of O. piceae  Proteolytic activity in cultures was assessed using the azocoll substrate and a peptide substrate. Azocoll is a non-specific protease substrate that is widely used for proteolytic activity determination (Chavira et al. 1984). Azocoll is an insoluble, powdered collagen (cowhide) to which a bright red azo dye is attached. Proteolytic cleavage at any of the peptide linkages results in the release of peptide-bound dye into the suspending medium. The amount of dye liberated over a certain amount of time was measured spectrophotometrically and was proportional to the amount of active protease in the solution. Figure 1 illustrates the growth and extracellular proteolytic activity of O. piceae in soya milk and inorganic nitrogen media. Both the growth and the extracellular proteolytic activity in the protein supplemented cultures were greater than those observed in the inorganic nitrogen supplemented cultures. When provided with protein, O. piceae grew exponentially/actively for approximately 3 days reaching a maximum biomass of 12 mg/mL (Fig. la). Concurrent with growth, proteolytic activity gradually increased in the culture supernatant of soya milk supplemented cultures during the active growth phase and demonstrated little variability during stationary growth phase. In medium supplemented with inorganic nitrogen, the fungus grew actively for about 2 days and reached a comparatively lower maximum biomass of 5.3 mg/ml (Fig. lb). Proteolytic activity in the culture filtrate of inorganic nitrogen cultures was very low reaching a maximum of only 0.002 Units per mg of dry weight early in the exponential phase of growth. Activity then dropped approximately 4 fold to remain unchanged during stationary phase. This extracellular activity seemed negligible, being 100 to 500 times lower than proteolytic activity detected in the protein supplemented cultures.  24  a. 1.4  H 1.2 1  -° !!•§• w ro  o 3^  0.8  ft)  -j 0.6 ^ ft qo.4 0.2  £ v <  0.0 2  4  6  8  10  Days after inoculation  b. 14  0.014  12  0.012  10  -] 0.010  8 .2 b 60  a  3* 2. 3 «•  ST  \  O  A 0.008  3^  I-  0.006  ^a  4U  0.004  2  0.002  6  r-  * El-  0.000 2  4  6  8  10  Days after inoculation  Figure 1: Biomass and proteolytic activity of O. piceae grown in organic (a) and inorganic nitrogen (b) supplemented minimal medium. Activity is expressed as the amount of enzyme required to give an absorbance increase of 0.1 optical density (OD) units (A525) per mL of supernatant per mg dry weight of fungus. In an attempt to improve the chance of detecting activity in the inorganic nitrogen culture samples, assays were conducted over several hours rather than minutes, as was done with the soya milk culture samples. Note the 100 times difference in the expression of activity between cultures. Although the protein concentration of the culture filtrate was different for the two growth conditions, the total amount of protein per mg of fungal biomass was almost equivalent. A larger volume of supernatant from inorganic nitrogen culture was used in the azocoll assay to compensate for this difference. Therefore, the volumes of supernatant assayed were comparable per mg of fungal biomass. Error bars indicate standard deviation (n=3). 25  To determine if any proteolytic activity was associated with 0. piceae cells, the pellets from O. piceae cultures were also assayed with azocoll. Figure 2 and Table 1 show the total proteolytic activity detected in unwashed cells, the wash of the cells, washed whole cells and the whole cell homogenate from cultures provided with soya milk or ammonium nitrate. In addition to the large amount of activity detected in the supernatant of the soya milk culture, a significant amount of activity was detected in unwashed whole cells and the wash of the whole cells. Less activity was detected in the washed whole and homogenized cells, approximately 32 times less activity than in unwashed stationary growth phase cells. Conversely, little activity was detected in all inorganic nitrogen culture samples. In comparison to the soya milk culture derived cells, there was 35 times less total activity in the unwashed inorganic nitrogen cells during stationary phase. Unlike in soya milk cultures, total activity in the inorganic nitrogen cultures was more or less equally distributed between cells and culture filtrate. The total activity in the washed soya milk and ammonium nitrate pellets were similar, being only 1 to 2 times different, which again indicated that most of the proteolytic activity detected in the soya milk culture cells was found extracellularly. Unfortunately, little quantitative information on proteolytic activity could be drawn from the intracellular and cell wall fractions of homgenized cells. There was greater than 50% loss in volume of homogenate as a result of the homogenization procedure. Since the recovery was so low, it was not possible to calculate the total amount of activity in the given fraction relative to the whole culture. It is for this reason that these results have not been presented.  26  1000 ,  Proteolytic activity in organic nitrogen cultures ofO. piceae •  Growth phase  Proteolytic activity in inorganic nitrogen cultures ofO. piceae 100  >  SH H D •  80  .— .< -*-»  u n3  supernatant unwashed pellet wash pellet  60  •U — •« 4—>  g Si o u  | H  40 20  E  S Growth phase  Figure 2: Total proteolytic activity in fractions from cultures of O. piceae that were provided with organic nitrogen or inorganic nitrogen. Samples were taken during exponential (E) and stationary (S) growth phases. Activity was determined for whole cultures of approximately equal biomass. Note that activity in the inorganic nitrogen culture samples is expressed on a scale ten times lower than the organic nitrogen culture samples. Also, activity in the "unwashed pellet" fraction from E growth phase cultures was not determined.  27  ai  co  ON ~  oo  t>.  '-i V  tN  C  1/1  rN  ro  ro  rH  r-H I — i  *  ••c g g 3 6 5  0) <n re  m in re  01  ro  LQ NO vq $ NO O CN « c  01  6 o  o -g s ^ -p vn ^- rr\ c o *-<  re  ^ 2 C3  pq  a» w  re _co  o re c o  tN VO . o q "p  to  O  Ttf  Xi  C  re in II CD  a;  cn X 33 in  O 0 N re <u  D-  O 60 re X! c 01 c  xs rS LO i - i  co  VO  0\ O O  fX-r  CO 0 0  ts O O O O  O O  TI  £  ?s s . 8  '%  01 II  Ml  01  C  pq  1^  •n  M  S m^"a.)£> TJ X >  •X!  CO  (NI  o "p  v-  co  N  N  If) 0 0 IT)  u 01_ c — 0) S u° | re ~* TJ g o oi 0) > aj 60 6J0 H 2 &" l-l  co  N O "tf O  CO CN  H  NOr o r oq "3"  r-H r-H  O  c  o & 60  °^ vO i<  0) T3  c  ^ ON ON "2 o o  pq  ON TJ LO NO ^2 O " O o  C TJ = ° g o^ X > L OO 01 <» 0) re ^ .P « c TJ S m01 O£ C 3 oi "re ll  a.  6  TS oi TJ '>  to  O  ON  ro  ,—i  CO o o  o n  01  O  O 3 •a '3 cr  3  CJ  o u  re H  N ^ NO ON r-H  _K  ^ O  Si o o re H  in <n m -< « — c nj a s w 2 1« TJ g g TJoi fi J o T I . S | < co &1§" g f X 5 in cn SJ S ^ re re §7 o S  u  w  3  & 0 01 o >.  EX  re H  CJ  -a  L^N  —  NO  LO  £ ' TJ O O J fv N a, . •-j Cu 0) 01 60 O in> •. o Ol01 " X, > 0) 0 C uy ^ >i 01 L' 0 -i: ai S  >  CJ 10  HH  ^2 c '  Xi (J re  re  fi  Si ^  pq  to *  >  X!  OJ  N  X!  CJ  .=>  LO NO LO  co  >-  CJ  re  "2 '> 0 &J in 01  s  >  trj  0)  Ol II  w  I—I  ^  >P2 & o  c Ol c  LO  .S2 r> Ol  "re X!  a,s X  TJ  6  LO  re  OJ in  0)  •4-J  in  cj  CX  X. (8 l-  j_,  OJ  OJ en re  re  CJ  6 o  in S  in  cn  33 0)  c oi a s VJ 3 u iii i t .  M  S a "S 7 .a a «  in  ^  in  MJ  o 28  cn 01  ^  S2  73  OJ  .s 01 H-* 01  TJ ±j O c II TJ G  The results obtained with the serine protease specific peptide substrate, succinyl-(Ala)2Pro-Phe-p-nitroanilide, provided information similar to the azocoll assay results (Table 2). This small substrate should allow for the detection of proteolytic activity in cells which might not have been detected by azocoll, which is an insoluble, relatively large substrate. Again, the soya milk culture derived samples demonstrated some proteolytic activity in the whole cells and cell wash and a relatively large amount of proteolytic activity was detected in the culture supernatant. The cells from inorganic nitrogen cultures showed a negligible amount of activity as indicated by a slight colour change over a relatively long incubation period of 5 days. The negative control, where no sample was added to the substrate, did not develop any colour even after 7 days of incubation at room temperature. In contrast, a positive control, the ammonium sulphate precipitate of supernatant proteins which contained the proteinase, changed colour within seconds following the addition of sample.  Table 2: Proteolytic activity, in liquid cultures of O. piceae , determined with the peptide substrate, succinyl-(Ala)2-Pro-Phe-p-nitroanilide. Samples taken from inorganic nitrogen or soya milk supplemented cultures of O. piceae at exponential growth phase Sample supernatant wash of intact cells pellet cell walls  Inorganic nitrogen* nd nd +  - control (substrate+buffer) + control (ammonium sulphate precipitate of nitrate proteins)  Organic nitrogen* +++ ++ ++  +++  * Symbols indicate intensity of colour after >24 hours incubation at room temperature - = no colour development; + = very pale yellow; ++ - medium yellow; +++ = dark yellow, nd=not determined  29  3.2 Production of the purified polyclonal antibodies  To meet the main objective of this work - to localize the extracellular proteinase of O. piceae by immunodetection methods - antibodies that specifically recognize the proteinase were needed. The initial difficulties encountered with purifying this enzyme led us to raise antibodies against proteinase K, an enzyme that is similar to the O. piceae proteinase (Abraham and Breuil 1995b). When the 33 kDa proteinase of O. piceae was purified, antibodies were generated against it. Both sets of polyclonal antibodies were purified and used to detect this proteinase in O. piceae cells. Before being used for this purpose, the production of antibodies against the corresponding proteinase was verified by ELISA and the specificity of the resulting antibodies was assessed by immunoblotting (Section 3.3). ELISAs were performed on test bleeds taken at various times after the initial immunizations. Over time, they showed an increasing reactivity of the sera to the corresponding antigen which indicated that the rabbits responded to the antigen as expected. The sera were recovered and purified to give a preparation of IgG class antibodies only. The amount of protein contained in the IgG fraction of the anti-proteinase K serum was 1.4 ug/uL. With 1 ug of proteinase K as coating antigen, the endpoint titration of the pKIgG was 1/250 000 and an optical density (OD) of 1.2 was obtained when the antibodies were used at a dilution of 1/5000. In contrast, at this antibody dilution, the reaction with the purified proteinase of O. piceae was much lower giving an OD of less than 0.1. Upon testing the pOpIgG with 1.0 ug of the HIC purified proteinase of O. piceae , the endpoint titration was 1/8000 and an OD of 1.3 was obtained with the antibodies diluted 1/400. The IgG fraction purified from the rabbit pre-immune serum, corresponding to the pOpIgG, was similarly tested and gave an OD equal to or less than 0.13 when used at the same IgG concentration. This indicated that the pOpIgG contained antibodies that had been generated against the O. piceae proteinase. In 1 uL of pre-immune serum IgG fraction, there were 6.8 ug of protein, while in the same volume of pOpIgG, there were 12.5 ug of protein.  30  3.3 Characterization of the purified polyclonal antibodies  The specificity of the purified anti-proteinase K and anti-O. piceae proteinase polyclonal antibodies was assessed by immunblotting methods. As described in section 2.7.1, proteinase K, ammonium sulphate precipitate of O. piceae supernatant proteins (semi-purified proteinase) and preparative HIC purified O. piceae proteinase as well as various other commercial enzymes were used for the antibody characterizations. These proteins were analyzed by gel electrophoresis , and used for dot blot and Western blot analysis.  3.3.2 Electrophoretic analysis of proteins used for antibody characterization  Figures 3 and 4 are silver stained gels of the proteinases separated by SDS-PAGE and IEF, respectively. As shown previously by Abraham and co-workers (1993), SDS-PAGE (Fig. 3) of the ammonium sulphate precipitate of supernatant proteins (lane 2) showed a major band corresponding to a protein of approximate molecular weight 33 kDa. When further purified by preparative HIC, a single band of the same molecular weight was observed (lane 3). Proteinase K (lane 4 and Fig. 5 lane 3) appeared as a very faint band at 31 kDa, slightly higher than 28 kDa, its reported molecular weight (Jany and Mayer 1985), and a wide band of low molecular weight. The low molecular weight staining indicated that the proteinase had degraded at some point during the sample preparation. In later experiments proteinase K was prepared with PMSF to prevent this autolysis (Bajorath et al. 1988) and SDS-PAGE analysis showed no degradation products (Fig. 3, lane 5) When separated by IEF (Fig. 4), the ammonium sulphate precipitate of supernatant proteins (lane 4) showed various bands with a major band that focussed at approximately pi 5.9. This corresponded to the proteinase purified by preparative HIC seen as the major band in lane 3. The pi of the proteinase has actually been more precisely determined to be 5.6 (Abraham and Breuil 1995b). The other band in lane 3 was believed to be a product of proteinase degradation. Proteinase K, with a pi of 8.9, migrated as a single, intense band (lane 2).  31  The other fungal and bacterial enzymes used for the antibody characterization were also analyzed by SDS-PAGE and silver staining (Fig. 5). When loaded at high concentration, various bands and smears appeared for all the enzymes except for a-amylase from Boehringer and Mannheim which appeared as a bold, single band (lane 7). Again, proteinase K appeared as a very faint band around 31 kDa with degradation products (lane 3). The preparative HIC purified proteinase from O. piceae (lane 2) also showed many bands. These lower molecular bands resulted from proteinase degradation during storage.  32  1 i  2 i  3 i  4 i  5 i  kDa 97.4 66.2 45.0 31.0 21.5 14.4  Figure 3: Silver stained SDS-PAGE Phast gel (8-25% gradient) showing O. piceae proteinase and proteinase K . The proteinase from O. piceae purified from soya milk culture supernatant by ammonium sulphate precipitation (semi-purified proteinase) is in lane 2 and the preparative HIC purified proteinase is in lane 3. Proteinase K is in lane 4 and appears degraded. However, in lane 5, PMSF inactivated proteinase K appears mainly as a single band of approximately 31 kDa. Lane 1, low molecular weight standard (Bio-Rad). Proteins were loaded at approximately 300 ng per lane. 33  1  2  3 4 JL  i.  pi  3.5 5.2  5.85 6.55 7.35  8.158.45 8.65 9.3  Figure 4: Silver stained IEF gel (pi 3-9) of O. piceae proteinase purified from soya milk culture supernatant by ammonium sulphate precipitation (lane 4) and further purified by preparative HIC (lane 3). The proteinase, focuses at pi 5.9. Proteinase K is in lane 2 and focuses at about 8.9. Pharmacia IEF standards are shown in lane I. Proteins were loaded at approximately 500 ng per lane.  34  1  2  3  4  5  6  7  8  kDa  Figure 5: Silver stained SDS-PAGE Phast gel (8-25%) of commercial preparations of various proteins that were used for screening the purified polyclonal antibodies for cross-reactivity. In lane 2, a sample of preparative HIC purified proteinase from O. piceae is shown after storage at -20°C without PMSF. The many lower molecular weight bands indicate proteinase degradation. Lane 3 shows proteinase K which appears as a faint band along with its low molecular weight degradation products. Protease type X (thermolysin) is in lane 4, followed by lysing enzymes in lane 5, cellulase preparation in lane 6, amylase in lane 7 and chitinase in lane 8. A low molecular weight standard (Bio-Rad) is shown in lane 1. Proteins were loaded at approximately 3 ug/lane.  35  3.3.2 Characterization  of the anti-proteinase K IgG  Figure 6A is a dot blot illustrating the reactivity of the pKIgG with the proteins described in section 3.3.1. When used at a dilution of 1/500, the pKIgG could detect as little as 0.15 ug of proteinase K. At the same pKIgG dilution, at least 0.38 ug of protein from the O. piceae semi-purified proteinase preparation was required to detect a signal and 0.75 ug of this protein was required to obtain a signal of similar intensity as 0.15 jig of proteinase K. The HIC purified proteinase blots gave a weaker signal than those from the ammonium sulphate precipitate. Cross-reactivity with cellulase was also noted but there was no reaction with other enzymes blotted. The secondary antibody did not cross-react with any of the proteins screened (Fig, 6B). In contrast, Western blots of proteinase K and of the O. piceae extracellular proteinase preparations, separated by SDS-PAGE, native-PAGE or IEF in Phast gels, gave no signal when probed with the pKIgG (Fig. 7). Even after long exposures, chemiluminescent bands did not appear on the blots. Varying the pKIgG concentration did not improve the results. Instead, the background signal increased when the antibody concentration was too high. It was not believed that the lack of reactivity was because the transfers were unsuccessful since Ponceau red staining of the blots prior to immunodetection showed protein bands (not shown). In a subsequent run using the Bio-Rad Mini-Protean II, the ammonium sulphate precipitate and HIC preparation of O. piceae proteinase were separated by native-PAGE and transferred to PVDF membrane. Upon coomassie blue staining of the gel, the semi-purified preparation of O. piceae proteinase appeared as four protein bands while only one band appeared in the lane containing the preparative HIC proteinase preparation (Fig. 8A). As seen in both lanes, some protein migrated with the dye front. Gel overlays onto gelatin coated X-ray film, indicated that the proteinase was still active following electrophoresis. Proteolytic activity was detected from 3 of the 4 bands in lane 1 and from the single band in lane 2 (Fig. 8B) after 4 minutes incubation at 37°C. When the Western blot of a similar gel was probed with the pKIgG, smears were detected near the stacking gel and separating gel interface (Fig. 8C) but no bands corresponding to the proteinase were detected in either lane. To determine if the transfer was  36  complete, both the gel and blot were stained for protein. Since no protein bands were visible in the gel and bands were detected on the blot by Ponceau red, the transfer had been deemed successful.  37  Figure 6: Immunodot blots of various proteins probed with the pKIgG (A) or buffer (B) in the primary incubation. Protein amounts are indicated in the left margin except for proteinase K, of which 10 times less protein was dotted. Lane 1, preparative HIC purified proteinase; lane 2, semipurified proteinase (from soya milk culture supernatant); lane 3, proteinase K; lane 4, thermolysin; lane 5, chitinase; lane 6, amylase; lane 7, cellulase and lane 8, lysing enzymes. 38  Figure 7: Western blots of proteins shown in Fig. 3 that were probed with the pKIgG. Proteins had been separated by SDS-PAGE (A) native-PAGE (B) and IEF (C). For all transfers, approximately 2.5 ug of protein, per lane, had been loaded onto the gel and the antibody was used at a dilution of 1/300 (A) or 1/500 (B and C). No bands were detected on any of the blots although Ponceau red staining of the membranes indicated that proteins had transfered onto the membranes (not shown). 39  Figure 8: Native-PAGE separation (Bio-Rad Mini-Protean II) of the semi-purified proteinase (lane 1) and the proteinase purified by HIC (lane 2). Half the gel was stained with Coomassie blue (A). The other half was used for an X-ray film overlay, as shown in (B) after 4 minutes incubation at 37°C. Clearings on the X-ray film represent gelatin hydrolysis i.e., proteolytic activity. Western blot of HIC purified proteinase and semi-purified proteinase from O. piceae soya milk cultures transferred from native-PAGE gel and probed with the pKIgG diluted 1/500 (C) or, the pOpIgG diluted 1/3000 (D). Note the smears at the start of the gel, or the top of the blot within which no bands were discernable. Amount of protein loaded per lane per gel was 11 ug (coomassie staining and overlay) and 13 ug (Western blot). 40  3.3.3 Characterization of the Anti-O. piceae proteinase IgG  Figure 9 illustrates the chemiluminescence obtained with dot blots that were probed with either the rabbit pre-immune serum IgG fraction or the pOpIgG. The pre-immune serum IgG fraction (Fig. 9A) gave a signal with the preparations of proteinase K (lane 4), protease type X (thermolysin, lane 5), cellulase (lane 6) and lysing enzymes (lane 9) from Sigma. Reactivity was also noted with an extract from O. piceae cells (lane 1) and slight reactivity with the ammonium sulphate precipitate (lane 2). But, there was no cross-reactivity with the purified extracellular proteinase (lane 3). Figure 9B shows the results obtained with the pOpIgG used at a dilution of 1/18 000 to probe an identical blot. The results were somewhat different to those obtained with the preimmune serum IgGs. Very faint reactivity was observed with thermolysin (lane 5) and cellulase (lane 6) blotted at 3 ug of protein. Reactivity with proteinase K (lane 4) and lysing enzymes (lane 9) was not evident. However, when the pOpIgG was used at higher concentrations, e.g. 1/7000, cellulase and proteinase K gave stronger signals (not shown). When these proteins were Western blotted and probed with the pOpIgG, chemiluminescent signals were only detected from proteins derived from O. piceae culture filtrate (Fig. 10A, lanes 1 and 2). For films exposed for a longer time period, a very faint signal was detected from proteinase K but not from the other commercial enzymes, unlike the dot blot result (not shown). There was no signal when the rabbit pre-immune serum IgGs were used on a Western blot of O. piceae proteinase and proteinase K (Fig. 10B). In addition, on a Western blot of a native-PAGE gel run in the Mini-Protean II, a long smear was observed at the top of the gel (Fig. 8D) but protein bands were not detected.  41  1 2  A  B  i  3.0-  0  1.5-  a  0.75-  o  0.40-  o  3.01.5-  •i  3 i>  4 1i  5  6  7  8  9 10  i  i  i  i  i  i  ••  ••  0.750.40-  Figure 9: Immunodot blot of various proteins probed with the purified rabbit pre-immune serum IgG fraction (A) or the pOpIgG (B). Primary antibody was diluted 1/9500 and 1/18 000 respectively. Protein amounts are indicated in the left margin with the exception of the O. piceae derived samples (lanes 1-3) for which ten times less protein was dotted due to the sensitivity of the antibody for the proteinase. Lane 1, extract of the cell homogenate from inorganic nitrogen culture taken at stationary phase; lane 2, semi-purified proteinase; lane 3, preparative HIC purified proteinase; lane 4, proteinase K; lane 5, thermolysin; lane 6, cellulase; lane 7, chitinase; lane 8, amylase; lane 9, lysing enzymes. The chemiluminescent signals can be compared with the blank, lane 10, where no sample was added. 42  -I  n 1  2  I  I  3  4  5  6  12  3  4  7  Figure 10: Western blot of various proteins probed with the pOpIgG (A) and the pre-immune serum IgG fraction (B). (A) Proteins were separated by SDS-PAGE (12%) and transfered onto PVDF in a Bio-Rad Transblot apparatus. Lanes 1 and 2 show the proteinase from O. piceae purified by preparative HIC and ammonium sulphate precipitation, respectively. Lane 3, proteinase K; lane 4, thermolysin; lane 5, cellulase; lane 6 amylase and lane 7, lysing enzymes. Proteins were loaded onto the gel at 6 ug per lane. Antibody was diluted 1 /15 000. (B) Rabbit pre-immune serum IgG fraction was used to probe a Western blot of O. piceae proteinase preparations and proteinase K that were separated by SDS-PAGE in the Phast-system. The lanes contained low molecular weight standard in lane 1, proteinase K in lane 2, preparative HIC purified proteinase and semi-purified from O. piceae in lanes 3 and 4, respectively. The serum was diluted 1/1000. 43  3.4 Localization of the proteinase of O. piceae using the pKIgG and the pOpIgG  The 33 kDa proteinase of O. piceae was detected and localized in the cellular fractions of liquid cultures primarily by immunodetection methods using the purified antibodies. Cells were harvested during exponential growth phase, while the fungus was actively secreting proteinase in the soya milk cultures, and at stationary growth phase, when extracellular proteolytic activity was at its highest. Cells were homogenized and protein was extracted from various fractions that were recovered (Section 3.4.1). Dot blots and Western blots of these extracts and other fractions derived from the cell disruption were probed with both purified antibodies (Section 3.4.2). Finally, the microscopic detection of the proteinase, in cells and cell wall fragments of O. piceae , was achieved by immunogold labelling with transmission electron microscopy as shown in Section 3.4.3.  3.4.1 Cell disruption and protein extraction  Prior to disruption (Fig. 11 A), cells appeared to be normal and exhibited cytoplasmic streaming. After disruption (Fig. 11B), many cells were empty and/or had a break in their wall, hyphae were fragmented and a large amount of cellular debris was observed in the background. Approximately 30-45 minutes of total disruption time was required to sufficiently break open the cells of O. piceae. It was found that brief disruption intervals of 10-20 seconds with cooling on ice between intervals kept samples cool, an important consideration for the prevention of protein denaturation. To analyze the whole cell and cell wall pellet fractions obtained from the cell disruption, proteins were extracted and solubilized by boiling with SDS.  Protein determinations of these  extracts and various other cellular fractions were performed so that samples could be compared and approximately equal amounts of protein could be loaded onto gels and blotted onto membranes. The extraction method employed was sufficient to remove up to 6 ug of protein per uL of cell wall fragments extract. The amounts of protein extracted from soya milk and inorganic nitrogen cultures were similar. More protein was extracted from the exponential phase cells than in cells taken  44  from stationary growth phase. Protein concentrations were also determined for the extracellular fractions where a relatively small amount of protein was measured in both the inorganic nitrogen and soya milk culture supernatants. As shown in Figure 12, fractions obtained from the cell disruption experiment contained numerous proteins. A fine band corresponding to a protein of approximately 33 kDa appeared in the pellet fractions from both culture conditions. Proteinase was detected by SDS-PAGE in extracellular samples from exponential phase and stationary phase cultures supplemented with soya milk (Fig. 13A). Samples of culture supernatants (lanes 1 and 4) and pellet washes (lanes 2 and 5) from two sampling times, each showed a single protein band of the same molecular weight as that of the HIC purified proteinase (lane 3). This suggested that the proteolytic activity detected in the supernatant and the pellet wash (Table 1) was due to the 33 kDa proteinase. Supernatant and cell wash from inorganic nitrogen culture showed no protein bands by SDS-PAGE (Fig. 13B). This was unexpected since the protein determination indicated that protein was present in the ammonium nitrate culture supernatant. It is posible that the BCA reagents reacted with peptides and amino acids, as well as protein, which would have affected the protein determination result.  45  Figure 11: Phase contrast photomicrographs of stationary phase O. piceae cells from soya milk culture before (A) and after (B) homogenization. Scale bars = 2.5 urn 46  o  1 2 3 4 5 6 7 8 i  I I  i  i  i  i  i  1 2 3 4 5 6 7 8 i  i  i  i  i  i  i  i  Figure 12: SDS-PAGE (8-25%) and silver staining of extracts and intracellular fractions from stationary phase cultures provided with organic nitrogen (O) or inorganic nitrogen (I). Electrophoresis and staining were performed in the Phast system. In all samples, various bands were observed. In some, there appeared to be a protein of the same molecular weight as the extracellular proteinase. Proteins were loaded as 500 ng per lane. Lane 1, low molecular weight standard from Pharmacia; lane 2, O. piceae proteinase purified by HIC; lane 3, extract of unwashed pellet; lane4, extract of washed pellet; lane 5, extract of cell homogenate; lane 6, intracellular filtrate; lane 7, wash of the cell fragments and lane 8, extract of the cell fragments. 47  A  V  Figure 13: Silver stained SDS-PAGE (8-25%) gels of extracellular protein from soya milk culture (A) and inorganic nitrogen culture (B). Samples were taken during exponential growth phase (E) and stationary phase (S). Lanes 1 and 4 culture supernatant; lanes 2 and 5, pellet washes; lane 6 low molecular weight standard (Bio-Rad) and lane 3, HIC purified proteinase of O. piceae. 48  3.4.2 Immunoblot detection of O. piceae proteinase using the purified antibodies  Figures 14 and 15 are dot blots of O. piceae fractions, from soya milk and inorganic nitrogen stationary phase cultures, respectively. Samples were dot blotted onto nitrocellulose and the blots were probed with either the pKIgG (Fig. 14A, 15A) or the pOpIgG (Fig. 14B, 15B) to determine in which fractions the enzyme might be located. Dot blotting also indicated the sensitivity of the antibodies when used at a specific dilution i.e., the minimum amount of blotted protein required to obtain a chemiluminescent signal with a low background signal. When probed with the pKIgG, samples from the two culture conditions gave mixed results. For both the soya milk (Fig. 14A) and the ammonium nitrate (Fig. 15A) supplemented cultures, signals were mainly detected from the various pellet extracts: washed whole cells (C), homogenized cells (H) and cell wall fragments (Cf). Similar reactivity was also detected with the washes of the cell fragments. With the soya milk culture, there was slight reactivity with the culture supernatant (Sn) and even stronger reactivity with the wash of the whole cells (WC). The intracellular fraction (F) gave a weak signal. With the same fractions from the inorganic nitrogen culture, no chemiluminescence was detected. When the pOpIgG were used on identical dot blots, relatively strong signals were observed for the various pellet extracts of both cultures (Fig. 14B and 15B). Other fractions that gave a strong chemiluminescent signal included the wash of the whole cells, the filtrate and the wash of the cell wall fragments from both cultures. Between the cultures, the only significant difference in reactivity was in the culture supernatant. Very low signal was detected with the supernatant from the soya milk culture while there was a stronger reaction with that from the inorganic nitrogen cultures. Only 0.25 ug protein from inorganic nitrogen culture supernatant was required to achieve a chemiluminescence intensity similar to that obtained with 2 ug protein from soya milk culture supernatant. When the pre-immune serum IgGs were used to probe similar blots, reactions were observed with all the fractions (see Figure 9 lane 1 for an example). When antibody was excluded from the primary incubation, no reactions were observed indicating that the secondary antibody did not cross-react with the samples (not shown).  49  i l l  if a  S  c i "53 u S , II  u  _ J2  O ~ fx <u oi CJ o •3 "§ TJ u  i  u  •<  S J-  «  m  0J  O- « 60 O O  6  Sll 1<!1 °  «  TJ  5 3 £ an  SiI t-j-l i  re  « 3 >  III II •  £  X  *<8.  I  CO  n  HJ  o reo a OH  2-S XI  -3  3 Eb fl3 ro X! fx,  0J  3 oi in <u  -  ^  re in  J 3  1  3 S0J •—3• c re oi °  § TJ  cn  £  P  I  o  i  I  L  O  g *> S o  N  I  O T-  1  O  "1  I LO  5  c <u 2 2 oo §2* X! co Jl | 3* o 6« O 3 i> o cx £ £ Q ?  T>  3 « £ 2 PH  50  T-I  O  01  so  •a P. 3  JJ  dj S3  9^2 u in  '1*8 01  N  '5 Ol  6J0  O "8 (fl o£  3 U  £ 8" if •§ 8 .2 2 js »s tfl . 2 3 a  o> g  •8 "8 a> g  C  0  c  OJ  •is* CL,  C -*3  Ol  OJ  OCX  tj  «  .-a a .s2«* 5 * QJ S .a U  u  o .g  .S u  T3  OJ N  2 M " o -a QJ * £ o £ SI o 4= •g * II g  DC  ? o  6  )•  o  w  Ifl  o  O  i  q  N  CM  CO  o  o  T-'  «?  o  m W  .  o  o o 3  I o A  OI  o oo  •s -  8 8 X  o oi 51  SDS-PAGE showed that the pellet extracts and filtrates from both cultures contained various protein bands (Fig. 12). These electrophoresed proteins were Western blotted onto PVDF membranes. Figure 16 shows that when the pOpIgG were used to probe these blots, chemiluminescence signals originated from a smear and a few bands, in all lanes at the higher molecular weight range of the gel (between 80 and 200 kDa). The soya milk filtrate also gave a slight chemiluminescent signal from a band (lane 4) at a position corresponding to a protein of molecular weight similar to that of the HIC purified proteinase from O. piceae (lane 5). Only slight chemiluminescent signals were detected in the high molecular weight range for all samples when the pre-immune serum IgG preparation was used to probe similar blots (not shown). Coomassie blue staining of gels after blotting indicated that the transfer was not complete since many bands were still visible in some lanes of the gel (not shown). This was observed for both BioRad transferred and semi-dry transferred gels. Although, Ponceau red staining of the membranes indicated that proteins were transferred, the transfer efficiency may have been improved with a longer transfer time.  52  Figure 16: Western blot of extract from cell wall fragments and intracellular filtrates from stationary phase cultures of O. piceae grown in medium containing organic nitrogen (O) or inorganic nitrogen (I). Samples were loaded at 25 ug protein per lane, separated by SDS-PAGE in the Bio-Rad Mini-Protean II and then transferred to PVDF membrane in the Trans-Blot apparatus. Blots were probed with the pOpIgG diluted 1/18 000 which resulted in the detection of high molecular weight bands and smears in all samples. Shown are the intracellular filtrate (lane 1) and the extract of the cell fragments pellet (lane 2) from inorganic nitrogen and soya milk cultures. After a long exposure of the X-ray film, a faint band was seen in the soya milk intracellular filtrate (lane 4). This band might have corresponded to the O. piceae proteinase since it had migrated as the HIC purified proteinase from O. piceae that had been stored with PMSF had (lanes 3 and 5). 53  3.4.3 Immunogold Labelling - Microscopic detection of O. piceae proteinase using the purified antibodies.  Before moving to immunogold labelling for TEM, unwashed O. piceae cells from soya milk culture were immunogold labelled and silver stained and observed by dark-field microscopy to assess the utility of the pKIgG for immunomicroscopy. As a preliminary assessment of fixation for preparing samples for TEM viewing, cells were fixed in three different ways to determine if sample processing would affect labelling of the cells (not shown). It was found that a combination of paraformaldehyde and glutaraldehyde (2.0% and 2.5% respectively) or glutaraldehyde (2.0%) alone resulted in strong labelling of the hyphae only but not of the yeast or spore cells (Fig. 17). In contrast, when fixed with 4.0% paraformaldehyde, labelling was not observed suggesting that fixation with paraformaldehyde alone was not sufficient for antigen retention (not shown). This might have been because fixation of proteins by paraformaldehyde is reversible (Hayat 1981). Based on these results, various combinations of fixation and embedment for transmission electron microscopy were tried on samples from soya milk supplemented cultures of O. piceae to define a processing method that would preserve cellular ultrastructure and allow for immunolabelling of the proteinase. Table 3 summarizes the results and Plates 1-4 illustrate them. The best ultrastructure preservation of O. piceae appeared to be achieved with methods 1A (Plate 1A), 3B (Plate 3B), and 4B (Plate 4B) since nuclei, cytoplasmic organelles as well as cell membranes were relatively, well preserved.  O. piceae cells processed by the other methods,  appeared to be lysed as indicated by detached cell membranes, "bundled" membrane structures and empty cells. By most treatments, the cell wall was electron translucent. Sometimes an extracellular sheath or mucilage surrounding the cells was preserved appearing either electron lucent (e.g. Plate 2A) or electron dense (e.g. Plate 1A).  54  Figure 17: Dark field photomicrograph of O. piceae cells labelled with the pKIgG for IGSS. Samples were taken from soya milk supplemented culture in exponential growth phase, and were fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde. Note that only the hyphae (arrow) were labelled while no gold appears on the spores or yeast cells (arrow head). Scale bar=5 um  55  TABLE 3: SUMMARY OF RESULTS OF TEM PROCESSING TRIALS  Fix No. 1 2 3 4  Fix Composition* 2.0% G 2.5% G + 2.0% P 2.0%G + 1.0%Os 2.5% G + 2.0% P + 1.0% Os  A. LR White ++ ++  -  +  B. EPON+ + ++ +++ +++  * Where G= glutaraldehyde, P= paraformaldehyde, Os= osmium tetroxide Resulting appearance: -= very poor, += poor to moderate, ++= moderate, +++= good +  56  Key to symbols  cm=cell membrane; cw=cell wall; m=mitochondrion; mvb=multivesicular bodies; N=nucleus; s=septum; sh=extracellular sheath or mucilage; sp=septal pore; V=vacuole.  Plates 1 through 4  Transmission electron micrographs of O. piceae cells from exponential phase of growth in soya milk medium. To test the effect of fixation on sample processing, the fungus was fixed in 4 different ways, methods 1 through 4, and embedded in two different media, LR White (acrylic) resin (A) and Epon (epoxy) resin (B). The best combinations appeared to be those of methods 1A, 3B and 4B which resulted in better preservation of cellular structures including, mitochondria, nuclei and plasma membranes. By most treatments, O. piceae's cell wall appeared to be electron translucent. Plate 1: O. piceae cells fixed with 2.0% glutaraldehyde (method 1) and embedded in LR White (A) or Epon (B) resin. Scale bar=0.25 um. (small arrow=sheath) Plate 2: O. piceae cells fixed with 2.5% glutaraldehyde plus 2.0% paraformaldehyde (method 2) and embedded in LR White (A) or Epon (B) resin. Scale bar=0.5 um. Plate 3: O. piceae cells fixed with 2.0% glutaraldehyde, post-fixed with 1.0% OsO^ (method 3) and embedded in LR White (A) or Epon (B) resin. Scale bar=0.25 um. Plate 4: O. piceae cells fixed with 2.5% glutaraldehyde plus 2.0% paraformaldehyde, post-fixed with 1.0% OsC>4 (method 4) and embedded in LR White (A) or Epon (B) resin. Scale bar=0.5 um.  57  1  60  4  To confirm that labelling with the purified antibodies would be possible following any of these processing methods, samples from all methods were immunolabelled with the pKIgG. The results showed that labelling was possible with all processing conditions, including those involving post-fixation with osmium (data not shown). For further experimentation, methods 1A (without osmium) and 4B (with osmium), were followed because of their superior preservation of the cells and because each represents a very different processing protocol, one that is harsh (4B) and another that is relatively mild (1A).  3.4.3.1 Immunogold labelling of the proteinase in O. piceae cells  For immunogold labelling with the purified polyclonal antibodies, samples from both inorganic nitrogen cultures and soya milk cultures were harvested at 2 different sampling times, exponential (day 2) and stationary (day 8) growth phases, and were processed by methods 1A and 4B. The ultrastructural preservation achieved by method 4B again appeared to be superior to that of method 1A. For this and other reasons described below, a greater emphasis was placed on the analysis of the Epon embedded samples. When the pKIgG were used, labelling was observed primarily in the cell wall of the fungus for both culture conditions at both sampling times (Plates 5-8). In the soya milk culture cells, there was no obvious difference in labelling intensity in cells sampled at the different growth stages (Plates 5 and 6). There was however, a noticeable difference in labelling intensity between exponential phase and stationary phase cells from inorganic nitrogen cultures (Plates 7 and 8). Cells from younger cultures appeared to be more heavily labelled. These observations were only qualitative since the gold particles were not quantified. Similar observations were made when the pOpIgG were used (Plates 9-12). Labelling was in the cell wall, but gold particles were also observed near the cell membrane (Plate 10). The wall labelling was predominantly of the outer portion of the wall and particularly in the darkly staining surface of the cell wall which was considered to be an extracellular sheath. This was  62  particularly noticeable in the samples taken later in growth (Plates 10 and 12) for which labelling was more localized to the outer portion and perimeter of the cell walls. In addition, cells from inorganic nitrogen cultures demonstrated a couple of unique features when labelled with the pKIgG. Firstly, when processed by method 1A, cells were mainly labelled intracellularly across the cytoplasm (Plate 13A). This observation was not made for similar samples labelled with the pOpIgG (Plate 13B). The reason for this differential labelling pattern was unclear, and is why a lesser emphasis was placed on samples processed by method 1A. Secondly, gold particles were found inside multivesicular bodies that were often found near to the cell membrane (Plate 14A). Again, this was not observed for samples labelled with the pOpIgG (Plate 14B). The negative controls did not result in labelling of the cell walls. Incubation with normal rabbit IgG sometimes gave background seen as random labelling in the cells and in the surrounding resin (Plates 15B and 16B). Pre-immune serum IgGs corresponding to the pOpIgG (Plate 9B) and buffer only (Plates 13C, 15A and 16A) incubations, served as true negative controls, showing no labelling or very few randomly distributed gold particles on some occasions.  3.4.3.2 Immunogold labelling of the proteinase in O. piceae cell wall fragments  Cell fragments obtained from the homogenization experiment were also processed for TEM and immunogold labelling. Cells were well broken apart as shown by the randomly sized cell wall fragments among cellular debris and fragmented sheath material. Immunogold labelling of the fragmented cells showed gold particles in the cell wall fragments when either the pKIgG (Plates 17 and 18) or the pOpIgG were used (Plates 19 and 20). As illustrated with Plate 17, washing of the cell fragments did not appear to reduce the labelling intensity. Washing did however, remove some of the debris that surrounded the cell wall pieces. Labelling did not occur when buffer was used in the primary incubation (Plates 19C and 20B).  63  Similar to the labelling observed with the pKIgG on the whole cells grown with inorganic nitrogen, cell wall fragments processed by method 1A (Plate 18B), were not labelled as extensively as those processed by method 4B (Plate 18A). There seemed to be more gold particles in the surrounding debris and the cell walls were less heavily labelled.  64  Plates 5 through 8  Transmission electron micrographs of O. piceae cells processed by method 4B and immunogold labelled with the pKIgG. Cellular ultrastructure was well preserved, particularly in the younger cells in which vacuoles, nuclei, mitochondria and multivesicular bodies were seen. The cells taken from stationary growth phase culture showed relatively little intracellular organization which was likely because cells may have been autolysing in this older culture. Alternatively, this may have resulted from improper fixation. Gold labelling was evident in all samples, including those from inorganic nitrogen culture, and was primarily localized in the cell wall, including septa, of the fungus. 5 and 6: Immunogold labelling of O. piceae grown in minimal medium supplemented with soya milk. Samples were processed for TEM when the cultures were in exponential growth phase (Plate 5) and stationary growth phase (Plate 6). The pKIgG antibody was diluted 1/50 (A) and 1/25 (B). Labelling of the cell wall was uniform and reproducible between samples with little or no background. The distribution of gold particles did not seem to vary between 2 day old and 8 day old cultures. Scale bars=0.5 um (Plate 5) and 0.25 um (Plate 6).  Plates  Immunogold labelling of O. piceae grown in minimal medium supplemented with ammonium nitrate. Samples were processed for TEM when the cultures were in exponential growth phase (Plate 7) and stationary growth phase (Plate 8). The pKIgG was used at a dilution of 1/25. Gold particles were found throughout the cell wall (Plate 7) and in multivesicular bodies (Plate 7A and C). Exponential phase cells were more heavily labelled than stationary phase cells. Note the prominent extracellular sheath surrounding the cell in Plate 7B in which some gold particles can be seen. Scale bar=0.5 um (Plate 7) and 0.25 (Plate 8). (arrowhead=woronin body; small arrow=multivesicular body) Plates 7 and 8:  65  5  6  68  g  Plates 9 through 12 Transmission electron micrographs of O. piceae cells processed by method 4B and immunogold labelled with the pOpIgG. Gold labelling was similar to that observed with the pKIgG i.e.,gold was found primarily in the cell wall in all conditions- with variation where specified. Gold was rarely found over the cytoplasm or nucleus. Plates 9 and 10: Immunogold labelling of O. piceae grown in minimal medium supplemented with soya milk. Samples were processed for TEM when the cultures were in exponential growth phase (Plate 9) and stationary growth phase (Plate 10). Plate 9A shows O. piceae cells from culture in exponential growth phase that were incubated with the pOpIgG diluted 1/100. Gold labelling was found in the cell wall and the septum. Gold particles were also seen in sheath-like material surrounding the cells and in electron, dense areas of the cell wall (arrowheads). O. piceae cells from stationary growth phase cultures were similarly labelled with the pOpIgG, diluted 1/100 (Plate 10A-D). Labelling was predominantly seen in the outer portion and surface of the cell wall as well as in electron dense cell wall coating or sheath (arrowheads). Gold particles can sometimes also be seen near the cell membrane (Plate 10 A-D). When O. piceae cells were treated with the pre-immune serum IgG fraction, very few gold particles were seen in the cells as illustrated in Plate 9B. Scale bars=0.25 urn. Plates 11 and 12: Immunogold labelling of O. piceae grown in minimal medium supplemented with ammonium nitrate. Samples were processed for TEM when the cultures were in exponential growth phase (Plate 11) and stationary growth phase (Plate 12). Again labelling was in the cell wall and in the extracellular sheath surrounding the cells. Labelling was not observed in multivesicular bodies however, as was observed in similar sections labelled with the pKIgG. The sheath appears both electron dense and translucent around some cells (11B and C). Cells from the older culture were not as heavily labelled and as was seen in the soya milk cultures, much of the gold was localized to the outer portion and surface of the cell wall. Few particles were found inside the cells, and some gold particles were seen where the cell membrane would be. Scale bar=0.25 urn. (arrowheads=labelling of sheath and outer cell wall)  70  71  72  11  73  12  Plates 13 through 16 Plate 13: Immunogold labelling of O. piceae cells, grown in minimal medium supplemented with ammonium nitrate, and processed for TEM by method 1A. Sections incubated with the pKIgG, diluted 1/25 (A) were heavily labelled intracellularly as well as in the cell wall. Sections incubated with the pOpIgG, diluted 1/100 (B), were only labelled in the cell wall. In the control condition, where the primary antibody was excluded (C), no gold particles were seen in the cells, indicating that the secondary antibody was not binding non-specifically to these samples. This plate illustrates differences in gold labelling obtained with the pKIgG and the pOpIgG which might have been an effect of fixation since these differences were not observed in cells processed by method 4B. Scale bars=0.25 um Plate 14: Immunogold labelling of method B processed O. piceae cells that were taken from exponential phase culture supplemented with ammonium nitrate. These figures illustrate that with the pKIgG (A), labelling was not only seen in the cell wall but also in multivesicular bodies (arrows) and this intracellular labelling was not observed with the pOpIgG (B). Scale bars=0.5 um. Plates 15 and 16: Negative controls for gold labelling of cells from soya milk culture (plate 15) and inorganic nitrogen culture (plate 16) processed by method 4B. With no antibody in the primary incubation, gold particles were not seen in the cells nor in the surrounding resin (A). When normal rabbit IgG was used, few gold particles were seen in the cells and some were seen in the resin, indicating background non-specific binding (B). Scale bars=0.5 um  75  76  77  15  78  79  Plates 17 through 20 Transmission electron micrographs illustrating gold labelling of O. piceae cell fragments. O. piceae was grown in both soya milk supplemented medium and ammonium nitrate supplemented medium. These fragments were of cells that were harvested and homogenized when cultures were in exponential growth phase, (small arrow=cell wall fragment; d=cellular debris) Plate 17: Transmission electron micrographs of cell wall fragments, from soya milk culture, that were processed for TEM by method 4B and immunogold labelled using the pKIgG diluted 1 /25. Figure A shows labelling of wall fragments prior to washing with buffer, while Figure B shows labelling of fragments after washing. Gold particles in the surrounding cell debris were mainly associated with electron translucent material, which were believed to be remnants of the extracellular sheath. Scale bars=0.25 um. Plate 18: Transmission electron micrographs of washed cell wall fragments, from ammonium nitrate culture, that were processed for TEM by methods 4B (A) and 1A (B) and immunogold labelled using the pKIgG diluted 1/25. Notice that the labelling of the cell wall pieces in A was different than in B. Labelling was much more pronounced in the cell walls that were processed by method 4B than those processed by method 1A. A similar difference was observed in whole cells (Plates 11 and 13 A). Scale bar=0.25 um. Plate 19: Transmission electron micrographs of washed cell wall fragments from soya milk culture, that were processed for TEM by method 4B and immunogold labelled using the pOpIgG diluted 1/100 (Figures A and B). Labelling was only seen in the cell wall fragments and sometimes appeared to be on the edges of these cell wall pieces. No gold particles were found in the background. Gold particles were not observed in sections that were incubated in buffer (Plate 19C), or without a primary antibody. Scale bar=0.25 um. Plate 20: Transmission electron micrographs of washed cell wall fragments, from ammonium nitrate culture, that were processed for TEM by method 4B. Sections were incubated with the pOpIgG diluted 1/100 (A) or with buffer only (B). Similar to observations made for the soya milk culture derived fragments (Plate 19), gold labelling was observed in the cell wall fragments only, with some gold particles found on the edges of these wall pieces. Scale bars=0.25 um.  80  17  81  83  84  4.0 DISCUSSION  Abraham et al. (1993) showed that O. piceae secretes a major extracellular proteinase when grown in a defined medium under specific nitrogen conditions. Proteolytic activity is detectable in the culture supernatant of liquid cultures supplemented with protein as the sole nitrogen source as well as when the fungus is starved for nitrogen. However, when provided with inorganic nitrogen (e.g., ammonia) or non-protein organic nitrogen (e.g., amino acids), very little, if any, extracellular proteolytic activity is detected in the culture filtrate. These findings regarding the extracellular proteinase of O. piceae were based on analyses of culture filtrates. In this work, an attempt was made to localize this enzyme in O. piceae cells from liquid culture. To achieve this goal, the fungus was grown in two media which differed in their nitrogen component. Soya milk was used as a source of organic nitrogen because it permits the generation of high biomass and a large amount of extracellular proteolytic activity. Also, it was chosen over other commonly used proteins, such as BSA or gelatin, since it is plant derived and thus may be a more appropriate substrate for a wood inhabiting fungus. Ammonium nitrate supplemented culture was used as a negative control. Since negligible amounts of extracellular proteolytic activity are detected in such cultures, it was assumed that there would be little proteinase if any in these cells. Localization of the proteinase was initially assessed by assaying for proteolytic activity in the whole cultures. Finally, antibodies were produced to localize the enzyme by immunoblotting and by immunogold labelling.  4.1 Growth and proteolytic activity of O. piceae in liquid culture Cultures that were supplemented with protein (soya milk) grew to a greater extent than those provided with ammonium nitrate. To obtain a fungal biomass similar to that achieved with the soya milk medium, three to four times more inorganic nitrogen medium was required. However, it is difficult to compare the growth. Although the media contained equal amounts of total nitrogen, the soya milk medium contained two times more utilizable nitrogen since O. piceae  85  does not utilize nitrate (Abraham et al. 1993). Therefore, growth may have been enhanced by the larger nitrogen supply in soya milk medium. Growth may also have been enhanced by the nature of soya milk. Aside from a large amount of protein, it contains various other nutrients, including lipids, carbohydrates, vitamins and trace elements, all of which might have contributed to fungal growth. In agreement with previous findings of Abraham et al. (1993), the results suggest that O. piceae can utilize protein efficiently, reflecting its adaptation to growth in wood where the limited amount of nitrogen is found mainly as protein (Langheinrich and Tischner 1991). Like growth, proteolytic activity differed significantly between the inorganic nitrogen and organic nitrogen supplemented cultures. Concurrent with the large amount of growth in soya milk medium, a relatively large amount of extracellular proteolytic activity was detected in these cultures. In contrast, only a small amount of activity was detected in O. piceae cultures provided with inorganic nitrogen. Moreover, the activity detected in the latter normally shows a slight increase later on in stationary growth phase, presumably as a result of the depletion of nitrogen in the culture medium (Abraham et al. 1993). Similar observations were made in this study, except that the peak in proteolytic activity observed for the inorganic nitrogen culture was detected early during growth rather than in a later stage of growth. Although this peak was greater than the activity observed for the remainder of the culture's growth, it may not have been a significant difference since the overall activity was quite low. As has been observed for other fungi (Klapper et al. 1973; Zhu et al. 1994), it appeared that production of the extracellular proteinase by O. piceae was dependent on the composition of the growth medium. Extracellular protease(s) are often produced in response to changes in the source and availability of nitrogen, carbon or sulphur in the growth medium (Drucker 1975; Cohen 1981). These characteristics are specific to the organism or the enzyme produced and are often indicative of a particular regulation mechanism.  According to Cohen (1980), two main  mechanisms have been reported for the regulation of extracellular enzyme production in fungi: "induced synthesis ^with constitutive secretion" and "derepressed synthesis with constitutive secretion". Although appropriate, stringent studies have not been performed for determining the  86  regulation of the O. piceae extracellular proteinase, we suspected that proteinase production resulted from derepression when easily assimilable forms of nitrogen were no longer available. This suggestion is supported by unpublished findings that O. piceae preferentially utilized inorganic nitrogen when provided with both inorganic and protein nitrogen since almost no activity was detected in the culture filtrate before the inorganic nitrogen source was depleted (personal communication, Abraham 1995). The cellular portion of the two types of liquid cultures also showed differences in proteolytic activity. The proteolytic activity in the pellet from soya milk culture was relatively high compared to that from the inorganic nitrogen culture. The activity associated with the soya milk pellet was mainly extracellular since most of the activity was recovered in the buffer used to wash the cells; both the washed whole cells and the cell fractions (not shown) showed much lower or negligible amounts of activity compared to that of the unwashed cells and the cell wash. Since washing the pellet prior to cell disruption was adequate to remove the proteinase, it seemed that the enzyme was only loosely associated with the cells, extracellularly. Washing is therefore, an important step towards accurately assessing cell-associated proteolytic activity in O. piceae cultures which contain a large amount of extracellular proteinase. According to these results, it seems that the proteinase remained associated with the cells, a characteristic observed in other fungi. Various basidiomycetes secrete enzymes that often remain associated with the hyphal surface in sheaths or similar structures (Wood 1985). The extracellular sheath or mucilage produced by O. piceae , when it grows in wood (Serge Gharibian, personal communication; Luck et al. 1990) or in liquid culture (e.g., Plate 7), may also serve to harbour extracellular enzymes. It is possible that the proteinase, and other extracellular enzymes, somehow adhere to the sheath or are contained within it. This interaction may be weak such that washing the cells disrupts this extracellular matrix and causes the release of the proteinase. Alternatively, the sheath may not be firmly bound to the cells and washing may have been sufficient to remove at least part of it and the enzyme along with it. This latter point might partly explain why proteolytic activity recovered in the wash and in the washed cells did  87  not sum to equal the original amount of activity in unwashed cells. If the enzyme had remained with the washed off sheath material, it may not have been detected with the azocoll substrate since insolubles were removed by centrifugation prior to assaying the wash for activity. Earlier studies showed that O. piceae cells are very difficult to break open. As demonstrated in this work, at least 30 or more minutes of agitation were required to sufficiently disrupt the cells. Because the procedure lasted so long, there were concerns regarding the heat lability and autolytic property of the enzyme. Therefore, precautions were taken to prevent proteinase denaturation or degradation during the disruption procedure. For example, cell breakage was carried out over short disruption intervals interrupted with cooling on ice, and C a  2 +  was included in the homogenization buffer, since this cation has been shown to improve the thermostability of the proteinase (Abraham and Breuil 1995a). Although these measures should have prevented degradation or denaturation of the enzyme during this destructive procedure, it is possible that they were insufficient to protect it. They would not have protected the proteinase from other cellular proteolytic enzymes that could have been released by cell breakage. These may have acted on the extracellular proteinase affecting its detection. Protease inhibitors could have been included in the homogenization buffer to prevent the action of other proteases, but these would have interfered with the subsequent detection of the extracellular proteinase in an active form. Alternatively, the release of naturally occuring protease inhibitors, found in the cytosol of fungal cells may have inactivated any proteolytic enzymes present (Cohen 1977). These factors might help to explain why, at least for the soya milk cultures, the homogenized cells showed only a small amount of proteolytic activity. Because of the difficulties involved with cell disruption and the resulting poor recovery, these preliminary cell-associated activity results could not be utilized quantitatively.  A  significant finding was that the proteinase was found to be cell-associated in O. piceae liquid culture, mainly when a large amount of proteinase was being secreted. Cellular proteolytic activity was hardly detectable but it was not clear if this was because there was no proteinase in the O. piceae cells. By SDS-PAGE a protein band of molecular weight similar to the proteinase  88  was detected in various fractions. If this band did correspond to the proteinase, perhaps its activity was not detected because it was inactivated during the homogenization procedure, or alternatively, it was not in an active form in these fractions. The low activity was not likely a function of the assay or size of the substrate since both the general substrate, azocoll, and the smaller more specific substrate, succinyl-(Ala)2-Pro-Phe-p-nitroanilide, provided similar results i.e. that activity was very low or not present. Further experimentation is required to better interpret these results and gain a better understanding of the enzyme in relation to the cells. An alternative method for detecting the proteinase in the cells could establish the validity of these preliminary findings. The antibodies produced to the proteinase could serve this purpose in localizing the proteinase in O. piceae cells.  4.2. Production and characterization of antibodies for proteinase localization Antibodies were produced to localize the major extracellular proteinase in O. piceae cells by immunoblotting and by immunogold labelling. However, as described below, this was not an easy task. Various attempts were made to obtain antibodies that would specifically detect the O. piceae proteinase. Three sets of antibodies were produced including: 1) anti-O. piceae proteinase monoclonal antibodies, 2) anti-proteinase K polyclonal antibody (pKIgG), and 3) anti-O. piceae proteinase polyclonal antibody (pOpIgG).  4.2.2 Anti-O. piceae proteinase monoclonal antibodies  Originally, since only a small amount of proteinase could be purified, monoclonal antibodies were raised against PAGE-purified O. piceae proteinase by intrasplenic immunization of mice. Unfortunately, the antiserum titres were low and the hybridomas prepared following these immunizations produced antibodies that were mainly of the IgM class. These reacted poorly with O. piceae proteinase and cross-reacted with other enzymes. Similar difficulties with producing monoclonal antibodies to an extracellular elastase, from Aspergillus fumigatus, were reported by Frosco et al. (1992) who found that the native 32 kDa enzyme was weakly  89  immunogenic in mice and did not induce a secondary immune response. The O. piceae proteinase also seemed to be weakly immunogenic probably because of its small size or its conformation upon immunizing the mice (Crumpton 1974). The poor antibody generation may also have been due to autolysis of the proteinase prior to eliciting an immune response. Since its stability was not yet understood, the necessary precautions to prevent autolysis had not been taken. Therefore, the proteinase may have self-degraded into smaller autolysis products. Since the lower limit for immunogenicity is considered to be approximately 12 kDa (Crumpton 1974), the autolysis products would have been even less likely to induce an immune response. Although small proteins can induce an immune response, the antibody titres are often quite low (Crumpton 1974). The production of antibodies to products of autolysis could also result in the weak recognition of the intact proteinase. These factors could explain the poor results. Based on these characteristics alone, it seemed that producing antibodies against this proteinase would be a difficult task.  4.2.2 Anti-proteinase K polyclonal  antibodies.  A positive aspect of these results was the cross-reactivity of the resulting monoclonal antibodies with proteinase K. As aforementioned, proteinase K and O. piceae proteinase are enzymes that have many characteristics in common (Abraham and Breuil 1995b). The fact that the monoclonal antibodies raised against the O. piceae proteinase recognized this well characterized subtilisin-like enzyme supported the classification of the 33 kDa proteinase as a subtilase. In consideration of these factors, it was suggested that antibodies raised against proteinase K might cross-react with the proteinase of O. piceae. The benefit of using proteinase K as the antigen was that it was readily available in unlimited quantity, unlike the O. piceae proteinase which, at the time, was still difficult to purify in large quantities.  Therefore,  polyclonal antibodies were raised against proteinase K (pKIgG) with the intention of using them to detect and localize the O. piceae proteinase. Upon screening the pKIgG, it was evident that they reacted more strongly to proteinase K than to the proteinase of O. piceae . By ELISA and dot blotting it was shown that the pKIgG  90  could detect much smaller amounts of proteinase K than O. piceae proteinase. The relatively low reactivity of the pKIgG with the HIC purified proteinase of O. piceae might again have been due to the autolytic property of the proteinase. The proteinase is not stable when exposed to high pH at 37°C, the conditions under which the enzyme was coated onto the ELISA plate. Reichard et al. (1990) also experienced difficulties with ELISAs due to autolysis of an Aspergillus extracellular serine proteinase. Unless the proteinase inhibitor PMSF was included in the coating buffer, their ELISA results on purified proteinase were irreproducible. Although including PMSF with the proteinase for dot blots did not enhance the dot intensity (data not shown), reactivity with the proteinase in an ELISA might be improved by using PMSF-inactivated proteinase. Alternatively, it is also possible that the binding of the proteinase to the membrane or the plate masked or buried the antigenic determinants of the proteinase that the antibodies would bind to. Although proteinase K and the proteinase preparations from O. piceae could be detected by the pKIgG on dot blots and in ELISA, they were not detected on Western blots. Initially, since the blots were of proteins separated by SDS-PAGE, it was considered that boiling the proteinases and their treatment with SDS modified them such that they were no longer recognized by the antibody. This hypothesis was supported by results obtained with an immunodot blot for which the blotted samples had been boiled in SDS-PAGE sample buffer to mimic the conditions of sample preparation for SDS-PAGE. Only a very low level of chemiluminescence was detected from the sample buffer treated proteins, compared to a strong positive reaction in samples prepared in PBS (not shown). However, transfer of the proteinases under non-denaturing conditions, from IEF and native-PAGE gels, did not change these results. Although contact print zymograms showed that the enzymes had migrated as expected and were still proteolytically active following electrophoresis, there was no detection of any distinct bands on Western blots. On some of the Western blots, though, the pKIgG reacted with unknown components which appeared as smears near the start of the separating gel. It is possible that the proteinase was somehow associated with these components that had not entered the gel and its migration through the gel may have been hindered due to the size, solubility or charge of these unknowns.  91  This would explain the smears and support the positive reacdons obtained by dot blot and ELISA. Alternatively, the smears that were observed might have been due to the non-specific binding of antibodies from the rabbit pre-immune serum. Such a phenomenon would also explain the crossreactivity that was observed with cellulase and lysing enzymes dot blots. Since the pKIgG is a polyclonal antibody, the possibility of the immune serum containing cross-reacting antibodies cannot be ignored. Unfortunately, this could not be verified because the pre-immune serum of the rabbit was not available  However, it is important to consider that, in  the pKIgG, the molar ratio of anti-proteinase IgGs to non-specific, reacting pre-immune IgGs was probably high. Consequently, the other IgGs of the pre-immune serum that might recognize O. piceae antigens should have had only a minimal effect on these results. Therefore, because of the ELISA and dot blot findings, it was assumed that the pKIgG could be used to localize the proteinase in O. piceae cells.  4.2.3 Anti-O. piceae proteinase polyclonal antibodies  Once the O. piceae proteinase stability properties were better understood and the enzyme could be purified in larger amount, a polyclonal antibody to O. piceae proteinase (pOpIgG) was produced. Since the proteinase is autolytic, rabbits were immunized with PMSF inactivated HIC purified proteinase to promote antibody production since, as mentioned earlier, antigen size and protein conformation are, among other things, important for protein immunogenicity. Both ELISA and dot blotting showed that the pOpIgG reacted strongly with the O. piceae proteinase preparations while the purified pre-immune serum did not. Therefore, the pOpIgG contained antibodies specific to the proteinase that were likely produced as a result of the immunization. Although the pre-immune serum did not appear to recognize the proteinase, it did react with an extract of O. piceae homogenized cells. This was also found for other O. piceae cell extracts from cultures grown under both nitrogen conditions (not shown). The reactions  *Due to a freezer breakdown, the pre-immune serum of the rabbit that was immunized with proteinase K was lost. 92  observed were likely due to antibodies that recognized other fungal components such as, carbohydrates, chitin or protein, that had probably been produced by the rabbit in response to prior exposure to a fungus. Accordingly, the reaction of the pOpIgG to O. piceae cell extract might have been to these other unknown components in addition to the proteinase. This is further illustrated by the fact that the pOpIgG had recognized the same commercially prepared enzymes as the pre-immune serum did. Even so, it seemed that the proteinase specific antibodies were relatively high in concentration in the pOpIgG, since its reactivity to the proteinase remained strong, while reactivity to the commercial enzymes was reduced when the antibody concentration was decreased. The advantages and disadvantages of using polyclonal antibodies to specifically detect an antigen were illustrated by these antibodies. Due to their heterogeneous nature, polyclonal antibodies sometimes cross react with antigens that are similar to the immunogen. This property can be exploited by using antibodies raised against a given antigen to detect a similar antigen. This was illustrated by the pKIgG which cross-reacted with the proteinase produced by O. piceae. These antibodies that were raised against the readily available proteinase K appeared to recognize not only proteinase K but also the proteinase of O. piceae , an enzyme that was difficult to purify. Similarly, others have used antisera to homologous proteins for their own immunolocalization studies (Blanchette et al. 1989; Benhamou et al. 1990; Nicole et al. 1993). A negative characteristic of polyclonal antibodies is that one can also obtain unwanted, non-specific cross-reactivity with a sample. Again, this can be due to similarity of antibody binding sites on the antigen of interest and on other components found in the sample being screened. Or, as was the case for the pOpIgG, cross-reactivity can occur due to antibodies in the pre-immune serum. In summary, the two polyclonal antibodies that were produced for this study were shown to recognize the proteinase of O. piceae. From their characterization, it was apparent that they primarily detected the O. piceae proteinase leading to their use for immunolocalizing the proteinase.  93  4.3 Detection of the extracellular proteinase in O. piceae cells by immunoblotting  To immunodetect the proteinase in both intact and homogenized O. piceae cells, pellet fractions were extracted by boiling with SDS in order to obtain a sample of soluble proteins that could be quantified and easily blotted onto nitrocellulose. The extracts were shown to contain a variety of proteins. Unfortunately, proteolytic activity was destroyed by boiling in SDS. Ideally, extraction would have also retained any proteolytic activity that was associated with the pellets. However, other milder extraction methods that were tried including, treatment at room temperature with high salt and/or Triton X-100, were not as efficient. Very little protein was recovered, and no activity was detected in those extracts (data not shown). Dot blots of the SDS extracts and other fractions were probed with the purified polyclonal antibodies. The results varied with respect to the two antibodies and the different cultures. The pKIgG reacted with pellet fractions from both the soya milk and ammonium nitrate cultures. However, a reaction, though very slight, was also observed with the intracellular filtrate of the soya milk culture, probably because these cells were producing the proteinase. In contrast, the pOpIgG reacted with all the fractions from both cultures which suggested that the proteinase was found in the cells and cell walls of both inorganic nitrogen and soya milk culture cells. Results of Western blot analysis of the pellet extracts and cell filtrates were similarly ambiguous. Only the intracellular filtrate from the soya milk culture showed a very faint band corresponding to a 33 kDa protein. The pOpIgG mainly detected high molecular weight bands and smears. It is possible that the high molecular weight staining was the detection of the proteinase in association with other cellular components, a characteristic of the enzyme that has not been previously observed. Smears on Western blots are often attributed to insoluble carbohydrate entering the gel in association with proteins (Banerjee et al. 1994). However, previous experiments have shown that the proteinase produced by O. piceae is not glycosylated (Abraham and Breuil 1995b) . Perhaps the proteinase was associated in a less specific manner with carbohydrates of the cell wall or the extracellular sheath. Dickerson and Baker (1979) showed  94  that extracellular enzymes that are bound to |}-glucans in the fungal cell wall can be difficult to isolate. If the proteinase was associated with carbohydrates in O. piceae , boiling in SDS may not have been sufficient to solubilize proteinase that was entrapped within the wall or sheath carbohydrate. Treating the cell extracts to modify or remove any carbohydrates associated with the enzyme might reduce or eliminate background labelling. The Western blot smears may also have been due to the reaction of non-specific antibodies with other O. piceae proteins or glyco-proteins. This cross reactivity to other cell components might be reduced if the antibody were used at a lower concentration as was observed in screening the commercially prepared enzymes. Reactivity to the proteinase should still be evident since the pOpIgG is probably predominantly composed of antibodies to the proteinase. Though the limit of detection of this antibody for blotted proteins is not yet known, it was found that as little as 150 ng of purified proteinase could be detected on a dot blot when the antibody was diluted by as much as 1 in 18 000. It is not known how much proteinase can be detected with less antibody nor how much can be detected when it is in a mixture of proteins as in the cell extracts. This has yet to be systematically determined. Nevertheless, the positive reactions of the pKIgG and the pOpIgG with the cell fractions indicated that the proteinase was associated with the cells and/or the cell walls of O. piceae whether or not it was cultured with protein as a nitrogen source. In combination with the results of the azocoll assays and assuming that the proteinase was not destroyed during the homogenization procedure, these findings might suggest that the antibodies detected proteinase that was not yet in an active form. The antibodies may have detected the proteinase before it was fully processed and accordingly, before it was active. Processing of many secreted enzymes involves specific, proteolytic cleavage of peptides from the immature and often, not yet folded enzyme. Extracellular proteases in bacteria have been shown to be processed as pre-pro-enzymes with pro-regions that are autocatalytically cleaved prior to or following transport across the cell membrane (Terada et al. 1990; Lee et al. 1994). This cleavage is required for the secretion (Lee et al. 1994) or proper folding of the enzyme  95  (Ikemura, et al. 1987; Silen and Agard 1989). The pro-region of subtilisin from Bacillus subtilis, as well as that of a-lytic protease have also been shown to have an inhibitory effect on the respective enzyme (Baker et al. 1992). Many enzymes depend on this cleavage for maturation and thus, activation. Siezen and co-workers (1991) found that nearly all of the subtilases are produced as pre-pro enzymes which are activated by cleavage of the pro-segment once the proteinase has translocated across a membrane. Such a mechanism might exist for production of the extracellular proteinase in O. piceae.  4.4 Proteinase localization by immunogold labelling and microscopy The main considerations for preparing samples for immunoelectron microscopy are to preserve the antigen structure, distribution and accessibility to the antibody. Ultrastructural preservation is also an important factor in localizing an antigen of interest. Unfortunately, these goals are often mutually exclusive since the traditional fixation and embedment methods used for ultrastructural studies are quite harsh and as a result, are often unsuitable for immunocytochemistry. Strong fixatives, such as glutaraldehyde and OSO4, can modify or denature proteins (Wolman 1955; Hayat 1981). This can affect their antigenic properties and drastically reduce their immunolabelling (Bendayan et al. 1987). Also, the commonly used embedding media are epoxy resins which are hydrophobic and heavily cross-link tissue, characteristics which can negatively effect immunolabelling (Wolman 1955; Causton 1985). The complete dehydration of tissues required for the proper infiltration of these resins can also damage the cells and introduce structural artifacts. Clearly, sample preparation can modify the antigen of interest and, effectively, disrupt its recognition and binding by antibody. Therefore, selecting an appropriate and effective processing protocol was an important step for immunolabelling of O. piceae. Initially, O. piceae cells from soya milk culture were processed and immunogold labelled (IGSS) with the pKIgG for viewing by dark field microscopy for a preliminary assessment of the effect of fixation on immunogold labelling. This experiment showed that the pKIgG could react  96  with fixed fungal cells and could therefore be useful for immuno-microscopy. It also illustrated that fixation could have an affect on the results since cells treated with paraformaldehyde only were not gold labelled. Similarly, for TEM observation of O. piceae, various combinations of fixation as well as embedment were attempted in order to obtain a method that would preserve the ultrastructure of O. piceae cells while maintaining the integrity of the proteinase for immunogold labelling. All the methods tried allowed for labelling in the cells with methods 1A, 3B and 4B resulting in relatively good ultrastructure preservation. Method 4B offered the best cellular preservation. This was not surprising since it included a combination-fixation including the rapidly penetrating paraformaldehyde and a rather high concentration of glutaraldehyde, a post-fixation with OsC>4 and embedment in Epon resin. The positive labelling, however, was surprising given these harsh conditions. Although OsC>4 is among the best fixatives for preserving cellular ultrastructure, its use for immunocytochemistry is usually avoided since it can mask antigens (Bendayan and Zollinger 1983; Stirling 1990; VandenBosch 1991). For example, Daniel et al. (1992) found that the amount of gold labelling in their fungal samples decreased by as much as 50% when osmium tetroxide was included in sample preparation. Fortunately, this did not seem to be the case with O. piceae. Labelling was possible with the osmium fixed and Epon embedded material and the pattern or amount of labelling in these cells was comparable to cells that were fixed without osmium (data not shown). These results suggested that the epitopes were not sensitive to strong fixation and embedment. These conclusions, however, were based only on a qualitative observation of the gold labelled samples. The gold labelling observed by TEM was mainly localized in the walls of O. piceae cells independent of the culture medium used to grow the fungus. Assuming that most of the labelling observed was of the extracellular proteinase, this indicated that the enzyme can be found in the fungal cell wall whether or not it was secreted into the growth medium in large quantities. This observation had also been made with immunoblots. Cell wall labelling in cells from soya milk culture seemed reasonable since the proteinase was being secreted at the time of sample processing  97  and was likely in transit across the cell wall (Cohen 1977). However, the heavy labelling of the cells from inorganic nitrogen cultures was unexpected. Since these cells exhibited relatively little proteolytic activity, it was presumed that these cells were only producing or secreting a minute amount of proteinase. It was for this reason that cells from this culture were to serve as a negative control for proteinase labelling. Similar observations were made by Gallagher and Evans (1990) when they immunolocalized p-glucosidase in the cell wall of Coriolus versicolor. In cultures where p-glucosidase activity was very low, they found that much of the enzyme was present in the cell wall, whereas cultures whose extracellular activity was much higher, demonstrated less labelling. The gold labelling observed in O. piceae suggested that the cells have a constitutive low level of proteinase production which was difficult to detect by activity assay. Perhaps the proteinase is produced as a zymogen that becomes activated only when it is required by the fungus. An example of such an activatable enzyme is chitin synthase, an intramural enzyme that functions in cell wall growth (Choi 1994). This suggestion could explain the gold labelling observed in the cells from inorganic nitrogen culture where very little proteolytic activity was detected. However, there is no other evidence to date, such as the existence of an "activator" proteinase, to support this suggestion. The cell wall labelling might be better understood by considering the theories that have been proposed for protein secretion in fungi. Though this process is still poorly understood, protein secretion, in both yeast and mycelial fungi, is believed to be mediated by vesicles (Brada and Schekman 1988; Bartnicki-Garcia 1990).  While vesicles supply new  membrane, they  simultaneously extrude their lumenal contents into the wall domain. These contents include enzymes and/or matrix polymers (Wessels 1993) which can either reside in the periplasmic space, the cell wall or extracellular sheath, and/or are further secreted into the growth medium (Trevithick and Metzenberg 1966; Bartnicki-Garcia 1990; Wessels 1993). Traditionally, the cell wall, in mycelial and yeast fungi, has been viewed as being porous, analogous to a sieve through which molecules of various sizes can pass (Trevithick and Metzenberg 1966; de Nobel and Barnett 1991). Presumably, secretion through pores occurs  98  throughout fungal growth, from any region of the hypha. But the pores in the mature, polymerized cell wall are only large enough to allow the release of smaller macromolecules by a sort of "molecular sieving" (Trevithick and Metzenberg 1966). Theories were then proposed to account for the secretion of larger molecules through the cell wall. From their studies of Neurospora crassa, Chang and Trevithick (1974) proposed that enzyme secretion occurs most readily at the hyphal tip where the wall is more elastic and porous. Secretion in this region would also account for the retention of extracellular enzymes in the cell wall and periplasmic space (Chang and Trevithick 1974). Since the subapical portion of the cell wall rigidifies first as the hypha extends, the porosity of the outer layer of the cell wall decreases first (Wessels 1990) and as a result, the macromolecules being secreted, get trapped and remain in the, eventual, lateral cell wall (Chang and Trevithick 1974). According to this theory , the gold labelling observed for the extracellular proteinase in the cell wall of O. piceae, could have been predicted, especially for the cells from the soya milk cultures in which proteinase secretion is high. The "bulk flow" hypothesis proposed by Wessels similarly predicts the trapping of apically secreted macromolecules in the polymerizing cell wall but it may also provide an explanation for the presence of proteinase in the cell walls of inorganic nitrogen culture cells. Wessels proposed that protein secretion across hyphal walls is a gradient phenomenon such that proteins that are produced at high rates would occur as steep gradients in the pathway and, in effect, would be added to the wall in a steep gradient. These would then be expected to occur at a higher concentration in outer wall regions and would also be found at higher concentrations in the surrounding medium. By the same reasoning, proteins that are produced in lower amounts create a shallower gradient and would less likely be found in the external medium. Immunogold labelling of the proteinase in O. piceae did not seem to illustrate this predicted pattern and distribution within the cell wall. But, the detection of proteinase in cells from inorganic nitrogen culture might be accounted for by this hypothesis, assuming, as suggested earlier, that O. piceae has a constant low level of enzyme production. The low level of proteinase production in these cells may  99  not have created a sufficient gradient, relative to other secreted macromolecules, to allow for its release into the surrounding medium. In summary, both theories suggest that proteins can be trapped in the polymerizing cell wall as the fungus is growing. Therefore, the observations made here are reasonable. The immunogold labelling in the cell walls was likely of the extracellular proteinase of O. piceae that, while in transit through the growing apex, was trapped as the cell wall polymerized. In addition to labelling in the cell wall, the pOpIgG gold labelling was mainly localized to the surface of the cell wall, in an electron dense layer or coating, particularly for cells in stationary growth phase. In many cells, this coating was quite thin, as has been previously observed in O. piceae from liquid culture (Luck et al. 1990). This distribution of gold labelling of the proteinase supported the azocoll assay findings, which showed that proteolytic activity could be detected extracellularly in a wash of the cells. Similarly, sheaths have been observed in other fungi grown in liquid medium (Gallagher and Evans 1990; Daniel and Nilsson 1992). We suspect that in wood, the sheath may serve, to house and protect extracellular enzymes secreted by O. piceae during growth. Various wood rotting fungi have been shown to produce extracellular sheaths or mucilage which often carry wood degrading enzymes (Palmer et al. 1983; Ruel and Joseleau 1991; Green III et al. 1992; Nicole et al. 1993). It is believed that they serve to protect enzymes from dessication and thus inactivation and have also been suspected of retaining extracellular enzymes in close proximity to the producing microorganism offering a nutritional competitive advantage to the fungus in vivo (Wood 1985; Kalisz 1987; Green III et al. 1992). An extracellular sheath may additionally serve to modify the extracellular ionic environment and pH (Green III et al. 1992) to create a buffered zone or a micro-environment for fungal enzymes to be active. Activity of the proteinase produced by O. piceae is inhibited by low pH and is optimal between pH 7 and 9 (Abraham and Breuil 1995a). When the pKIgG were used, gold particles were also often seen in what appeared as multivesicular bodies. Whether or not they serve a function in fungi is not known but others have speculated that they are involved in extracellular enzyme secretion (Calonge et al. 1969; Daniel  100  et al. 1992). In Sclerotina fructigena the presence of these structures was correlated with high levels of pectinolytic enzyme secretion (Calonge et al. 1969). In O. piceae, these multivesicular bodies were detected mainly in cultures provided with inorganic nitrogen. Since these structures are predominantly found in vegetative cells exhibiting a high level of enzyme secretion, they probably resulted from production of other enzymes and not the proteinase. Interestingly, the pOpIgG did not label these vesicle containing structures. This difference in labelling pattern between the antibodies might suggest that the antibodies have different specificities for the proteinase. The pOpIgG were the result of immunization with native protein so perhaps the antibody is specific for antigenic determinants found only on mature proteinase. Its avidity for the immature enzyme might then be quite low. Conversely, the pKIgG were raised against proteinase K which was not protected from autolysis prior to the immunization. Therefore, the resulting antibodies may be able to detect both sequential antigenic determinants, on linearized portion of degraded proteinase K as well as the whole enzyme. A more simple explanation would be that localization of proteinase in these vesicles might be background labelling only or even be an artefact. Since the structures were mainly seen when the cells were processed with osmium tetroxide, they may have been artifacts of sample processing. By the same token, fixation with osmium may have allowed for the preservation of these structures, that were otherwise lost during sample processing.  101  5.0 CONCLUSION  Previous studies of O. piceae grown in liquid medium showed that when this fungus is provided with protein as a nitrogen source, its major extracellular protein is a 33 kDa proteinase. This enzyme has been isolated and purified from liquid culture supernatant and has been classified as a subtilisin-like serine proteinase. In this work, for the first time, this proteinase was studied with respect to the cellular portion of O. piceae culture. Proteolytic activity was assayed in whole cells and in disrupted cells of O. piceae grown in liquid media. In addition, the major extracellular proteinase was localized in the cells from these cultures by immunological detection methods, including immunoblotting and immunogold labelling. Since proteolytic activity is much higher in the supernatant of cultures provided with protein than in cultures supplemented with alternative forms of nitrogen, the production and/or secretion of the major extracellular proteinase produced by O. piceae is considered to be dependent on the nutrients in the culture medium (Abraham et al. 1993). Similarly, as shown in this work, more proteolytic activity was detected in cells from organic nitrogen culture than in the cells from inorganic nitrogen culture. However, the cellular proteolytic activity was much lower than that of the culture supernatant. In addition, the proteolytic activity detected in cells from soya milk culture was found to be mostly due to enzyme that was loosely bound to the cells. Once this cellassociated extracellular activity was removed, the proteolytic activity of the soya milk culture cells was approximately equal to that of the inorganic nitrogen culture cells. Very little activity, if any, was found in isolated cell walls. Therefore, proteolytic activity was mainly extracellular, particularly in cultures producing large amounts of proteinase. Polyclonal antibodies were produced and characterized to specifically detect and localize this proteinase in O. piceae cells. Due to difficulties with proteinase purification and antibody production, a polyclonal antibody was raised against proteinase K before one was raised against the O. piceae proteinase. Both polyclonal antibodies recognized the purified proteinase, as  102  shown by ELISA and immunoblotting.  Also, the pKIgG cross reactivity with the O. piceae  proteinase supported O. piceae's proteinase subtilase classification. These antibodies were used to localize the proteinase in O. piceae cells by immunoblotting and immunogold labelling for TEM. Dot blots of cell fractions showed that the proteinase could be detected in cells from both inorganic nitrogen and organic nitrogen culture. By immunogold labelling, the proteinase was primarily localized in the cell wall and in an extracellular sheath or matrix that surrounded the cells taken from both cultures. This was accepted as a reasonable finding since the proteinase is an extracellular enzyme which was likely in the process of crossing the cell wall. In addition, the pKIgG also labelled multivesicular bodies while the pOpIgG did not which suggested that the antibodies may have different specificities for the proteinase. For the first time, the cells from inorganic nitrogen culture were shown to contain proteinase in an amount comparable to that found in cells from organic nitrogen culture. This suggested that a basic level of proteinase is produced even when inorganic nitrogen is available to the fungus. Furthermore, since the proteolytic activity detected in the inorganic nitrogen cells was minimal while the immunogold labelling was quite strong, it was suggested that the enzyme was present in the cell wall in an inactive form. There are still questions to be answered and much to learn about the production, regulation and localization of O. piceae's major extracellular proteinase. In particular, the processing and activation mechanisms of the proteinase and the apparent low constitutive production of proteinase should be addressed. The possible role of the extracellular sheath for proteinase activity should also be investigated. Such factors may need to be considered for targeting the proteinase in wood infected by O. piceae . Preliminary results on immunolocalizing the proteinase in infected wood indicated that the major extracellular proteinase is in fact produced during growth on wood (personal communication, S. Gharibian). This finding supports the contention that the same major proteinase is produced by O. piceae in liquid medium and in wood.. Therefore, determining the  103  production and activation mechanisms of this proteinase would be key to the development of specific method of proteinase disruption and effectively, O. piceae growth inhibition.  104  6.0 L I T E R A T U R E C I T E D  Abraham, L.D. 1995. Functions of a serine proteinase produced by the sapstaining fungus Ophiostoma piceae. PhD thesis, University of British Columbia, Vancouver, B.C., Canada (In preparation). Abraham, L.D. and Breuil, C. 1993. Organic nitrogen in wood: Growth substrates for a sapstain fungus. ERG Doc No. IRG/WP10019 Abraham, L.D. and Breuil, C. 1995a. Factors affecting autolysis of a subtilisin-like serine proteinase secreted by Ophiostoma piceae and identification of the cleavage site. Biochim. Biophys. Acta (In press). Abraham, L.D. and Breuil, C. 1995b. 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