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Production of labeled DNA probes for the rapid diagnosis of disseminated candidiasis in immunocompromised… Cheung, Lori 1987

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PRODUCTION OF LABELED DNA PROBES FOR THE RAPID DIAGNOSIS OF DISSEMINATED CANDIDIASIS IN IMMUNOCOMPROMISED PATIENTS by LORI CHEUNG B.Sc, (Microbiology), University of British Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY 'We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 23, 1987 © Lori Cheung, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of 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 by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6G/81) - i i -ABSTRACT The increasing incidence of disseminated (invasive) candidiasis i s probably attributable to iatrogenic factors and to improved pre and postmortem evaluation. Premortem diagnosis of such infections have seldom been made early enough for successful treatment. In order to increase the l ikelihood of successful antifungal chemotherapy, rapid diagnosis of such infections i s v i t a l . However, present diagnostic procedures for invasive candidiasis are insensitive and often do not re l iably differentiate superf ic ial from invasive infections. This study was undertaken to produce DNA probes and to optimize conditions for rapid and eff ic ient detection of Candida DNA. Seven random Candida albicans DNA fragments (2-7 kbp) were cloned into plasmid pACYC 184. These recombinant plasmids were labeled with either P or biot in and used as probes. Two of the four recombinant plasmids tested were genus speci f ic . The other two were s l ight ly cross reactive with other yeasts (Saccharomyces cerevisiae and Hansenula anomala). Probes labeled with 32 32 P were twice as sensitive as the biot in probes. One P labeled recombinant (#66) detected 7 Pg of target DNA , which corresponds to approximately 2 X 10 C.albicans ce l l s . With refined simple DNA extraction procedures for C.albicans (in serum), these recombinant probes could possibly be suitable for c l i n i c a l application. - i i i -ACKNOWLEDGEMENTS I would lik e to express my gratitude to my supervisor, Dr. J.B. Hudson, for providing me with the opportunity to carry out this project in his laboratory, and for his support and guidance throughout the course of the study. I would also like to thank the members of my supervisory committee, Drs. J.A. Smith, B. D i l l , M.T. Kelly, G. Weeks, and A. Eaves, for their guidance and helpful suggestions. My sincere thanks also go to Dr. M. Altamirano-Dimas, Ms. Jessica Boname and Mrs. Nina Vellani for helpful discussions, to Ms. Ruth Poehlke for secretarial assistance and to the St a t i s t i c a l Consulting and Research Laboratory (SCARL, U.B.C.) for s t a t i s t i c a l input. - i v -TABLE OF CONTENTS INTRODUCTION I. Properties of C.albicans pi - 5 I I . Virulence factors of C.albicans P5 ~ 7 III. Defences against C.albicans infection in the competent host p7 - 12 IV. Predisposing factors for candidal infections pl2 - 17 V. Types of candidiasis pl7 - 19 V I . Diagnosis (in compromised patients) pl9 - 24 V I I . Purpose of this study p25 - 26 ABBREVIATIONS p27 MATERIALS I. Cells p28 II. DNA p28 III. Enzymes p29 - 30 IV . Miscellaneous materials p30 V. Solutions p31 - 33 METHODS I. Isolation of C.albicans rRNA and DNA p34 - 37 II. Nucleic acid quantitation p37 _ 38 III. Large scale amplification and isolation of cosmid pJB8 p38 - 39 IV. Large scale amplification and isolation of plasmid pACYC 184 p39 - 40 V. Small scale amplification and isolation of plasmid pACYC 184 p40 - 4l VI. Agarose gel electrophoresis p4l -v-VII. Cloning with cosmid pJB8 p4l - 44 VIII. Cloning with plasmid pACYC 184 p44 - 47 IX. Probe production p47 X. DNA spotting on to nitrocellulose p47 - 48 XI. Hybridization of probes to DNA on nitrocellulose p48 - 49 XII. Detection of hybrid on nitrocellulose p49 - 51 XIII. Cell quantitation p51 XIV. Growth curve of C.albicans p51 - 52 XV. Treatment of patient samples p52 RESULTS I. Large scale isolation of C.albicans r-RNA and DNA p53 - 54 II. Small scale isolation of yeast DNA p54 III. DNA cloning with cosmid pJB8 p54 - 55 IV. DNA cloning with plasmid pACYC 184 p55 V. DNA denaturation and spotting onto nitrocellulose p55. 58 VI. Comparison of small scale c e l l l y s i s methods p58 VII. Cross reaction of probes with other yeast DNAs and unrelated DNAs p58, 61, 68 VIII. Comparison of biotin detection methods p68 IX. Comparison of hybridization efficiencies p68, 77 X. Sensitivity of 32P labeled probes p77 XI. Sensitivity of biotin labeled probes p77 XII. Comparison of hybridization temperatures p77. 84 XIII. Comparison of hybridization times p84 XIV. Probing patient serum specimens p84 XV. Growth curve and DNA recoveries at various time intervals p95 XVI. DNA recoveries p95. 112 - v i -CALCULATIONS D I S C U S S I O N REFERENCES P115 pll6 - 126 pl27 - 149 - v i i -LIST OF FIGURES 1. Recombinant plasmids 2. Methods of c e l l l y s i s 3. Cross reaction between recombinant probes and other yeast and unrelated DNAs 4. 5-6. Cross reaction between plasmid probe and unrelated DNA's 7. Comparison of biotin detection methods 8. 9. Plasmid hybridization versus C.albicans DNA hybridization 10. Yeast self hybridizations 11. Sensitivity of 32P probes 12. Sensitivity of biotin probes 12. Comparison of hybridization temperatures 13. Various hybridization times 14. (densitometer tracing) 15. 16. 17. Leukemic patients' serum samples 18. C.albicans DNA isolated from c e l l culture at various time intervals - C.albicans probe 19. (densitometer tracing) 20. C.albicans DNA isolated from c e l l culture at various time intervals - #66 and #70 probe 21. (densitometer tracing - #66 probe) 22. (densitometer tracing - #70 probe) - v i i i -23. Cell culture plot pl06 2k. Cell DNA recoveries with lyticase and SDS treatment pl08 25. (densitometer tracing) pllO 26. Cell DNA recovery plot pll3 -1-INTRODUCTION I. PROPERTIES OF CANDIDA ALBICANS A. Classification, Replication and Ploidy Candida albicans belongs in the order of Cryptococcales, class of Deuteromycotina (fungi imperfecti) and genus of Candida [174]. There are more than 150 species of Candida, but C.albicans represents 50-60$ of the c l i n i c a l yeast isolates [174]. It i s a unicellular eucaryotic organism which replicates asexually by budding into yeast cells referred to as blastoconidia. Candida has no known sexual cycle and the ploidy of this organism i s uncertain. However, the concensus i s that C.albicans i s diploid and contains 12 chromosomes, with an - 1 4 absolute DNA content of 3-79 X 10 g per c e l l [82,184,238]. B. Morphology Candida i s a white (nonpigmented) asporogenous yeast which also exists i n a mycelial form. Its colonies are usually visible within 24 hours of incubation at 25-37° C. Although C.albicans i s normally referred to as a dimorphic organism, i t i s actually polymorphic [161,162] because i t coexists in several morphologies. C.albicans has four morphological growth forms : blastocondia, true hyphae, pseudohyphae, and chlamydospores. The most commonly encountered form i s the blastoconidium (or yeast). Blastoconidia are ovoid to nearly spherical cells whose size range from 2.7 x 2.0 um to 10 x 6.0 um [8,228]. Certain conditions favour a shift in morphology from the blastoconidial to the hyphal -2-form. True hyphae are cells which elongate continuously while laying down septa at intervals behind the growing tip. This results in almost perfectly cylindrical structures in which each cellular element i s connected to i t s neighbour by a septal pore [174,198]. In contrast, pseudohyphae are blastoconidial c e l l s , elongated to a variable extent by an apical budding process, which remain unseparated. This results in a filamentous c e l l chain appearance that i s quite often confused with a true hypha. This close relationship between the blastoconidia, pseudohyphae, and true hyphae, has made separation of the different morphological forms quite d i f f i c u l t . Some people believe that pseudohyphae are probably the intermediate form between blastoconidia and true hyphae. Within blastoconidial and true hyphal cultures, pseudohyphae are always present as a minor component [162]. The fourth fundamental growth form of C.albicans i s the chlamydospore. It i s regarded as a dormant or storage form and i s a product of a nutrient poor environment. Chlamydospores are thick walled, highly re f r a c t i l e spheres of approximately 10 um in diameter, and are usually extended from pseudohyphae via suspensor (tapered c e l l adjoining the hyphae and the chlamydospore) cells [162]. In addition to the four growth forms already mentioned, C.albicans also exhibits germ tube formation in serum within two hours of incubation at 37° C. Germ tube formation i s the i n i t i a l stage in the yeast to hyphae transition. Germ tubes are quite often mistaken for pseudohyphae, but can be distinguished by the absence of a constriction at the mother c e l l junction (ie. not pinched at the base but are attached by a wide base) [155.211]. The a b i l i t y to form true hyphae, chlamydospores, and germ tubes i s unique to C.albicans in the genus Candida [162,174]. Cell Wall The organization of the c e l l wall determines the morphology of the c e l l . It i s approximately 200-300 nm thick and composed of lipids (2%), protein (3-6%), chitin (0.6-2.7%), mannoprotein (20-23%) and glucan (48-60%). The amount of chitin increases four times with germ tube formation, whereas the amount of mannoprotein and glucan does not vary much with stage of growth or germ tube formation [174,221]. The fungal c e l l wall was originally considered a static structure but recent studies have shown that i t i s a site of continual changes by which the fungus controls i t s relationship with the environment. Certain c e l l wall structures of C.albicans are influenced by the growth medium and the age of the cells and are therefore unstable [196]. The c e l l wall i s a v i t a l part of the cellular structure. Its superficial layers are responsible for the adherence of Candida cells to host [73i174,188] c e l l s . The microfibrils arranged radially perpendicular to the c e l l wall provide the mechanical forces required for invasion of the host cells or tissue (eg. cornified layers of the epidermis) [94,174]. In addition, the permeability of the c e l l wall to host enzymes or to products of host humoral or cellular immune system determines the survival of the fungus [174]. D. C.albicans Enzymes There are more than 40 enzymes known for C.albicans [161]. Some of these are secreted and some are found in the c e l l wall. In the case of the enzymes present in the c e l l wall, i t i s d i f f i c u l t to determine whether they are integrated into the wall or just transitory during their secretion from the cytoplasm into the external environment [174]. Amongst the enzymes that have been investigated are phospholipases [195]i which are associated with bud sites [176] and are also secreted into the growth medium [175]- These phospholipases may play a role in tissue invasion. Different strains of C.albicans may produce varying amounts of these enzymes [175]-Another enzyme, trehalase, i s usually located in the vacuole in many yeasts and fungi [225] but in C.albicans, 90% of this enzyme i s found outside the plasma membrane [177]- -glucanases which are present in the c e l l wall may play a role in morphogenesis, c e l l wall turnover, and regulation of glucan structure [6,23,236]. There i s also an inducible secreted acid proteinase of about 40-46 kilodaltons [83,134,189,193] that has been implicated in pathogenicity [161,216]. More recently, a collagenolytic enzyme was found to be produced by C.albicans which degrades human dental collagen [101]. E. Distribution Candida species are present as saprophytes in the environment (eg. s o i l , fresh and salt water plants, insects, and fowl) but pathogenic species are isolated frequently only from humans and other mammals [90]. The pathogenic species C.albicans i s seldom an -5-environmental contaminant. It has been suggested that C.albicans i s obligately associated with warm-blooded animals [50]. C.albicans i s part of the normal flora of humans. It is normally present in the gastrointestinal, genital and urinary tracts but never present as a saprophyte in blood or other internal fluids or in tissues. For example, the rate of isolation from asymptomatic healthy individuals i s 10-30$ in stool, 50$ in the oral cavity and 10-50$ in the vagina. Certain factors such as pregnancy and age appear to increase the frequency of isolation from the vagina and skin respectively, of healthy individuals [90]. II. VIRULENCE FACTORS OF C.ALBICANS Although host factors are important determinants of Candida species pathogenicity, virulence factors of the organism i t s e l f also contribute to the pathogenic potential of the fungus. C.albicans i s the most virulent species of the genus Candida [1]. A. Adherence Properties As seen with other microbial infections, C.albicans tissue invasion i s preceded by colonization of epithelial surfaces [24]. Studies have suggested that adherence i s mediated through an interaction between glycoproteins of the fungal c e l l wall [227] and epithelial c e l l s . Adherence of C.albicans to host cells requires the yeast to rearrange components of i t s c e l l wall [227]. Subsequent penetration of the epithelial c e l l membrane may be aided by phospholipase C or other hydrolytic enzymes [176]. The organism grows -6-and multiplies within the epithelial c e l l layer before penetrating into the endothelium [93]• B. Enzymes Acid proteinases secreted by pathogenic Candida species such as C.albicans, C.tropicalis, and C.parapsilosis [191,194] are believed to be involved in Candida pathogenicity as these enzymes are localized around invading Candida cells [134,136]. Under acidic conditions similar to those of the in vivo environment of injured tissues, these proteinases can cleave human IgM and secretory IgA [191]. In vivo proteolytic attack by Candida proteinase has not been identified, but possible targets may include zymogens of regulatory serine proteinases (i.e. coagulation Factor X), and angiotensinogen [190,191]. Other Candida enzymes (secreted) have been observed [7,101] but their roles are unknown at this time. C. Evasion or Suppression of Host Defences Candida blastoconidia have been demonstrated to resist intracellular k i l l i n g by phagocytes [181], but the mechanisms or determinants involved in this resistance are not clear. However, i t i s known that there are substances released by k i l l e d Candida hyphae which bind to the surfaces of neutrophils, hence, contact between neutrophils and l i v e hyphae i s inhibited [47]- Furthermore, there appears to be a component of Candida c e l l s , possibly a c e l l wall glycoprotein or polysaccharide, which induces immunosuppression of the T-lymphocyte population [25,185] or generates suppressor T-lymphocytes [172,219]. -7-D. Dimorphism In vivo, under normal conditions, C.albicans i s almost always in the blastoconidia form, but tissue invasion i s always associated with mycelia as well as blastoconidia. Comparison of virulent and avirulent strains of Candida in mice indicates that virulence i s associated with the ab i l i t y of the fungus to form germ tubes [182]. C.albicans strains which i n i t i a l l y grow predominantly i n the filamentous form, are non-pathogenic for mice [244], This suggests that the mycelial form cannot successfully i n i t i a t e infection hence, the yeast needs to be able to transform into mycelia in vivo in order to be pathogenic or invasive. I I I . DEFENCES AGAINST C.ALBICANS INFECTION IN THE COMPETENT HOST Candida species are opportunistic pathogens as they are usually benign colonizers of mucosal surfaces. The noncompromised host has a variety of mechanisms to resist infection by such organisms. Both specific and nonspecific mechanisms help to protect, but much disagreement exists with respect to the relative importance of these mechanisms. A. Nonspecific Mechanisms The nonspecific mechanisms are usually the f i r s t lines of defence against infection. These are often present from birth and are effective against a wide range of potential microbial pathogens. Some of the determinants of competency in terms of microbial resistance include the genetic constitution, age, and the nutritional and -8-hormonal balance of the host [183]-One of the more important passive defences i s the skin. The cornified and st r a t i f i e d squamous epithelium of the skin i s a relatively inhospitable site for C.albicans colonization. One reason for this may be because skin lipid s are probably inhibitory to the growth of C.albicans [2b]. In addition, regular sloughing and replacement of skin cells result in effective eradication of attached microorganisms [212]. Therefore, when intact, the skin serves as a mechanical barrier to penetration by Candida [183]. The intact mucosal surface often serves as a barrier against tissue invasion but i t i s much less well defended when compared to the skin. Candida and other microorganisms colonize these surfaces but subtle changes in the environment or in the host defences can result in increased colonization and possibly lead to invasion and disease [212]. Inflammation i s the most important nonspecific mechanism of the host. The i n i t i a l inflammatory reaction i s the release of chemical mediators resulting in the accumulation of f l u i d and cells at the site of infection. The f l u i d brings with i t a number of nonspecific serum factors such as properdin, lactoferrin and transferrin which aid in the elimination of the invading microorganism. Some of the accumulated cells are phagocytic cells which have traveled from the circulation into the i n t e r s t i t i a l spaces at the sit e . Phagocytosis may be the earliest and most efficient nonspecific mechanism for preventing the establishment of C.albicans. - 9 -The polymorphonuclear leucocytes and the macrophages are the principal phagocytic c e l l types involved [183]. There is disagreement among researchers as to the degree of effectiveness of phagocytic cells in the k i l l i n g of yeast or hyphal forms of C.albicans. There is evidence that human neutrophils are capable of k i l l i n g C.albicans cells [170,217]. This candidicidal activity i s associated with oxidative mechanisms that depend on the interactions of the C.albicans c e l l surface with hydrogen peroxide, myeloperoxidases and possibly also the lysosomal enzymes from specific granules [47,121,124]. There i s also evidence which suggests that mononuclear cells are capable of k i l l i n g cells of C.albicans. Murine hepatic macrophages and human peripheral blood monocytes have been shown to ingest and k i l l this organism [46,122,148]. Human alveolar macrophages are phagocytic for C.albicans [215] and may be also candidicidal [57]- A myeloperoxidase-linked fungal mechanism has been demonstrated in monocytes [46,120]. It appears that neutrophils and mononuclear cells may have some limited candidicidal activity but there are also suggestions that these phagocytic cells help to spread rather than contain C.albicans infection [64,200]. Although C.albicans blastoconidia are readily ingested by phagocytes [83], quite often the blastospores are not k i l l e d and have been seen in vitro to produce germ tubes resulting in the hyphae rupturing out of the phagocytes [129,181,217]. Mouse macrophage cultures have been destroyed within 24 hours after culture with C.albicans [166,217]. Clearly, more work i s required before the candidicidal activity of PMN and MN cells can be determined. -10-Serum complement components have been shown to augment the elimination of C.albicans. Furthermore, blastoconidia stimulated greater migration of human neutrophils than did mycelia. Since a greater amount of mannan is exposed in the blastoconidium, i t was suggested that surface mannans and serum components were together chemotactic for neutrophils [39]• Confirmation of the chemotactic effect of mannan came from Escobar et a l . [62]. This was followed by the demonstration that mannan induced complement activation through the alternative complement pathway [178,179], obviating the need for the presence of specific anti-C.albicans IgG or IgM. Complement activation results in the production of the C5 component which is responsible for the migration of neutrophils. Support for the value of complement activation came from the observations that C5 was required for the production of the acute neutrophilic responses observed in murine superficial candidiasis [179] and that murine disseminated infection with C.albicans was enhanced i f the alternative complement pathway was depleted [71]. Complement also appears to play a role in enhancing neutrophil phagocytosis i n the presence of anti-C.albicans IgG and i s c r i t i c a l for phagocytosis in the absence of these antibodies [65,156,214]. In addition, in the absence of C3, human neutrophils can ingest but not digest C.albicans cells [247]. B. Specific Defense Mechanisms Specific defense mechanisms are acquired and are dependent upon the development of a specific lymphocytic response that i s directed against C.albicans. This response consists of two antigen recognition-elimination mechanisms: the humoral response and the -11-cell-mediated response. Macrophages also play a role in terms of antigen processing and presentation to lymphocytes. The specific humoral immune system, which involves the production of immunoglobulin by macrophage-stimulated B-lyphocytes, may play a role in host defences against C.albicans, but i t i s of lesser importance than the phagocytic and T-lymphocyte mediated systems [100]. Low levels of anti-Candida IgG in normal human serum have been reported to enhance phagocytosis of Candida pseudohyphae by human neutrophils [ 4 8 ] . However, controversy exists regarding the importance of anti-Candida IgG as opsonins for phagocytosis of Candida c e l l s . The observations that addition of specific rabbit antibodies to an i n vitro system containing C.albicans and mouse neutrophils did not augment the rate of phagocytosis or k i l l i n g , and that high-titers of anti-Candida antibodies may in fact inhibit phagocytosis and k i l l i n g , suggest that immunoglobulins may be of no value [99]. This i s supported by the demonstration that sera from patients with disseminated candidiasis inhibited phagocytosis by human neutrophils [115] and that the defect in neutrophil candidicidal activity in three patients with chronic mucocutaneous candidiasis was mediated by Candida antibodies in the serum [230]. Overall, there i s an uncertainty as to the value of anti-Candida immunoglobulins. Protection from C.albicans correlated more with cell-mediated immunity than with humoral immunity [84,100]. Circulating T-lymphocytes involved in cell-mediated responses are usually inactive but become activated when presented with a specific antigen. Antigen stimulation results in differentiation and proliferation of the -12-T-lytnphocytes. The activated T-cells release various chemical mediators, some of which result i n chemotaxis and activation of macrophages, and the recruitment of additional uncommitted T-cells [183]. Animal studies and recognition of specific defects of cell-mediated immunity (CMI) in patients with chronic mucocutaneous candidiasis [56], or in patients receiving immunosuppressive drugs (individuals with increased susceptibility to Candida infections) [68,l6l], have provided evidence for an important role for T-lymphocytes in defense against Candida infections. However, as with other host defence components (PMN, macrophages and antibodies), there i s uncertainty regarding the value of T-cells. Although immunity to C.albicans has been associated with passive transfer of immune lymphocytes but not with immune serum [153]. nude mice that were deficient in T-cell mediated immunity are actually more resistant to systemic candidiasis [36]. IV. PREDISPOSING FACTORS FOR CANDIDAL INFECTIONS Candida species are opportunistic pathogens and hence are normally of low pathogenicity. The state of the host i s of primary importance as very minor and subtle defects in the host defences could allow these organisms to invade and cause ill n e s s . Various factors can compromise an individual such as : humoral or cellular immunological impairment, age, breakdown of mechanical barriers (skin or mucous membranes) and iatrogenic factors. A. A n t i m i c r o b i a l agents The invasive and disease causing potential of C.albicans i s often -13-related to the magnitude of colonization. The most important environmental factor affecting the degree of colonization of mucosal surfaces by Candida species i s the interaction between Candida species and other microbial flora [212], Indigenous mucosa-associated organisms compete with C.albicans for attachment sites in the intestinal wall. This suppresses C.albicans colonization and dissemination from the gastrointestinal tract [104]. Lactobacilli have been demonstrated to compete with C.albicans for cellular binding sites [197.213] and other organisms may compete for available nutrients such as glucose [109]. Bacterial secretion of toxic products or substances that inhibit Candida adhesin (possibly volatile fatty acids and bil e salts), could also play a role in this suppression of C.albicans [103,104,167]. Antimicrobials, such as tetracyclines and aminoglycosides, that are broad spectrum and inhibit gram negative enteric bacteria, are most l i k e l y to augment colonization with Candida species. Candidiasis has been frequently associated with the use of antimicrobial agents [161,204]. Both human and animal studies have associated antimicrobials with increased C.albicans colonization [85,98]. These drugs may help Candida to colonize through the loss of the suppressive effect of the normal flora [85] or through the decreases in host defences such as leukocyte function (due to the toxic effects of antimicrobials) [66]. B. Cancer A number of malignant hematological conditions involve neutrophil, and T- and B-cell, defects [86,231]. Individuals with -14-these conditions are susceptible to infection by opportunistic organisms, with fungi being a major contributor [81,95.201]. Studies of patients with neutropenia or impaired neutrophil function provide evidence for an important role for neutrophils against Candida. The high incidence of disseminated candidiasis i n leukemics has been associated with neutropenia [15,43,248]. Furthermore, individuals with leukemia have defects in neutrophil phagocytosis and digestion of Candida [125]. Patients with myeloperoxidase deficiency and chronic granulomatous disease, both of which involve isolated defects in neutrophil function, appear to have increased susceptibility to disseminated candidiasis [106,124]. The i n i t i a l defects in neutrophil and lymphocyte responses are further enhanced with cancer chemotherapy. C. Immunosuppressive treatment and cancer treatment Systemic candidiasis occurs in neoplastic patients and individuals such as organ transplant recipients, who have been immunosuppressed with irradiation, corticosteroids and anti-neoplastic drugs [205]. Immunosuppressive drugs like corticosteroids play an important role in the spread of Candida in compromised patients especially i f these drugs are given in combination with antibiotics [81]. This effect of corticosteroids could be due to the suppression of the neutrophilic response to Candida [205]- Irradiation and anti-neoplastic drugs impair the lymphocytic immune response as well as the mononuclear c e l l response. Cancer patients subjected to intermittent chemotherapy f a i r better than those that have continuous treatment [87]. T- and B-cell function i s abolished for prolonged -15-periods during continuous chemotherapy. In contrast, intermittent treatment allows the immune system to recover from the depressant effect, during the periods of rest from chemotherapy [28,87.127]. The prognosis i s often much better for patients who, despite treatment, retain their immunologic responsiveness. In addition, most of the agents used in cancer chemotherapy can cause ulceration of the mouth, esophagus or of the intest inal tract [231]. This effect on the mucosal epithelium, combined with the debil i tated condition of the patient and the antibiotic suppression of the normal microbial population, s ignif icantly influences the frequency of candidiasis in cancer patients [16,61,160,231] and often results in a fatal disseminated infection. D. Disruption of the mechanical barrier Another important factor predisposing to candidal infection i s the mechanical breakdown of normal skin due to trauma or to increased moisture and laceration of the skin. Medical and surgical care procedures have also contributed to the mechanical seeding of Candida either subcutaneously or direct ly into the blood c i rculat ion. Patients who are subjected to the prolonged use of indwelling intravenous catheters, part icular ly with total parenteral nutri t ion (TPN), are at a greater risk for Candida septicemia. Goldman and Maki [77] showed that greater than 53% of septicemias i n patients receiving TPN were fungal infections. This high incidence of infection could be due to contamination of infusion fluids at the time of manufacture or during infusion. Infection could also be a result of the contamination of the cannula at the moment of insertion [42,70,145]. -16-In addition, patients on TPN are usually on broad spectrum antimicrobial agents which eliminate or reduce bacterial competition with the fungus [107]• The incidence of candidemia increases with the length of catheter usage [35.240]. Urinary catheters are associated with transient local fungal infections [199] but occassional ascending infections do occur and can lead to disseminated candidiasis in compromised patients [231]. Drug addicts are at high risk of systemic candidiasis due to the use of contaminated drugs and injection paraphernalia and the use of saliva to ease the injection [231]. Compromised individuals who undergo dental surgery are also at risk of candidiasis. Diabetics [173] and leukemics [45] have died from disseminated candidiasis following teeth extractions. E. Major Surgery Surgery, in particular that involving the alimentary tract, predisposes an individual to systemic candidiasis. Several studies have shown that greater than 57$ of patients develop either systemic or disseminated candidiasis following gastrointestinal surgery [12,78,243]. This can be partly explained in terms of the use of antimicrobials and rupture of mechanical barriers. Surgical patients are often given antimicrobial agents to suppress the bacterial content of the gut prior to the operation. Since the yeast population is unaffected by such treatments, the fungi grow uninhibited and are seeded into the circulation at the time of surgery [231]. Cardiac surgery is the most common iatrogenic factor in Candida endocarditis [206,207,208]. Yeasts may gain access to the circulation -17-during the operation, during intratracheal intubation and through indwelling intravenous catheters, resulting in the deposition of yeasts on traumatized tissues or foreign intracardiac material. The immune responses may also be affected as surgical stress has been found to depress phagocytosis [2] and humoral defence mechanisms [80]. F. Infancy Immune competence varies with age and hence age i s associated with susceptibility to candidiasis. Neonatal infants are not immunologically mature enough to resist yeast present in an infected birth canal [205]• Unborn infants are also susceptible to intrauterine infection. V. TYPES OF CANDIDIASIS Despite the existence of numerous Candida species, C.albicans and C.tropicalis account for the majority of the candidal infections. There are three types of human candidiasis: superficial, locally invasive and deep or systemic. The latter two forms are usually encountered only in immunocompromised individuals. A. Superficial The superficial form i s the most common type of candidal infection. It i s limited to surface linings such as the skin, oropharynx, gastrointestinal tract and upper and lower respiratory tracts. The surface epithelium i s totally or part i a l l y destroyed and hyphae or pseudohyphae may extend into the underlying tissue [133]• In the normal host, superficial candidiasis i s banal and responds -18-rapidly to treatment. A superficial infection that i s unresponsive to treatment may be indicative of abnormalities in the host defense system [205]-B. L o c a l l y Invasive Severe localized invasive candidiasis may occur in a variety of manifestations including pneumonia, c y s t i t i s , esophagitis, endocarditis, pyelonephritis, or intravenous catheter-induced phlebitis. Ulcerations of the intestinal tract, respiratory, or genitourinary tract are the most frequently encountered invasive localized lesions. This type of infection involves invasion beneath the superficial membranes leading to submucosal growth of fungi and necrosis of the overlying mucosa. A subsequent systemic infection may occur in the event that the fungi extend into the deeper layers [133]-C. Systemic (Disseminated) The systemic form of candidiasis i s the most severe of the three types. It i s defined as "an invasive infection involving the parenchyma of two or more visceral organs, excluding the mucosa of the gastrointestinal respiratory or genitourinary tract " [158]. Any organ may be involved but the most common sites are the heart, kidneys, l i v e r , spleen, lung and brain [133]- Almost a l l forms of visceral Candida infection result from hematogenous spread of yeasts. The exceptions are the digestive, respiratory and urogenital tracts in which the organism may invade directly through the epithelial surfaces [161]. The site of hematogenous dissemination and the immune status of the host determines the extent and distribution of organ involvement. -19-Patients most susceptible to systemic candidiasis are : (a) patients with hematologic malignancies; (b) patients who have tumours or other immunosuppressive disease or are on immunosuppressive therapy; and (c) surgical patients (post operative) [102,160]. Patients with disseminated candidiasis have high mortalities unless diagnosis of the fungal infection i s made early. This i s due to the immunocompromised state of the individuals and to the fact that antifungal drugs are ineffective during the latter stages of deep fungal infections. VI. DIAGNOSIS (in compromised individuals) Although a l l three types of candidiasis occur i n compromised patients, only the locally invasive and systemic forms are problematic in terms of diagnosis. The superficial type i s readily diagnosed on the basis of c l i n i c a l symptoms and direct smears and scrapings from lesions [205]. Unfortunately, characteristic c l i n i c a l manifestations are often not apparent in patients with deep fungal infections and serological tests are usually confusing and inadequate for confident diagnosis [92]. A. Isolation of C.albicans Since C.albicans i s part of the normal flora in humans, recovery of this organism from superficial areas or secretions of the body i s not indicative of a deep fungal infection. However, recovery of C.albicans from normally s t e r i l e sites such as blood, cerebrospinal f l u i d or pleural f l u i d i s a reliable indication of infection [63]. -20-Early reliable detection of disseminated candidiasis has not been very successful. As many as 40-60% of the patients with evidence of widespread visceral infection at autopsy, had negative blood cultures [15,81,160] un t i l the terminal stages of the infection, when they became positive. In addition, a positive culture may just represent an intravenous catheter-induced candidemia which could be self limiting [26,59,224,248]. Hence, the compromised patient may be needlessly subjected to toxic antifungal drugs such as amphotericin B, which i s associated with nephrotoxicity [ l 6 l ] . B. Detection of Anti-Candida Antibodies A number of serological tests have been developed for antibody detection including whole-cell agglutination [67,89,113,149], agar-gel diffusion [67,76,89,112,113,139,149], latex agglutination [67,89,113.149], counterimmunoelectrophoresis (CIE) [40,41,79,91.113,138,139,149,159]. indirect immunofluorescence [112], passive hemagglutination (HA) [112], radioimmunoassay (RIA) [31,158] and enzyme immunoassay (EIA) [111,112,146]. Reliable anti-candidal antibody detection i s possible in the immunocompetent host since western blotting with human serum has allowed the demonstration of antibodies to at least 18 different candidal antigens [220]. One of the more commonly used antigens in antibody tests i s the c e l l wall mannan component of Candida species. However, IgG to C.albicans mannan are often seen at similar levels in normal and colonized or infected individuals [110,119,146], indicating a need for a more specific antigen. Cytoplasmic antigens are also employed for antibody detection -21-(CIE and agar-gel diffusion) since i t was hypothesized that the host makes contact with these antigens only during an invasive disease [58]. Unfortunately, contamination of cytoplasmic antigens with mannan during antigen preparation [113] has also limited the use of these antigens. MacDonald and Odds [134,135] have shown that a purified inducible acid proteinase enzyme of C.albicans may be a promising diagnostic antigen. In their studies, patients with deep candidasis had higher titre s of antibodies to this enzyme than patients without proven infection. In general, measurement of antibody titres in immunocompromised patients for diagnosis of disseminated candidiasis has proven so far to be insensitive, as the incidence of false negatives i s 27~70$ in these individuals [67,79,91.159]- This may be partly explained by the fact that many immunocompromised patients are unable to mount an adequate humoral response [67]. In addition to false negatives, false positives are also seen in compromised individuals such as burn patients [89] and patients who have had cardiac surgery [157]-C. Candidal Antigen Assays Assays for direct detection of candidal antigens which do not depend on an adequate host humoral response, have also been developed. Such methods include hemagglutination inhibition [150,234], RIA [218,233], and ELISA [3,128,146,209,232]. The interest in circulating candidal antigens has focused mainly on c e l l wall mannan. The presence of mannan in serum was f i r s t described in 1973 in a patient with chronic mucocutaneous -22-candidiasis[4]. Mannan i s believed to be the principal antigen circulating in patients with candidiasis [233.234]. Unfortunately, there are factors which complicate the detection of mannan in serum. One major problem i s sensitivity, as mannan i s often found in amounts that are near the lower limit of detection of most serological tests (i.e. ng/ml) [44,92]. In addition, antigen clearance from the circulation i s rapid [128]. This clearance in the normal host i s thought to be mediated by anti-candida antibodies and mannan-specific receptor-mediated endocytosis by macrophages [9]. However, there have been reports of antibodies blocking phagocytosis by binding to circulating mannan. This results in soluble immune complexes which must be dissociated in order to get reliable antigen detection [92,223]. Various methods have been developed to dissociate these antigen-antibody complexes [128,180]. Despite the addition of a dissociation step, mannan has been detected in only 50-70% of patients with disseminated candidiasis by ELISA or RIA [146,233]. and even fewer (30-50%) by hemagglutination inhibition [150,234]. Latex particle agglutination (LPA) tests suitable for rapid detection of candidal antigens other than mannan are also available [5.72]. Unfortunately these tests either give a significant number of false negatives [5,22] or are not sensitive enough. These tests do not allow detection of antigen un t i l late in the course of infection (i.e. at approximately two weeks before death of the patient) [5]-A method for the detection of a C.albicans acid proteinases has been developed which involves an ELISA test. This test has a -23-sensitivity of 0.1 ng/ml of the enzyme and, with further investigation, may eventually be a useful diagnostic test [192], D. Gas Liquid Chromatography Gas liquid chromatography has been used to detect and quantitate metabolic products or constituents of Candida in serum of individuals with suspected infection. Miller et a l . [152] showed that serum from patients with deep candidiasis had characteristic GLC profiles which were different from those of patients with bacterial or other fungal infections. This observation was confirmed by Maliwan et a l . [137]• GLC profiles indicate that two candidal metabolites, arabinitol [60,105,186] and mannose [140,154], are present at elevated levels in patients with deep candidiasis. However, patients with impaired renal function [60,105,186], or patients who have blood glucose concentrations greater than 3000 mg/dl (diabetics), have similar raised levels of arabinitol and mannose. To deal with the arabinitol problem, Wong et a l . [245.246], measured the arabinitol/creatinine ratio instead of just the arabinitol value. This was based on the fact that clearance of arabinitol from the kidney i s quantitatively identical to that of creatinine. Even though raised a/c ratios may be indicative of candidal infections, the ratios do not discriminate between superficial and invasive forms of the disease [88]. It appears that the use of GLC for the quantitation of fungal metabolites may aid diagnosis but whether elevated levels can be detected in time to influence c l i n i c a l decisions has not yet been established [92]. -24-£. Biopsies At the present time, diagnosis of deep candidal infections i s ultimately dependent upon obtaining histopathological or microbiological confirmation of tissue invasion. This requires invasive procedures which are problematic, particularly in patients with malignant hematological conditions making biopsy procedures hazardous. -25-VII. PURPOSE OF THIS STUDY In recent years, there has been an increase of fungal infections in hospital patients. Prior to I960, Candida species and related organisms were rare hospital acquired pathogens [15,16]. Since then, these organisms are the fourth most frequently recovered blood-cultured isolate at many major medical institutions. Only 3$ of patients with acute leukemia showed indications of disseminated candidasis at autopsy in the 1940's as opposed to the 30$ since the I960's [15,16,57.81,114]. This increase i s probably due to a number of factors including increased recognition, improved pre and postmortem evaluation, increased use of antibiotics and immunosuppressive treatments, complicated medical and surgical care procedures such as organ transplants, and the higher survival rate of individuals with compromising illnesses. One of the most prominent and pathogenic fungal infections in these individuals i s systemic candidiasis. Such an infection in compromised patients i s often fatal since i t i s d i f f i c u l t to treat and often goes undetected unt i l the terminal stages of the disease. One possible solution to this problem i s early diagnosis with subsequent chemotherapeutic elimination of the organism before the infection overwhelms the compromised patient. Only 15~40$ of patients with invasive candidiasis have premortem diagnosis made early enough for treatment [57]-Since the existing rapid diagnostic methods are inefficient, the objective of this project was to construct labeled DNA probes as a -26-means of rapid diagnosis of candidal infections. The production of these probes could be accomplished by cloning unique fungal DNA sequences and labeling them with radioactive or biochemical molecules for detection. These probes could then be added to patients' blood or tissue samples. Theoretically, i f the patient has an infection (the causative agent being identical to the organism used for the production of the probe), then hybridization of fungal DNA i n the patient's sample with the DNA probe w i l l occur. The labeled hybrid could be detected rapidly using various techniques. The detection procedure of choice i s dependent upon the type of "reporter" molecule used for the labeling of the probe. -27-ABBREVIATIONS 1. BRL : Bethesda Research laboratories 2. DEPC : diethylpyrocarbonate 3. DTT : dithiothreitol 4. EDTA : ethylenediamine tetraacetic acid 5. EtBr : ethidium bromide 6. GITC : guanidinium isothiocyanate 7. NCF : nitrocellulose f i l t e r 8. SDS : sodium dodecyl sulfate 9. DNase : deoxyribonuclease 1 0 . RNase : ribonuclease 1 1 . Tris : tris(hydroxymethyl) aminomethane -28-MATERIALS Cells The C.albicans (strain 10231) cells were originally purchased from the American Type Culture Collection. The c e l l stock was lyophilized and stored at 4°C. A continuous culture was established on Sabouraud cerelose agar plates kept at 4°C and subcultured every two to three months. A l l other fungi used in this study were obtained from Dr. B. D i l l , Department of Microbiology, U.B.C. These cells were stored in Sabouraud cerelose agar tubes covered with paraffin o i l . The mouse 3T3 (CCL 92.1) cells were originally purchased from the American Type Culture Collection. This c e l l type i s a continuous line of 3T3 (Swiss albino) mouse embryo fibroblasts. DNA The Pseudomonas syringae DNA was obtained from Dr. R. Hancock, Department of Microbiology, U.B.C. The Bacillus subtilis phage 29 DNA was obtained from Dr. G. Spiegelman, Department of Microbiology, U.B.C. Lambda DNA was purchased from Bethesda Research Laboratories. -29-Enzymes 1. Alkaline phosphatase : calf intestinal enyzme preparation - purchased from Boehringer Mannheim 2. Chitinase : preparation from Streptomyces griseus - purchased from Sigma Chemical Company 3. DNase 1 (RNase-free) : purified from bovine pancreas - purchased from Bethesda Research Laboratories k. B-Glucuronidase : (B-D-Glucuronide glucuronosohydrolase) preparation from Helix pomatia - purchased from Sigma Chemical Company 5- Lysing Enzyme : preparation from Rhizoctonia solani - purchased from Sigma Chemical Company 6. Lysozyme : (muramidase;mucopeptide) preparation from chicken egg white - purchased from Sigma Chemical Company 7. Lyticase : crude preparation from Arthrobacter luteus - purchased from Sigma Chemical Company -30-8. RNase Tl : purified from Aspergillus oryzae - purchased from Bethesda Research Laboratories 9. Protease : (pronase E) purified from Streptomyces griseus - purchased from Sigma Chemical Company 10. T4 DNA ligase : preparation from E.coli (BNN67) - purchased from Pharmacia 11. Proteinase K : purchased from Bethesda Research Laboratories Miscellaneous Materials 1. Ribosomal RNA 28S and 18S : isolated from Saccharomyces cerevisiae (ATCC 976) - purchased from Pharmacia 2. Nick Translation reagent k i t : purchased from Bethesda Research Laboratories 3. Oligo(dT)-Cellulose Type 7 : purchased from Pharmacia 4. Radioactive deoxynucleotide triphosphates : purchased from Dupont 5. Biotin-ll-UTP : purchased from Bethesda Research Laboratories -31-Solutions (not described in the METHODS section) 1 . Denharts ( 1 0 0 X ) - 2% f i c o l l 2% polyvinylpyrrolidone 2% bovine serum albumin (BSA) 2. Low Tris/EDTA buffer - 1 0 mM tr is-HCl 1 mM tetrasodium salt dihydrate (EDTA) pH 8 3. TBE (gel running buffer) - 89 mM tris-borate 89 mM boric acid 2 mM EDTA (pH 8 . 0 ) 4. STE - 1 0 0 mM NaCl 1 0 mM t r i s - C l (pH 8) 1 mM EDTA 5. Luria-Bertani Medium (LB) - 0.5% Bacto-yeast extract 1 . 0 % Bacto-tryptone 1 . 0 % NaCl •(PH 7.5) 6. Sabouraud Cerelose Broth - 1.0% proteose peptone 4.0% cerelose (D-glucose) (pH 5-3) 7. SSC - 0.150 N NaCl 0.015 M NaoC.H,_0r7(2H_0) 5 o 5 / <L (pH 7-0) 8. TEK - 50 mM KC1 10 mM t r i s - C l (pH 8.1) 1 mM EDTA 9. SM - 100 mM NaCl 80 mM MgS0Zl(7H20 50 mM t r i s - C l (pH 7-5) 0.01% gelatin 10.SMCK - 1.20 M Na2HP04 0.55 M KH2P0jj 11.PBS - 0.13 M NaCl 2.70 mM KC1 8.10 mM Na2HP04(7H20) 1.50 mM KH2P04 1.00 mM CaCl 2 0.50 mM MgCl 2(6H 20) -33-12.Biotin Detection Solutions and Buffers a) Buffer I - 0.1 M t r i s - C l (pH 7.5) 0.1 M NaCl 2.0 mM MgCl (6H 0) 0.05$ (v/v) triton X-100 Buffer II - 3$ (w/v) BSA in buffer I Buffer III - 0.1 M t r i s - C l (pH 9.5) 0.1 M NaCl 0.05 M MgCl (6H 0) b) Solution I - 2.0 X SSC 0.1$ SDS Solution II - 0.20 X SSC 0.1$ SDS Solution III - 0.16 X SSC 0.1$ SDS c) Solution A - 0.1 M NaCl 0.1 M t r i s - C l 3.0 mM MgCl (6H 0) 0.5$ Tween 20 (v/v) (pH 7-5) Solution B - 0.1 M NaCl 0.1 M t r i s - C l 3.0 mM MgCl (6H 0) 0.05$ Tween 20 fv/v) (pH 7-5) Solution C - 0.1 M NaCl 0.1 M t r i s - C l 10.0 mM MgCl (6H 0) (PH 9-6) METHODS ISOLATION OF C.ALBICANS r-RNA AND DNA Protoplast Formation Protoplasts formation was performed as described by Torres-Bauza and Riggsby [226]. Pretreatment : Candida albicans cells were grown overnight at 37° C (C represents celsuis) with aeration in Sabouraud broth. Cells were harvested by centrifligation at 2,200xg for two minutes and washed twice with st e r i l e d i s t i l l e d water. The c e l l pellet was resuspended in 50 mM DTT, 5 mM Na2EDTA, 100 mM t r i s (pH 8.9), and protease (1 mg/ml). The suspension was then incubated at 32° C with gentle agitation for 60 to 70 minutes before harvesting and washing once with s t e r i l e water. Treatment : Cells were washed once with 0.6 M KC1 and then resuspended at 200 mg ( wet wt/ml ) in a solution containing 0.6 M KC1, 0.04 M phosphate buffer (pH 6), 7 mM 2-mercaptoethanol, chitinase (0.25 mg/ml) and /?-glucuronidase (10 units/ml). The c e l l suspension was incubated for two hours at 37° C. Conversion to protoplasts should have occurred within two hours. -35-B. Isolation of rRNA Method I : Protoplasts collected by centrifugation at 2,200xg for 15 minutes and resuspended in 0.25 M sucrose, 1 mM EDTA, and 5 mM tris-HCl (pH 7-5). The c e l l suspension was then homogenized with 15 strokes in a hand homogenizer and allowed to stand for 30 minutes for maximum l y s i s . The pH of the suspension was adjusted to 7-5 [24l] before centrifuging at 7,800xg for 30 minutes to remove whole ce l l s , nuclei, mitochondria and other cellular debris. The supernatant was then centrifuged for another two hours at l40,000xg to collect the crude ribosomes. The pellet was resuspended in 10 mM tris-HCL (pH 7.4), 5 mM Mg acetate, 50 mM NH^Cl and 5 mM 2-mercaptoethanol and c l a r i f i e d at 12,000xg for 10 minutes. The pellet was discarded and the supernatant layered on a 10 ml cushion of 50$ w/v sucrose, 10 mM tris-HCl (pH 7.4), 5 mM Mg acetate, 50 mM NH^Cl, and 5 mM 2-mercaptoethanol. This mixture was centrifuged at 100,000xg for 15 hours to collect the purified ribosomes [10]. The ribosomal pellet was resuspended in 0.01 M CH^COONa, 0.1 M NaCl, 0.001 M MgCl^, and 0.5$ w/v SDS. Hot phenol extraction was used to extract the rRNA from the ribosomes. The aqueous phase from this extraction was combined with two volumes of 95$ ethanol to precipitate the rRNA [171]. Method II : Protoplasts resuspended in PBS were lysed with 8 volumes of 5-7 M GITC, 50 mM lithium citrate, 0.1 M 2-mercaptoethanol, 0-5$ sarkosyl (pH 6.5). and 10 mM EDTA (pH 8.0). Cesium chloride (0.2 g/ml) was added to the lysate which was then placed on a 5-7 M CsCl cushion and centrifuged at -36-132,000xg for 24 hours at 20° C. After centrifugation, the RNA pellet at the bottom of the tube was resuspended in DEPC treated s t e r i l e water and then precipitated with ethanol. The RNA was then passed through an oligo-dT-cellulose column to remove RNA with poly-A t a i l s (mRNA). C. Isolation of DNA Method I : The method used for rRNA isolation involving GITC/CsCl was followed except that the DNA band suspended just above the CsCl cushion was removed instead of the RNA pellet after centrifugation. The DNA fraction was extracted with phenol/chloroform 3_4 times to remove unbroken ce l l s , proteins and other cellular debris. The aqueous fraction from the f i n a l extraction was precipitated with ethanol, treated with of RNase TI (1000 units) for 15 minutes at 37° C, treated with proteinase K (50 ug of enzyme/ml of reaction solution) for 1-2 hours at 55 -65° C, dialysed against low tris-EDTA buffer (10 mM t r i s and 1 mM EDTA) for 24 hours and precipitated with ethanol again to obtain the purified DNA. Method II : C.albicans cells were grown overnight in a 5 ml Sabouraud broth at 37° C with aeration. Cells were washed with 1 M sorbitol and resuspended in 0.5 ml of 1 M sorbitol, 50 mM potassium phosphate (pH 7-5). 14 mM 2-mercaptoethanol, and 25~30 units of lyticase or lysing enzyme. The c e l l suspension was incubated at 30° C for 60 minutes and then sedimented (at 13000xg for three minutes) before resuspending in 0.5 ml of 50 mM Na_EDTA (pH 8.5) and 0.2$ SDS to lyse the protoplasts. The -37-lysed c e l l suspension was then cooled on ice for at least 30 minutes. After cooling, the tubes were centrifuged at 13000xg for 15 minutes. The supernatant was removed, ethanol precipitated and f i n a l l y incubated at 37°C for 15 minutes with RNase Tl (10 units) to obtain RNA-free DNA. Method III : C.albicans cells were prepared and treated with lyticase as described in method II . After the enzyme treatment, the cells were combined with 0.813 volumes of supersaturated (12.2 molal) Nal and then heated at 90° C for 10 minutes before the lysed c e l l suspension was spotted through the minifold apparatus. NUCLEIC ACID QUANTITATION  Method I : A small volume (30-40 ul) of the DNA sample was diluted in water. The optical density (260 nm) of this diluted sample was obtained from a Gilford spectrophotometer 250. The DNA concentration of the original sample was calculated on the assumption that one O.D. unit was equal to 50 ug/ml of DNA solution [33c]. Method II : The ethidium bromide fluorescent quantitation of DNA was performed as described in the Cold Spring Harbor Molecular Biology manual [33c]. Briefly, ethidium bromide was added (1 ug/ml of sample) to a small volume of the DNA sample. The sample was then diluted to form various concentrations before spotting (in 10 ul volumes) onto Saran wrap. Upon viewing over an ultra-violet light box, fluorescence of varying -38-intensities was seen as a result of the interchelation of ethidium bromide into the DNA. The concentration of the DNA spots was determined by comparing their intensities with those of a series of identically treated lambda DNA solutions (commercially obtained) of known concentrations. I I I . LARGE SCALE AMPLIFICATION AND ISOLATION OF COSMID DNA The large scale amplification and isolation of cosmid DNA was performed as described in the Cold Spring Habour manual [33a]- Cosmid pJB8 i s 5-2 kilobases in length and has a Bam HI site within i t s ampicillin resistance gene. It i s possible to insert a 30 - 4 0 Kb nucleic acid fragment into this cosmid to produce a 35-50 Kb recombinant which i s preferentially packaged into a lambda phage head and used for transfection. E.coli containing cosmid pJB8 was inoculated into 5 mis of Luria-Bertani (LB) medium with ampicillin and incubated overnight at 37° C with aeration. Two mi l l i l e t e r s of this 5 ml culture was used to inoculate 500 mis of LB medium containing ampicillin ( 4 0 ug/ml). After incubating for 2.5 hours ( O . D . ^ Q Q approx. 0 . 4 ) , chloramphenicol (170 ug/ml) was added and incubated at 37° C for a further 12-16 hours. The bacterial cells were harvested by centrifugation at 2,300 rpm for 10 minutes at 4 ° C and washed with ice-cold STE (0.1 M NaCl, 10 mM t r i s pH 7-8, and 1 mM EDTA). The cellular pellet was resuspended in a 10 ml solution of lysozyme (5 mg/ml), 50 mM glucose, 25 mM tris-HCl (pH 8), and 10 mM EDTA. This suspension was then transfered to a Beckman SW27 polyallomer tube and allowed to stand for 5 minutes at room temperature - 3 9 -before the addition of 20 mis of 0.2 N NaOH and 1% SDS. After mixing, the tube was placed on ice for 10 minutes and then combined with 15 mis of ice-cold 5 M potassium acetate (pH 4.8). The tube was cooled on ice for another 10 minutes before centrifugation at 52,800xg for 20 minutes. The cellular DNA and bacterial debris were pelleted out and the supernatant containing the cosmids was precipitated with 0.6 volumes of isopropanol. The cosmid pellet was washed with 70$ ethanol (to remove salts) and dissolved in low tris-EDTA buffer (10 mM t r i s and 1 mM EDTA). This cosmid preparation was purified by layering onto a CsCl density gradient. After centrifuging for 40 hours at 130,000xg (20° C), two bands were visible (due to the interchelation of ethidium bromide into the DNA helices). The top band was the contaminating cellular DNA (nicked circular or linear DNA) and the bottom band was the cosmid DNA (closed circular DNA). The bottom band was aspirated from the gradient and extracted with iso-amyl alcohol to remove the EtBr. This purified cosmid preparation was then dialysed in low tris-EDTA buffer to remove salts and f i n a l l y ethanol precipitated to concentrate the cosmid preparation. IV. LARGE SCALE AMPLIFICATION AND ISOLATION OF PLASMID Plasmid pACYC 184 i s 4.0 kilobases in length and has chloramphenicol and tetracycline resistance genes. Within the tetracycline gene, there are restriction enzyme sites (eg. Bam HI and Hind III) which could be used to insert up to 16 Kb nuclei acid fragments and s t i l l have efficient transformation. -40-The amplification and isolation procedures for pACYC 184 are the same as those used for the cosmid pJB8 except for the types of antibiotics used. For the plasmid, chloramphenicol (10 ug/ml) was added to the growth medium instead of ampicillin which was used for the cosmid. In addition, for the amplification of the plasmid, streptomycin was used to interfere with cellular protein synthesis (as opposed to the use of chloramphenicol in the case of the cosmid). V. SMALL SCALE AMPLIFICATION AND ISOLATION OF PLASMID The small scale amplification of plasmid was performed as described in the Cold Spring Habour manual. E.coli containing plasmid pACYC 184 was inoculated into 5 mis of LB medium with chloramphenicol (10 ug/ml) and incubated overnight at 37° C with aeration. A small volume (1.5 ml) of this culture was centrifuged at 13,000xg for one minute and the resulting pellet was resuspended in 100 ul of an ice-cold solution of 50 mM glucose, 10 mM EDTA, 25 mM tris-HCl (pH 8), and lysozyme (4 mg/ml). This suspension was l e f t at room temperature for 5 minutes before adding 200 ul of an ice-cold solution of 0.2 N NaOH and 1% SDS. After mixing, the suspension was stored on ice for 5 minutes and then 150 ul of an ice-cold solution of 5 M KAc (pH 4.8) was added. The resulting suspension was mixed by vortexing the tube in an inverted position for 10 seconds. After storing on ice for 5 minutes, the suspension was centrifuged for 5 minutes at 13,000xg to pellet out the cellular DNA and debris. The supernatant was then extracted with phenol/chloroform twice and ethanol precipitated at room temperature for 5 minutes. The pellet from this precipitation was washed once with 70% ethanol, dried in a -41-vacuum d e s i c c a t o r , d i s s o l v e d i n low t r i s - E D T A b u f f e r and t r e a t e d w i t h RNase T I b e f o r e s t o r i n g . V I . AGAROSE GEL ELECTROPHORESIS A. Denaturing Gel For rRNA I s o l a t e d rRNAs were d e n a t u r e d by i n c u b a t i n g a t 55° C f o r 15 minutes i n the p r e s e n c e o f 17$ formaldehyde and 50$ formamide. A f t e r i n c u b a t i o n , the rRNAs were e l e c t r o p h o r e s e d on a 1$ agarose formaldehyde g e l u s i n g a s o l u t i o n c o n t a i n i n g 0.2 M MOPS (pH 7), 50 mM sodium a c e t a t e , 1 mM EDTA (pH 8) and e t h i d i u m bromide (0.5 ug/ml), as the r u n n i n g b u f f e r . The p r o g r e s s i o n o f t h e rRNA th r o u g h the g e l was d e t e c t e d w i t h 0.04$ bromophenol b l u e and 0.4$ x y l e n e c y a n o l . A f t e r e l e c t r o p h o r e s i s , the g e l was washed w i t h two changes o f 0.1 M ammonium a c e t a t e f o r one hour and then s t a i n e d f o r one hour w i t h E t B r (0.5 ug/ml) i n 0.1 M ammonium a c e t a t e s o l u t i o n . Exposure o f g e l t o UV l i g h t p e r m i t t e d v i s u a l i z a t i o n o f rRNA bands. B. Gel For DNA I s o l a t e d DNA was e l e c t r o p h o r e s e d on a 0.7$ ( n o n - d e n a t u r i n g ) agarose g e l u s i n g a s o l u t i o n c o n t a i n i n g 89 mM t r i s - b o r a t e (pH 8.5), 89 mM b o r i c a c i d , 2 mM EDTA and E t B r (0.5 ug/ml) as t h e r u n n i n g b u f f e r . V I I . CLONING WITH COSMID pJB8 A. P r e p a r a t i o n o f Vector DNA Cosmid pJB8 d i g e s t e d w i t h 2-3X e x c e s s o f r e s t r i c t i o n e n d o n u c l e a s e Bam H I and r a n on a 0.7$ agarose g e l t o check e x t e n t o f d i g e s t i o n . The -in-digested cosmid was extracted with phenol/chloroform, chloroform, ethanol precipitated and resuspended in low tris-EDTA buffer (pH 8). The linear cosmids were then treated with calf intestinal alkaline phosphatase to prevent reannealing of cosmid ends. The cosmids were again extracted with phenol/chloroform and chloroform before passing through a spun column of sephadex G-50 to remove unincorporated phosphates. The eluate was ethanol (95%) precipitated overnight, washed with 70% ethanol and resuspended in low tris-EDTA buffer (pH 8). The Sephadex G-50 column was prepared by packing Sephadex G-50 into a 1.0 ml tuberculin syringe. The column was washed twice with STE (pH 8). A f i n a l wash with TE (pH 7-5) was performed prior to the addition of the cosmid sample to the top of the Sephadex column. The loaded column was then centrifuged for three minutes in a HN-S centrifuge at 5,000xg. Passage of the cosmid sample through the column during centrifugation, resulted in the adherence of unincorporated phosphates to the sephadex G-50 molecules. B. Preparation of C.albicans DNA for cloning Candida albicans DNA was digested with restriction endonuclease Mbo I (1 unit/ug DNA) at 37° C for 30 seconds to generate a range of fragments 23 kbp or under. It was expected that some of these fragments would ligate to form fragments of 30-40 Kb (suitable size for insertion in cosmid) with single stranded t a i l s generated by the Mbo I digestion. The C.albicans fragments were extracted with phenol/chloroform, chloroform, precipitated with (95%) ethanol, washed with 70% ethanol and -43-resuspended in s t e r i l e water. The DNA was electrophoresed on a 1$ agarose gel to check the size of the digested fragments. C. Ligation of Cosmid DNA and C.albicans Fragments Restriction endonucleases Bam HI and Mbo I generate complementary nucleic acid sequences at the site of cleavage. Hence, Bam HI cleaved cosmids and Mbo I cleaved C.albicans DNA were ligated together with T4 DNA ligase at 15° C for 25~50 hours. The ligated DNA preparation was electrophoresed on a 0.7$ agarose gel to check the sizes of the recombinant cosmids. D. Packaging Extracts For Transfection In vitro E.coli C Su /Lambda packaging extracts were produced in our laboratory according to the method described by Rosenberg et a l . [187]. E. T r i a l Packaging and Transfection of Lambda DNA i n E . c o l i C600 Indicator bacteria (E.coli C600) were grown overnight in LB medium containing 0.4$ maltose to generate lambda phage receptors on the bacterial membrane. Bacteria were washed once with SM solution and resuspended in SM. Sheared or unsheared lambda DNA was added to a frozen packaging extract. This was followed by the addition of TEK solution and gentle mixing with a glass rod. The mixture was then incubated at 28° C for 90 minutes before the addition of DNase (10 ug/ml) and SMCK solution. The resulting mixture was incubated for a further 15 minutes at 37° C, treated with chloroform and vortexed to eliminate clumps. Serial dilutions of the packaged cosmids were -44-combined with bacteria in the SM solution and incubated at 37 C for 20 minutes to allow transfection to occur. After incubation, transfected bacteria added to soft agar (45° C) and poured immediately onto LB agarose plates. The s o l i d i f i e d plates were incubated at 37° C overnight to allow for lambda plaque formation. F. Packaging and Transfection of Recombinant Cosmids into E.coli Recombinant cosmids were packaged as per packaging method used for lambda DNA. The packaged recombinant cosmids were combined with E.coli (at a ratio of 1:2 in terms of recombinant to bacteria) and incubated at 37° C for 20 minutes. After this incubation, 1 ml of LB medium (37° C) was added to the infected culture and incubated for a further 45 minutes at 37° C to allow expression of the ampicillin resistance gene. The cells from this culture were then spread plated onto ampicillin (30 ug/ml) agar plates. The plates were incubated at 37° C u n t i l colonies appeared. VIII. CLONING WITH PLASMID pACYC 184 A. Preparation of Vector DNA Plasmid pACYC 184 was cut with the restriction endonuclease Hind III and electrophoresed in 0.7% agarose gel to check extent of digestion. The digested plasmid was extracted with phenol/chloroform, chloroform, precipitated with ethanol (95%). washed with 70% ethanol and resuspended in low tris-EDTA buffer (pH 8). -45-B. Preparation of C.albicans DNA for cloning C.albicans DNA was digested with restriction endonuclease Hind III (1 uni t/ug DNA) at 37 C for one hour to generate random sized fragments. The C.albicans DNA fragments were extracted with phenol/chloroform, chloroform, precipitated with ethanol, washed with 70$ ethanol and resuspended in st e r i l e water. C. Ligation of Plasmid and C.albicans DNA Fragments Since both the vector and target DNAs were digested with Hind III, the two types of DNA had complementary nucleic acid sequences at the ends of the fragments. Hence the plasmid and C.albicans fragments were ligated together with T4 DNA ligase at 17° C overnight. The ligated preparations were electrophoresed in 0.7$ agarose gels to check for recombinant plasmids. D. Production of Competent E.coli Cells For Transformation F i f t y m i l l i l e t e r s of LB broth was inoculated with 1 ml of an overnight culture of E.coli (DH1). Cells were grown at 37° C with vigorous y shaking to a density of approximately 5 x 10 cells/ml (O.D. of 0.4-0.5). The culture was chilled on ice for 10 minutes and then centrifuged at 5.800xg for 5 minutes at 4° C. The c e l l pellet was resuspended in half the original culture volume of an ice-cold solution of 100 mM CaCl 2 and 10 mM t r i s - C l (pH 8). After s i t t i n g on ice for 15 minutes, the suspension was centrifuged at 5.800xg for 5 minutes at 4° C and this time, i t was resuspended in 1/15 of the original volume of an ice-cold solution of 100 mM CaCl_ and 10 mM t r i s - C l . This -46-concentrated c e l l suspension was then dispensed in 0.2 ml aliquots into pre-chilled tubes and stored at 4° C for 12-24 hours before use. Transformation of Recombinant Plasmids into Competent E.coli (DH1) Cells Ligated plasmids (40 ng) were added to 0.2 ml of competent cells and mixed gently before storing on ice for 45-60 minutes. The transformation mixture was then transferred to a 42° C waterbath for four minutes, after which one ml of. LB broth was added before incubating at 37° C for at least one hour to allow expression of the chloramphenicol resistance gene. The cells from this culture were then spread plated onto chloramphenicol (10 ug/ml) agar plates. These plates were incubated at 37° C u n t i l colonies appeared. Screening Colonies For the Recombinant Plasmids E.coli cells which grew on the chloramphenicol plates had taken up either a non-recombinant or a recombinant pACYC 184 plasmid. Therefore to screen for the cells containing the recombinant plasmids, colonies from the chloramphenicol plates were picked and spotted onto tetracycline (12 ug/ml) agar plates. Since the Hind III site i s situated within the tetracycline resistance gene, recombinant plasmids have a defective tetracycline gene and hence the cells with the recombinants w i l l not grow on tetracycline plates. However, there is also the possibility that a plasmid reannealed to i t s e l f in such a fashion that the tetracycline gene i s inactivated even though the plasmid does not contain an insert. To discriminate between the latter two types of plasmids, the colonies which grew on the chloramphenicol -47-plates but not on the tetracycline plates were selected and put through the small scale isolation procedures. The isolated plasmids were digested with Hind III and electrophoresed in a 0.7% agarose gel. Nonrecombinant plasmids with the inactivated tetracycline gene were seen as one single linear plasmid band on the gel whereas the recombinants appeared as two bands. One band was the linear plasmid and the second band consisted of the inserted C.albicans DNA. IX. PROBE PRODUCTION Deoxyribonucleic acid molecules (0.5 _1«0 ug aliquots) were labeled with the use of a BRL nick translation k i t . Biotinylated dUTP or 32 P-dTTP was used, in combination with unlabeled dCTP, dGTP, and dATP, as substrates for the DNA polymerase in the nick translation k i t . The labeled DNA's were put through a spun column of Sephadex G-50 to remove unincorporated nucleotides (as described in VIII-A of this section). 32 The act i v i t i e s of the P probes were determined by s c i n t i l l a t i o n counting of one ul volumes of each probe solution. X. DNA SPOTTING ONTO NITROCELLULOSE Method I A nitrocellulose f i l t e r (NCF) was soaked in either 2X, 4X, 6X, or 8X SSC before being spotted with 5 ul volumes of DNA solution. The NCF was allowed to dry before the DNA spots were denatured with 0-5 M NaOH for 7 minutes. After denaturation, the NCF was soaked twice in 0.6 M NaCl / 1 M t r i s (pH 6.8) for one minute each time and once in 1.5 M NaCl / 0.5 M -48-t r i s (pH 7-4) for 5 minutes. The NCF was then dried and baked at 80° C for at least two hours after which the DNA was irreversibily bound to the NCF. Method II DNA suspended in TE (pH 7) buffer was combined with 0.3 volumes of 1 M NaOH and incubated at 70° C for 30 minutes to denature DNA. The denatured DNA was neutralized with one volume of 2 M NH^ OAc (pH 1) and kept on ice u n t i l spotted onto a NCF using a minifold apparatus. The minifold was attached to a vacuumn pump so that the DNA solution was sucked through the f i l t e r leaving the DNA attached to the f i l t e r in a concentrated defined spot. After spotting, the NCF was washed in 6X SSC brief l y and dried before baking for at least two hours at 80° C. XI. HYBRIDIZATION OF PROBES TO DNA ON NFC A. Prehybridization Prehybridization was done to reduce nonspecific hybridization of probe by f i l l i n g in the unoccupied spaces on the DNA spotted NCF with salmon testes DNA. The prehybridization solution consisted of 45% formamide, 5X SSC, 5X Denhardts, 0.25 M phosphate ions (pH 6), and heat denatured salmon testes DNA (5 mg/ml of hybridization solution). The prepared NCF (as described in method II) was soaked in the prehybridization mixture for one to two hours at 42° C. B. Hybridization After prehybridization, the NCF was placed in a solution containing 45% formamide, 5X SSC, IX Denhardts, 0.25 M phosphate ions (pH 6), and heat -4g-denatured salmon testes DNA (5 mg/ ml of solution) and labeled probe D N A (0.3-0.7 ug/ml). Hybridization was allowed to proceed at e i t h e r 20°, 30°, 42° or 50° c e l s i u s f o r at l e a s t 16 hours. C. Post Hybridization Washes Stringency of h y b r i d i z a t i o n was regulated by post h y b r i d i z a t i o n washes. The NCF was washed twice with s o l u t i o n I f o r three minutes (each time) at room temperature, twice with s o l u t i o n II f o r three minutes at room temperature and twice with s o l u t i o n I I I f o r 15 minutes at 50° C. The decreasing s a l t concentration of each successive s o l u t i o n and the increased temperature increases the stringency of h y b r i d i z a t i o n . The NCF was given one f i n a l r i n s e with 2X SSC at room temperature before the a p p l i c a t i o n of detection procedures. XII. DETECTION OF HYBRID ON NCF A. Detection of Radioisotope-Labeled Probes The d r i e d NCF was exposed onto Kodak X-0MAT AR X-ray f i l m at -70°C f o r various time periods. B. Detection of Biotin-Labeled Probes Method I : O r i g i n a l BRL method The n i t r o c e l l u l o s e f i l t e r that had undergone the post-hybridization washes was washed i n buffer I f o r one minute, incubated i n buff e r II at 42° C f o r 20 minutes and then incubated with gentle a g i t a t i o n i n buffe r I containing s t r e p a v i d i n (2 ug/ml) f o r 10 minutes. The s t r e p t a v i d i n has strong a f f i n i t y f o r b i o t i n and hence i t attaches to the -50-biotinylated DNA hybrids on the NCF. After the streptavidin incubation, the f i l t e r was washed (three times) with an excess of buffer I at room temperature before incubating with biotinylated calf intestinal alkaline phosphatase (AP) at a concentration of 1 ug/ml for 10 minutes. The AP binds to the streptavidin (SA) resulting in DNA-biotin-SA-biotin-AP complexes. The NCF was again washed (two times) with buffer I for three minutes before i t was washed (two times) with buffer III for three minutes. The biotinylated DNA was visualized by incubating the f i l t e r i n a solution of buffer III, nitro-blue tetrazolium (33 ug/ml of solution), and 5-bromo-4-chloro-3-indolyl phosphate (18 ug/ml of solution) for up to four hours. After colour development, the f i l t e r was rinsed with 20 mM t r i s (pH 7«5) and 5 m M EDTA and then dried. Method II : Chan et a l . Method [27] The NCF that had undergone the post-hybridization washes was washed in solution A for 90 minutes with vigorous shaking, and then incubated for 10 minutes in solution B containing SA (2 ug/ml of solution). After the SA incubation, the f i l t e r was washed in solution A for 5 minutes (three times) before incubating for 10 minutes in solution B containing AP (1 ug/ml). This was followed by three (times) 5 minute washes in solution A and than two (times) 5 minute washes in solution C. The remaining steps of this procedure are the same the those of the original BRL method. Method III : Blugene BRL Method The NCF that had undergone the post-hybridization washes was washed in buffer A for one minute , incubated in buffer B at 65° C for one hour -51-and then incubated with gentle agitation in buffer A containing SA-AP conjugates (1 ug/ml) for 10 minutes. In this method the SA and the AP are added together and not separately as in method I. After incubation, the f i l t e r was washed (twice) with buffer A for 15 minutes and then washed once in buffer C for 10 minutes. The biotinylated DNA was visualized by incubating the f i l t e r in a solution of buffer C, nitro-blue tetrazolium (33 ug/ml of solution), and 5-bromo-4-chloro-3-indolyl phosphate (18 ug/ml of solution) for 20-120 minutes. After colour development, the NCF was rinsed with 20 mM t r i s (pH 7-5), and 5 mM EDTA and dried. XIII. CELL QUANTIFICATION Method I Serial dilutions of the C.albicans c e l l culture were made with PBS and loaded onto a hemacytometer. Cells were counted under a light microscope at the 40X magnification Method II Serial dilutions of the c e l l culture were made with PBS and 100 ul volumes were spread plated in duplicates onto Sabouraud agar plates (pH 5.4). The plates were incubated overnight at 37° C and the colonies were then counted. XIV. GROWTH CURVE OF C.ALBICANS A flask containing 100 mis of Sabouraud broth was inoculated with 1.0 ml of a 5 ml overnight culture of C.albicans c e l l s . A small volume (0.7 ml) was removed from the flask after inoculation and checked for -52-i t s optical density (O.D.) value. The flask was incubated at 37 C with vigorous shaking (1500 rpm). At various time intervals (total time up to 21 hours), a 0.7 ml volume of culture was removed from the flask for a O.D. reading. Small volumes were also removed for colony counting and DNA extraction. DNA extraction was performed as described i n isolation of DNA-method II. XV. TREATMENT OF PATIENT SAMPLES Serum specimens from cancer patients were obtained through Dr. John Smith at the Vancouver General Hospital. The status of these patients in terms of the presence or absence of candidiasis was unknown at the time of sampling. A small volume (1.3 ml) of each patient's serum specimen was centrifuged at ll.OOOxg for four minutes to pellet out any C.albicans cells that might be present. The pellet was given the lyticase l y s i s treatment mentioned earlier [Isolation of DNA-method II ] and the supernatant was ethanol precipitated in the event that there was naked DNA present. Prior to ethanol precipitation of the supernatant, the supernant was diluted 4 times with EDTA-PBS and boiled for 10 minutes to gelatinize serum proteins which may interfere with DNA precipitation. The liquid portion of this sample was removed and then ethanol precipitated and resuspended in low tris-EDTA buffer. -53-RESULTS Large Scale Isolation of C.albicans rRNA and DNA The fungal c e l l C.albicans i s enclosed within a r i g i d c e l l wall consisting of glucan, chitin and proteins. The presence of this r i g i d structure makes the conventional methods for nucleic acid extraction from bacterial or mammalian cells insufficient for C.albicans. The extraction methods used in these studies involved the formation of protoplasts with enzymes (protease, -glucuronidase and chitinase), prior to the physical or chemical disruption of ce l l s . Several attempts at various procedures for 10 large scale (greater than 2 X 10 cells) l y s i s of protoplasts, indicated that the GITC/CsCl centrifugation method (Isolation of DNA-method I) was the most eff i c i e n t , with kj% c e l l l y s i s (according to c e l l counts). Ribonucleic acid i s significantly more labile than DNA because of i t s greater susceptibility to hydrolysis and degradation by nucleases. Cell death ultimately results i n contact between RNases and RNA. Hence, isolation of RNA i s d i f f i c u l t in the absence of RNase inhibitors. The fact that GITC i s a nuclease (DNase and RNase) inactivator as well as a potent chaotropic agent [29,3^], made i t an attractive reagent for RNA extraction. The GITC/CsCl gradient method allowed for simultaneous extraction of separate RNA and DNA fractions. The buoyant density of RNA in CsCl i s much greater than that of other cellular macromolecules [33b]. After centrifugation, the RNA formed a pellet on the bottom of the tube. The majority of the DNA floated just above the CsCl cushion. Agarose gel electrophoresis of various C.albicans DNA preparations, -54-together with commercial lambda DNA molecular weight standards, indicated molecular weights of greater than 23 kbp for C.albicans DNA Agarose gel electrophoresis of various C.albicans rRNA preparations, together with commercial yeast 0.72 Md (18S) and 1.2 Md (28S) rRNA showed RNA molecules below the 0.72 Md band. This suggested that digestion of rRNA had occurred. Undigested rRNA was not obtained even after several attempts with this extraction procedure. Eventually, larger rRNA molecules were obtained after including a freezing (liquid nitrogen) and pulverizing step prior to centrifugation in the GITC/CsCl gradient. Small Scale Isolation of yeast DNA The DNA used for cloning was extracted by means of the GITC/CsCl gradient method. This procedure required at least two to three days to complete and involved multiple steps during which DNA loss could have occurred. Since this method was not suitable for rapid and efficient small scale DNA isolation, another procedure (method II) was used for a l l the yeast DNA extractions other than those used for cloning. I n i t i a l l y , this small scale isolation method resulted in 90% l y s i s of C.albicans while u t i l i z i n g SDS and only one enzyme preparation (lyticase). However, subsequent experiments with this method produced much lower percentages (10-34%) of c e l l l y s i s . This discrepancy may be due to inaccurate c e l l counts (refer to the discussion section). DNA Cloning with Cosmid pJB8 Several attempts at packaging different ligated mixtures (recombinant cosmid-Ampr) into phage heads and subsequent transfection into E.coli C 6 0 0 -55-yielded no colonies from ampicillin agar plates. Switching the transfection recipient to E.coli DH1 had no effect (no colonies). DNA Cloning with pACYC 184 The failure to clone C.albicans DNA with cosmid pJB8 led to "shot-gun" cloning with plasmid pACYC 184 (Cm1* Tet r). Transformation of E.coli DH1 with the ligated C.albicans DNA and pACYC 184 mixture produced a total of 325 colonies from 21 chloramphenicol agar plates. Results from the screening of these colonies with tetracycline agar plates indicated that 20/325 colonies haboured either a recombinant plasmid (insertional inactivation of the tetracycline gene), or a nonrecombinant plasmid with a defective tetracycline gene. Plasmids isolated from these 20 clones were digested with Hind III to cleave the plasmid at the site of possible insertion (tetracycline gene). Subsequent agarose gel electrophoresis revealed the presence of only 7 clones with recombinant plasmids. Of the 1 different recombinants, two had C.albicans DNA inserts of less than 4.3 kbp and five had inserts of greater than 4.3 kbp , but less than 6.4 kbp (Figure 1). The value of 4.3 kbp i s used here because the lambda (molecular weight standard) DNA band nearest the plasmid band on the gel is 4.3 kbp. DNA Denaturation and Spotting onto NCF DNA denaturation and spotting onto nitrocellulose in the i n i t i a l experiments were performed as stated in method I. The switch to method II (minifold apparatus) was made because i t offered several advantages over the previous method. The minifold apparatus method produced compact, distinct -56-FIGURE 1 : RECOMBINANT PLASMIDS Agarose gel electrophoresis of recombinant plasmid isolates. A l l DNA digestions were performed with Hind III. The arrow indicates the linear pACYC 184 plasmid (4.0 kb) bands. The Lambda band closest to the arrow (4.3 kbp) was used as the molecular weight marker for the plasmid and inserts. 1) digested Lambda DNA - molecular weight markers 2) undigested pACYC 184 - closed circular DNA (top band) - supercoiled DNA (bottom band) 3) digested (linear) pACYC 184 - nonrecombinant 4) digested recombinant #17 ( undigested plasmid) 5) " " #70 ( <4.3 kb ) 6) " " #18 ( >4.3 kb ) 7) " " #60 ( " ) 8) " " #79 ( " ) 9) " " #74 ( " ) 10) " " #22 ( <4.3 kb ) 11) '* " #66 ( >4.3 kb ) Plasmid #17 was probably undigested due to the accidental omission of enzyme in the digestion mixture. -57-Recombinant Plasmids 12 3 4 5 6 7 8 9 IO II -58-and evenly spaced spots on the NCF. In addition, the DNA was denatured prior to spotting through the minifold apparatus. This avoided the possib i l i t y of contamination of DNA spots or loss of DNA from the f i l t e r , which could occur i f DNA denaturation was accomplished on the NCF (method I). These factors a l l contributed to reduce background signals and to improved sensitivity of the system. Comparison of Small Scale Cell Lysis Methods Small scale isolation of C.albicans DNA with lyticase and SDS treatment (DNA isolation-method II), and eventual spotting of DNA onto the NCF, involved two separate procedures: In an effort to combine the two procedures, SDS was omitted and 12.2 molal Nal was incorporated into the lyticase treatment procedure (method III). By means of this method, the lysi s solution was spotted through the minifold directly after the addition of Nal. Nal i s a chaotropic salt and an effective solvent for macromolecules which simultaneously promotes DNA denaturation and DNA-NCF interactions [20]. The results shown in figure 2 indicated that the lyticase and SDS treatment procedure recovered more DNA than the lyticase and Nal procedure. The negative control groups (without C.albicans cells) did not show any hybridization signals. Cross Reaction of Probes with Other Yeast DNAs and unrelated DNAs In order to determine the specificity of the recombinant C.albicans probes, i t was necessary to establish the extent of their cross hybridization with other yeast DNAs and DNAs of unrelated organisms. The -59-FIGURE 2 : METHODS OF CELL LYSIS Equal numbers of cells were treated with i) lyticase and SDS; i i ) proteinase K, freezing/thawing, and 12.2 molal Nal; i i i ) proteinase K, and 12.2 molal Nal; iv) lyticase and 12.2 molal Nal. PBS instead of c e l l culture was used in the negative controls. Three different concentrations of each treatment group were spotted through the minifold. The membrane bound DNAs were probed with biotin-labeled DNA's (0.2 ug DNA/ml of reaction mixture). -60-METHODS OF CELL LYSIS B-CAN B-66 CELL TREATMENT Lyticase • SDS • • • • • • • • « w A B C A B C Prot K • F/T • Nal Prot K • Nal Lyticase • Nal Controls Controls : A. Nal • PBS B. Proteinase K + Nal + PBS C. Sorbitol • Nal + PBS Test Spots : A). 8 . 5 X 1 0 ? c e l l s B) . 4.3 X 1 0 ; c e l l s C) . 2 . 8 X 1 0 c e l l s -61-results of two cross hybridization experiments are shown in figures 3. 4 and 5-The C.albicans whole c e l l DNA probe hybridized well with the C.albicans DNA spot and the C.intermedia DNA spot (figures 3 and 5). The hybridization signal of the C.intermedia DNA spot was almost as intense as the C.albicans DNA spot. The C.albicans whole c e l l DNA probe also hybridized slightly with a l l the other yeast DNAs in this study. However, this probe did not hybridize with mouse (3T3) DNA, pACYC 184, Aspergillus fumigatus DNA, Pseudomonas syringae DNA or Bacillus subtilis phage 29 DNA (figures 3 and 5). The recombinant #66 probe hybridized well with DNAs from pACYC 184 (as expected - refer to discussion), C.albicans, C.intermedia, C.tropicalis and C.pseudotropicalis (figure 3)- Less intense spots were seen with Hansenula anomala, Saccharomyces cerevisiae, C . u t i l i s , C.guillieromondii and C.santamariae (figure 3). Similar results were obtained with the recombinant #70 probe. However, recombinant #70 reacted more strongly with S.cerevisiae and C.guilliermondii DNA than did recombinant #66 (figure 5)• Significant hybridization signals were obtained with the DNAs of C.tropicalis, C.albicans, C.pseudotropicalis, and C.intermedia, hybridized to either recombinant #74 or #22 (figure 3)• These two probes produced weak signals with H.anomala and C.guilliermondii. There were no cross hybridizations between recombinant probes and any non-yeast DNA such as Aspergillus fumigatus, mouse (3T3). Pseudomonas  syringae and Bacillus subtilis phage 29. There was also no significant hybridization by the nonrecombinant pACYC 184 probe with any yeast or other -62-FIGURE 3 : CROSS REACTIONS BETWEEN RECOMBINANT CANDIDA PROBES AND OTHER YEAST AND UNRELATED DNA Thirteen different DNAs were spotted through the minifold 32 apparatus. F i l t e r bound DNAs were hybridized with P-labeled probes (4.5 X 10 cpm/ ug DNA). Fi l t e r s were subsequently exposed to X-ray film for 24 hours. - 6 3 -CROSS REACTION BETWEEN RECOMBINANT CANDIDA PROBES AND OTHER YEAST AND UNRELATED DNA'S 1. C.albicans 8. C.guilliermondii 2. A.fumigatus 9. C.intermedia 3. Mouse 3T3 10. C.santamariae 4. H.anomala 11. C.pseudotropicalis 5. S.cerevisiae 12. C.tropicalis 6. K.marxianus 13. Water 7. C.u t i l i s 14. pACYC - 6 4 -FIGURE 4 : CROSS REACTIONS BETWEEN RECOMBINANT CANDIDA PROBES AND OTHER YEAST AND UNRELATED DNA Fifteen different DNAs were spotted in 10 ng amounts through 32 minifold apparatus. F i l t e r bound DNAs were hybridized with P-labeled 5 probes (2.3 X 10 cpm/ug DNA). Fi l t e r s were subsequently exposed to X-ray film for three hours. -65-CROSS REACTION BETWEEN RECOMBINANT CANDIDA PROBES AND OTHER YEAST AND UNRELATED DNA'S SPOTS : 1. C.albicans 2. H.anomala 3- S.cerevisiae 4. K.marxianus 5. C . u t i l i s 6. C.guilliermondii 7- C.intermedia 8 . C.santamariae 9. C.pseudotropicalis 10. C.tropicalis 11. A.fumigatus 12. Mouse 3T3 13. P.syringae 14. Bacillus phage 29 15. Water 16. pACYC 184 10 ng spots 3 Hours Exposure -66-FIGURE 5 : CROSS REACTIONS BETWEEN RECOMBINANT CANDIDA PROBES AND OTHER YEAST AND UNRELATED DNA F i f t e e n d i f f e r e n t DNAs were spotted i n 10 ng amounts through 32 m i n i f o l d apparatus. F i l t e r bound DNAs were hybridized with P-labeled probes (2.3 X 10 cpm/ug DNA). F i l t e r s were subsequently exposed to X-ray f i l m f o r 2k hours. The plasmid spot (#16) was removed p r i o r to placement onto f i l m to avoid over exposure. -67-CROSS REACTION BETWEEN RECOMBINANT CANDIDA PROBES AND OTHER YEAST AND UNRELATED DNA'S *P-CAN *p-66 *p-70 *P-pACYC SPOTS : 1. C.albicans 2. H.anomala 3. S.cerevisiae 4. K.marxianus 5. C . u t i l i s 6. C.guilliermondii 7. C.intermedia 8. C.santamariae 9- C.pseudotropicalis 10. C.tropicalis 11. A.fumigatus 12. Mouse 3T3 13. P.syringae 14. Bacillus phage 29 15. Water 16. pACYC 184 10 ng spots 24 Hours Exposure -68-DNA in this study (figure 6). Of the three recombinant probes shown in figure 3, recombinant #66 produced the strongest signal when hybridized to C.albicans DNA, while recombinant #74 produced the weakest signal. This w i l l be discussed further in the discussion section. The probes used in the study represented by figure 3 have much higher g specific a c t i v i t i e s (4.5 X 10 cpm/ug DNA) than that used in the study represented by figure 4 (2.5 X 10"* cpm/ug DNA). This may account for the greater spot intensities in figure 3 than in figure 5. Comparison of Biotin Detection Methods Sensitivity comparisons of the original BRL biotin detection system with either the Chan et al [27] (figure 7) or the BRL blugene detection (figure 8), favoured the original BRL system. In a l l cases, the original BRL method produced stronger spot intensities regardless of the diluent of the spotted DNA (figure 1 and 8). For example, with C.albicans whole c e l l DNA probe, the original BRL method allowed the detection of 11.8 ng of C.albicans DNA (diluted in water) wheras the blugene system did not produce a significant signal u n t i l the 117 ng spot (figure 8). The negative serum controls (without DNA), shown in figure 8 spot BI, produced background signals whereas the negative water controls did not. Comparison of Hybridization Efficiencies The hybridization signals of C9 (15-7 ng) and P2 (0.15 ng) shown in figure 9. are of approximately equal intensities. However, the amount of C.albicans DNA on C9 i s almost 160 times greater than the amount of pACYC 184 on P2. -69-FIGURE 6 : CROSS REACTIONS BETWEEN PLASMID PROBE AND UNRELATED DNA Fifteen different DNAs were spotted in 10 ng amounts through 32 minifold apparatus. F i l t e r bound DNAs were hybridized with P-labeled rr nonrecombinant plasmid pACYC 184 (2.3 X 10 cpm/ug DNA). Fi l t e r s were exposed to X-ray film for two or six days. -70-CROSS REACTION BETWEEN PLASMID PROBE AND UNRELATED DNA 2 days exposure 6 days exposure SPOTS : 1. C.albicans 9-2. H.anomala 10. 3. S.cerevisiae 11. 4. K.narxianus 12. 5. C.utilis 13-6. C.guilliennondii 14. 7. C.intermedia 15-B. C.santamariae 16. C.pseudotropicalis C. tropicalis A.fumigatus Mouse 3T3 P.sy r i n g a e Bacillus phage 2 9 Water pACYC 184 -71-FIGURE 7 : A. COMPARISON OF BIOTIN DETECTION METHODS Two methods of biotin detection were compared to determine sensitivity. Three dilutions of either DNA from known c e l l numbers of C.albicans or known DNA quantities of C.albicans or plasmid pACYC 184, were spotted onto NCF. The plasmid spots were probed with biotin-labeled plasmid. The other spots (C.albicans) were probed with either biotin-labeled C.albicans whole c e l l DNA or recombinant #66. -72-Comparison of Biotin Detection Methods pACTC Probe S P O T S CAN Probe C M * C A N 66b Probe CAN • • • H i ; »• • Tri ton X-100 Tween 20 BSA P= 70 ng C= 23 ng CAN= 2.5 X 10^ c e l l s 360 118 5-0 X 10? 720 237 i .o x i o ; 2.0 X 10 -73-FIGURE 8 : B. COMPARISON OF BIOTIN DETECTION METHODS Two methods of biotin detection were compared to determine sensitivity. Column A of each f i l t e r strip contains spots with C.albicans whole c e l l DNA and water. Column B contains spots with C.albicans whole c e l l DNA and serum. DNA samples were spotted without the minifold apparatus. Membrane bound DNA was probed with either biotin-labeled C.albicans whole c e l l DNA or recombinant #66. - 7 4 -Jomparison of Biotin Detection Methods ORIGINAL DETECTION BLUGENE DETECTION B-Can B-66b »• m 1 3 • 3 • ; ( » ; B-Can M 66b DNA d i l u t e d i n water - A serum - B 21°C C.albicans DNA 1) 0.0 4) 23.5 ng 2) 5-9 ng 5) 117.7 3) 11.8 6) 235.4 -75-FIGURE 9 : PLASMID HYBRIDIZATION VERSUS C.ALBICANS DNA HYBRIDIZATION Various amounts of C.albicans DNA and (nonrecombinant) plasmid were spotted through the minifold apparatus. F i l t e r bound DNAs were probed with biotin-labeled "self" DNA (0.2 ug DNA/ml of reaction mixture). Biotin detection was performed with the original BRL method. -76-PLASMID HYBRIDIZATION VERSUS C.ALBICANS DNA HYBRIDIZATION C l 0 4 3 # 6 B-CAN 8 $ # 1 2 • 9 # # 1 3 10 0 #14 B-pACYC P i ' # 5 • 2 # # 6 3 # # 7 4 # # 8 CAN SPOTS : 1) 0.5 ng 9) I5.7 ng pACYC SPOTS : 1) 0.05 ng 3) 2.0 11) 22.5 2) 0.10 6) 12.0 14) <J9.4 5) 0.80 -77-Comparison of the hybridization signals of the various yeast self hybridizations (figure 10), showed that C.albicans self hybridization produced the lowest signal intensity. The order of signal intensity (of DNA spots with comparable DNA quantities) from highest to lowest i s , C.tropicalis, C.intermedia, C.guillieromondii, Hansenula anomala, Saccharomyces cerevisiae and C.albicans. This w i l l be discussed further in the discussion section. 32 Sensitivity of P Labeled Probes 32 C.albicans whole c e l l DNA, labeled with P, detected as l i t t l e as 0.2 ng of C.albicans DNA (figure 11). Labeled recombinant #66 was able to 32 detect down to 8.0 ng of C.albicans DNA. Self hybridizations of P labeled recombinants (#66, #22, and #74) or lambda DNA, were detectable at 0.03 ng (and at possibly even lower amounts). Sensitivity of Biotin Labeled Probes C.albicans whole c e l l DNA labeled with biotin was able to detect down to 4.0 ng of C.albicans DNA (figure 12). Biotin labeled recombinants (#66 and #70) did not detect less than 14.0 ng of C.albicans DNA. Recombinant #22 was unable to detect up to 14.0 ng of C.albicans DNA. Self hybridizations of biotin labeled recombinants #66, #70, and #22 , were detectable at less than 0.03 ng, 0.06 ng and 0.06 ng respectively. Comparison of Hybridization Temperatures A l l hybridization reactions were allowed to proceed at a temperature of 42° C. as described by Meinkoth and Wahl [147]. As seen i n figure 12, -78-FIGURE 10 : YEAST SELF HYBRIDIZATIONS Various amounts of yeast DNAs were spotted the through minifold 32 appartus. F i l t e r bound DNAs were probed with P-labeled "self" DNA (10^ cpm/ml of reaction mixture). The amount of various yeast DNA's on the corresponding numbered spot i s not consistent. This i s due to errors made in the i n i t i a l calculations. The arrows point to the spots with comparable quantities of DNAs. C.albicans H.anomala S.cerevisiae 1) 11 ng 5) 23 l) 14 ng 5) 56 1) 17 ng 5) 72 2) 13 6) 40 2) 17 6) 67 2) 21 6) 86 3) 16 7) 45 3) 22 7) 90 3) 29 7) 114 4) 19 8) 50 4) 28 8) 112 4) 36 8) 143 C.guilliermondii C. intermedia c tropicalis 1) 8 ng 5) 30 1) 6 ng 5) 26 l) 4 ng 5) 17 2) 9 6) 36 2) 8 6) 31 2) 5 6) 20 3) 12 7) 48 3) 10 7) 42 3) 7 7) 27 4) 15 8) 60 4) 13 8) 53 4) 8 8) 34 -79-YEAST SELF HYBRIDIZATION CONTROLS 3 0 2 6 • 3 • ? • • • 4 8 • • • C.albicans #3) 16 ng H.anomala S.cerevisiae #2) 17 ng #1) 18 • • • • • • • • • - • • • • C.guilliermondii C.intermedia C.tropicalis !5ng #k) 13ng #5) i 7 n g -80-FIGURE 11: SENSITIVITY OF ^ 2P PROBES F i l t e r bound DNAs of various quantities were probed with 3 2P-labeled "self" DNA (2.2 X 10 8 cpm/ug DNA). Four spots of C.albicans 32 whole c e l l DNA (C5-C8) were probed with P-labeled recombinant #66. F i l t e r s were subsequently exposed to X-ray film for five days. [An exposure of 20 hours produced similar but less intense spots.] The arrow indicates the detectable spots for the C.albicans whole c e l l DNA probe and the recombinant #66 probe. -81-SENSITIVITY OF * P PROBES *CAN * 6 6 *70 * 2 2 Rl Rl Rl C 2 I R2 R2 R2 '1 R3 C7 R3 R3 C4 0 R4 C8 R4 R4 CAN SPOTS Cl) 0.2 ng C2) 0.4 C3) 0.6 C4) 4.0 C5) 6.0 ng C6) 8.0 C7) 12.0 C8) 14.0 RECOMB SPOTS: Rl) 0.03 ng R2) 0.06 R3) 0.08 R4) 0.12 42° C 5 days exposure -82-FIGURE 12 : COMPARISON OF HYBRIDIZATION TEMPERATURES [Sensitivity of Biotin Probes] Various quantities of C.albicans whole c e l l DNA were probed with biotin-labeled C.albicans whole c e l l DNA (0.2 ug DNA/ml of reaction mixture). Biotin-labeled recombinants (0.2 ug DNA/ml of reaction mixture)were used to probe "self" DNAs and also C.albicans whole c e l l DNA (C5-C8). Hybridizations were allowed to proceed at 42°C or at 20°C. Biotin detection was performed with the original BRL method. COMPARISON OF HYBRIDIZATION TEMPERATURES B-CAN *»2°C B-66 Cl 1 C5 • Rl C2 C6 R2 C3 - C7 R3 • C4 <• C8 Rt * C6 C7 B-70 Hll C 5 R<4 C 6 C7 C8-Rl R2 R3 R'4 B - 2 2 i C5 C6 C 7 C8 20°C • C8 C5 C6 C7 Rl R2 R3 Rl C5 C6 C 7 C8 Rl R2 R3 Rl 1 Rl R2 R3 R4 C7 C 8 RECOMB SPOTS : Rl) 0.03 ng R2) 0.06 R3) 0.08 R4) 0.12 CAN SPOTS : Cl) 0.2 ng C2) OA C3) 0.6 C4) 4.0 C5) 6.0 ng C6) 8.0 C 7) 12.0 C8) 14.0 -84-hybridization was more efficient at 42° C than at 20° C. Comparison of Various Hybridization Times DNA hybridizations were allowed to proceed in the hybridization reaction mixture for various time periods. The degree of hybridization was determined by densitometer tracings (figures 14, 15, and 16) of the spots on the autoradiogram (figure 13)-Recombinant #70 "self" hybridization reached a maximum after three days. In contrast, C.albicans " self" hybridization was s t i l l increasing after 7 days in the hybridization reaction mixture. Recombinant #70 hybridization with C.albicans DNA peaked after five days. This experiment was not repeated and hence the decreased signal intensities seen with the recombinant plasmid hybridization i s not conclusive. Probing Patient Serum Specimens Sera from 6 cancer patients (high risk group for systemic candidiasis) were probed with either the C.albicans whole c e l l probe or the recombinant #66 probe. These two probes failed to detect C.albicans DNA in either the serum supernatants or pellet fractions (figure 17). The positive pellet controls (pellet from serum spiked with a known quantity of c e l l s ) , exhibited hybridization signals with the C.albicans probe and with the recombinant #66 probe. The positive supernatant controls (supernatant from low speed centrifugation of serum spiked with DNA), which contained as much as 20 ng C.albicans DNA, were undetectable with either probe. -85-FIGURE 13 : VARIOUS HYBRIDIZATION TIMES Twelve spots of C.albicans whole c e l l DNA and six spots of 32 5 recombinant plasmid DNA were probed with P-labeled DNA (2.5 X 10 cpm/ug DNA). Column A = C.albicans whole c e l l DNA probed with "self" DNA B = C.albicans whole c e l l DNA probed with recombinant #70 C = recombinant #70 probed with "self" DNA Hybridization reactions were allowed to proceed for various time intervals. A l l spots were exposed together to X-ray film to obtain relative hybridization intensities. -86-VARIOUS HYBRIDIZATION TIMES AO Hours Exposure A = CAN-(*PCAN) 6 Hours Exposure B * CAN-(*P70) C = 70-(*P70) -87-FIGURE 14 : VARIOUS HYBRIDIZATION TIMES (DENSITOMETER TRACING) The autoradiograms were read with a densitometer to determine the relative amounts of DNA hybridization. This figure corresponds to column A in Figure 13. -89-FIGURE 15 : VARIOUS HYBRIDIZATION TIMES (DENSITOMETER TRACING) This figure corresponds to column B in figure 13. -90-VARIOUS HYBRIDIZATION TIMES -91-FIGURE 16 : VARIOUS HYBRIDIZATION TIMES (DENSITOMETER TRACING) This figure corresponds to column C in Figure 13--92-o VARIOUS HYBRIDIZATION TIMES 1 DAY 6 DAYS 7 DAYS |jji«iffiinn;i|'{[ 1! !C!:::!::::;I. ; i'''.1 V— Hnnsa:::; .! .L. / - ._. •i \ "T - — r —••' i f - •'- ti; \ j liiniKiiiii^ n - v -'••--•.-.urn 1 — \ -93-FIGURE 17 : LEUKEMIC PATIENTS' SERUM SAMPLES Six serum samples from four patients, who subsequently had positive blood cultures for Candida, were treated for probing. The spotted 32 5 samples were probed with P-labeled (2.8 X 10 cpm/ug DNA) C.albicans whole c e l l DNA (top figure), or recombinant #66 (bottom figure). The supernatant positive controls are indicated by the arrows in column G. The nitrocellulose was exposed to X-ray film for 6 days. -94-LEUKEMIC PATIENTS1 SERUM SAMPLES A B C D E F G A * CANDIDA DNA : 1) 0.005 ug 2) 0.010 3) 0.016 B • PELLET CONTROL : 1) 5 X 10^  2) 1 X l o ' 3) 2 X 10' C and D = PATIENT PELLET E and F = PATIENT SUPERNATANT G = SN CONTROL : 1) 0.005 ug 2) 0.010 -95-Growth Curve and DNA Recoveries at Various Time Intervals Small aliquots of a C.albicans c e l l culture were removed at various stages of growth. The DNAs isolated (method II) from portions of these aliquots were probed with C.albicans whole c e l l DNA, recombinant #66, and recombinant #70. Densitometer tracings (figures 19, 21 and 22) were made from the autoradiograms (figures 18 and 20). The densitometer values and the O.D.gQQ values were plotted against the age of the culture (figure 23-bottom). Cell numbers, as determined by colony counting, were also plotted against the age of the culture (figure 23-top). Increasing O . D . ^ Q Q n m correlated with increasing c e l l numbers unt i l after approximately 11 hours, when O.D..;__ continued to increase oUU nm slightly (0.3 units) while c e l l numbers decreased (2k%) before the O.D. value stabilized at a constant value. According to the C.albicans whole c e l l DNA probe, the amount of DNA recovered decreased progressively after 8 hours (late log phase). The C.albicans whole c e l l DNA probe detected more C.albicans DNA than did the recombinant probes. DNA Recoveries An overnight 5>0 ml culture of C.albicans was aliquoted to create a series of tubes with varying c e l l numbers. The DNA extraction (method II) of cells from each of these tubes was performed separately. Subsequently, 32 the extracted DNA was probed with P labeled C.albicans whole c e l l DNA. Densitometer tracings (figure 25) were made from the autoradiograms (figure 24). Known quantities of cells that had undergone the extraction procedure, and known amounts of C.albicans DNA, were plotted separately against densitometer values (figure 26). Hence, a given O.D. value corresponds to a -96-FIGURE 18 : C.ALBICANS DNA ISOLATED FROM CELL CULTURE AT VARIOUS TIME INTERVALS Cells were removed from culture after various periods of growth. Cell DNA was isolated by the lyticase and SDS method and spotted on f i l t e r s . Various known amounts of C.albicans DNA were also spotted 32 (C1-C4). Both sets of spots were probed with P-labeled C.albicans whole c e l l DNA (2.8 X 10 5 cpm/ug DNA). -97-C.ALBICANS DNA ISOLATED FROM CELL CULTURE AT VARIOUS TIME INTERVALS -98-FIGURE 19 : C.ALBICANS DNA ISOLATED AT. VARIOUS TIME INTERVALS (DENSITOMETER TRACING) The autoradiogram (figure 18) was read with a densitometer to determine the relative amounts of DNA hybridization between the f i l t e r bound DNA and the labeled C.albicans whole c e l l DNA probe. C.ALBICANS DNA ISOLATED FROM CELL CULTURE AT VARIOUS TIME INTERVALS -100-FIGURE 20 : C.ALBICANS DNA ISOLATED FROM CELL CULTURE AT VARIOUS TIME INTERVALS The f i l t e r bound spots are the same as those in Figure 18. 32 5 However, the spots were probed wi th either J P-labeled (2.8 X 10J cpm/ug DNA) recombinant #66 or #70. C.ALBICANS DNA ISOLATED FROM CELL CULTURE AT VARIOUS TIME INTERVALS F l 13 16 P-66 Cl) 20 ng 2) 50 ng 3) 100 ng It) 150 ng P-70 Fl) 1.1 X 105 CELLS F5) 4.7 X 106 F 9) 5.8 X 107 F13) 39 X 107 2) 9-0 X 10 3) 1.0 X 10 4) 2.8 X 106 6) 7.2 X 10 10) 4.7 X 10' 14) 3-6 X 10' 7) 2.0 X 10 11) 4.1 X 10' 15) 3-8 X 10' 8) 2.5 X 106 12) 3.7 X 107 16) 4.0 X 107 -102-FIGURE 21 : C.ALBICANS DNA ISOLATED AT VARIOUS TIME INTERVALS (DENSITOMETER TRACING) The autoradiogram (figure 20) was read with a densitometer to determine the relative amounts of DNA hybridization between the f i l t e r bound DNA and the labeled recombinant #66 probe. C.ALBICANS DNA ISOLATED FROM CELL CULTURE AT VARIOUS TIME INTERVALS -104-FIGURE 22 : C.ALBICANS DNA ISOLATED AT VARIOUS TIME INTERVALS (DENSITOMETER TRACING) The autoradiogram was read with a densitometer to determine the relative amounts of DNA hybridization between the f i l t e r bound DNA and the labeled recombinant #70 probe. C.ALBICANS DNA ISOLATED FROM CELL CULTURE AT VARIOUS TIME INTERVALS -106-FIGURE 23 : CELL CULTURE PLOT DNA quantities, c e l l numbers and optical density values at various times were plotted to correlate the amount of ce l l s and DNA recovery with different stages of growth. 32 C O = C.albicans whole c e l l P-probe 32 • . . . . - . . • 0 = recombinant #66 P-probe 32 A..». . . . . .A = recombinant #70 P-probe ^ = optical density \ J (600 nm) • = c e l l numbers O . D . C 6 0 0 ) -LOT-- 1 0 8 -FIGURE 2k : CELL DNA RECOVERIES WITH LYTICASE AND SDS TREATMENT The spots represent various known qua n t i t i e s of C.albicans DNA. F i l t e r B corresponds to DNA from various known numbers of C.albicans c e l l s . Both f i l t e r s were probed with P-labeled C.albicans whole c e l l DNA ( 2 . 8 X 1 0 cpm/ug DNA). Arrows i n d i c a t e the lowest amounts of f i l t e r bound DNA detected. -109-C E L L DNA RECOVERIES WITH LYTICASE AND SDS TREATMENT A. KNOWN DNA QUANTITIES : B. KNOWN CELL NUMBERS : 1) 0.00 ng 7) 4.00 n S 1) 5-0 X 10? 7) 1.0 X 10^  2) 0.25 8) 5-00 2) 7-5 X 10^  8) 2.0 X 10? 3) 0.50 9) 6.00 3) 1-0 X 10;? 9) 4.0 X io£ *») 1.00 10) 8.00 4) 2.5 x io£ 10) 6.0 x io£ 5) 2.00 11) 9.00 5) 5.0 X 10;? 11) 8.0 X 10, 6) 3-00 12)10.00 6) 7-5 X 105 12) 1.0 X 107 4 Days Exposure -110-FIGURE 25 : CELL DNA RECOVERIES WITH LYTICASE AND SDS TREATMENT (DENSITOMETER TRACINGS) The autoradiograms were read with a densitometer to determine the amount of DNA recovered from various numbers of C.albicans. The top section corresponds to f i l t e r A and the bottom represents f i l t e r B of figure 2k. -111-C E L L DNA R E C O V E R I E S W I T H L Y T I C A S E A N D S D S T R E A T M E N T -112-certain number of cells and DNA quantities. This provided an estimate of the amount of DNA recovered from a known number of ce l l s , using this particular DNA extraction procedure (method II). The percentage of DNA recovery was calculated with the assumption of a theoretical value of 3.79 X 10~±li ug C.albicans DNA per diploid c e l l [82,184,237]. The plot of c e l l numbers against percentages of DNA recovered increased slightly (up to 5%) as the number of cells increased. The amount of DNA recovered never exceeded more than 16%. -113-FIGURE 26 : CELL DNA RECOVERY PLOT This i s a comparison of the densitometer values obtained with various q u a n t i t i e s of DNA and with DNA from various q u a n t i t i e s of c e l l s . The amounts of DNA recovered from c e r t a i n q u a n t i t i e s of c e l l s were predicted from the two l i n e s p l o t t e d on t h i s graph. The predicted values were compared with the t h e o r e t i c a l DNA contents f o r the corresponding c e l l numbers. The percentages of DNA recovered were then p l o t t e d (figure 26-inset) against the number of c e l l s treated f o r DNA ex t r a c t i o n . O = DNA (ng) vs densitometer units • = DNA ( l o g i n c e l l numbers) vs densitometer units A = l o g 1 0 c e l l numbers vs DNA recovery Densitometer Units -115-CALCULATIONS -14 C.albicans genome = 3-79 X10 g 1 bp = 660 g/mole 1 mole = 6.023 X 1023 bp C.albicans DNA insert in recombinant #66 i s between 4.3 and 6.4 kbp - assume i t i s approximately 5 kbp A. Insert size : (5 X 103 bp) x 660(g/mole) / 6.023 X 1023 = 3.3 X l O - 1 7 g % of genome : (100) x 3-3 X 10"17 g / 3-79 X 10"14 g = 8.71 X 10"2 % B. Biotin probes detected : 14.0 ng spot x 8.71 X 10~2 % = 12.2 pg of target DNA 32 C. P probes detected : 8.0 ng spot x 8.71 X 10~2 % = 6.97 Pg of target DNA or 8.0 X 10~9 gram / 3.79 X lO - 1** gram per c e l l = 2.11 X 105 cells -116-DISCUSSION Every organism has i t s own unique DNA sequence. This property allows for specific detection of organisms through nucleic acid hybridizations. DNA probes consisting of unique base sequences labeled with radioisotopes or non-radioactive biotin have been used in both research and in diagnostic virology [19,30.51.69,97,168,202]. Judging from the success of probes in virology, there appears to be great potential for the application of hybridization techniques for the detection of Candida DNA in-patients with systemic candidiasis. The fact that two strands of DNA may be physically separated and specifically reassociated [52,l4l], provided the basis for nucleic acid hybridization techniques. This reaction is generally assumed to be sequence specific. Annealing occurs only between two DNA strands in areas representing corresponding sequences or between a DNA strand and an RNA molecule transcribed from i t . Duplex formation of DNA strands is a function of the kinds of related base sequences which exist in the particular DNAs [144]. The original proposal was to use C.albicans rRNA as probes, and this prompted the isolation of rRNA. However, literature reviews indicated that in Saccharomyces [14], bacteria [50,53,155]. plants [143], Drosophila [116], and protozoa [74,75], the rRNA sequences are more conserved than the rest of the genome. Thus the probability of cloning a unique sequence of rRNA was less than that of DNA. Since diagnostic probes require specificity, C.albicans DNA rather than rRNA was used in probe production. -117-Early observations indicated that virtually no hybridization (less than 16%) occurs between DNA strands of distantly related organisms such as Neurospora, E.coli, mouse and wheat [ 5 5 ] ' This result i s supported here by the absence of hybridization between the C.albicans whole c e l l DNA probe and the DNAs of mouse (3T3). Aspergillus fumigatus, Pseudomonas syringae, and Bacillus subtilis phage 29. Intergeneric DNA-DNA hybridizations in fungi suggest homology values of less than 20% [54,126,165]. Furthermore, sequence homology among yeasts i s only approximately 10% [ I 6 5 ] . This estimate of homology among yeasts has beeen contradicted by Segal and Eylan [210]. They obtained homology values of 25-28% from hybridizations between C.albicans and various Saccharomyces species. Sequence homology within fungi would account for the low levels of intergeneric DNA hybridizations of C.albicans with Saccharomyces cerevisiae, Hansenula anomala, and Kluveromyces marxianus reported here. The C.albicans whole c e l l DNA probe also hybridized with members of other species within the genus Candida. The most intense hybridization signals were with Candida intermedia, Candida pseudotropicalis, Candida  tropicalis, and Candida guiliermondii. Segal and Eylan [210] have reported high sequence homologies of C.albicans with C.tropicalis (86.6%), C.pseudotropicalis (76.1%), and C.guilliermondii (76.2%). Lower values have been described by Lek Bak and Stenderup [126]. The efficient hybridizations of the recombinant plasmids with the DNA's of C.albicans, C.intermedia, C.pseudotropicalis, and C.tropicalis suggest that the DNA inserts of the recombinants contain one or more similar or identical sequences common to these four species. -118-Recombinants #66 and #70 exibited low homologies with more organisms (H.anomala, S.cerevisiae, C.utilis and C.santamariae) than recombinants #22 and #7^. This observation may be a reflection of the fact that different parts of the genome are represented by the various inserts. Another factor may be the size differences between the inserts within each recombinant. The inserts in order of decreasing size, are : #70, #66, #74 ( a l l > 4.3 kb), and #22 ( < 4.3 kb). Long nucleic acid fragments presumably have greater varieties of base sequences, which allow for increased p o s s i b i l i t i e s for regions of complementarity [144]. Cross reactivity of the recombinant C.albicans probes with other Candida species w i l l not decrease the value of the recombinants as diagnostic probes. Although C.albicans i s the most frequent cause of candidiasis, other members of the genus Candida are also involved. C.parapsilosis, C.krusei, C.stellatoidea, C.tropicalis, C.pseudotropicalis, and C.guilliermondii, have a l l been shown to cause candidiasis in humans [17]-In fact, C.tropicalis has been isolated more frequently than C.albicans, in patients with hematologic malignancies [151,242]. Furthermore, the mere presence in serum of Candida, regardless of species, i s indicative of disease conditions. Recombinant #66 appears to be the most sensitive of the probes tested, but i t s lower specificity may require more stringent hybridization conditions. The lower intergeneric reactivity of recombinant #22 (the smallest of the probes) and i t s increased sensitivity over recombinants #70 and #74, warrant further investigation into the use of #22 as a diagnostic probe. -119-The cross reactivity of the recombinant C.albicans probes to the non-recombinant pACYC 184 i s expected since the recombinants contain 4 kbp of this plasmid. The whole recombinant plasmid was used as a probe because there were no cross hybridization of the non-recombinant plasmid with any other DNAs. The renaturation rate of denatured DNA i s proportional to the amount of f i l t e r bound DNA when hybridization proceeds under low concentrations ( <10 ug/ml) of labeled DNA [144]. The DNA of interest i s not total DNA, but the complementary sequences. In the presence of equal absolute DNA concentrations (i.e. total nucleotide concentrations), the probability of a co l l i s i o n between complementary single-stranded DNA fragments of smaller genomes i s greater than that of larger genomes [38,142]. In addition, intermolecular heterogeneity increases the frequency of unstable hybridizations. Transient hybrids can dissociate and be replaced by hybrids with greater s t a b i l i t y because of the avai l a b i l i t y of a large number of base sequences [142], Thus under comparable conditions, the reaction occurs more rapidly with DNA of more simple genomes than with that of complex genomes [144]. Optimum "self" hybridization of C.albicans DNA required a longer period (over 7 days) than "self" hybridizations of recombinant plasmids (3 days). This could be explained by the higher complexity of C.albicans 4 (approximately 3-5 x 10 kbp) over the recombinant plasmid (less than 10 kbp). The probability of the formation of a stable hybrid from the interaction of any two DNA strands in the recombinant plasmid hybridization solution i s 1:2 (50%). On the other hand, assuming that the 12 chromosomes -120-of (diploid) C.albicans were not sheared during isolation, only 1/24 strand collisions would form stable hybrids comparable to those of the recombinant plamids. This does not exclude the possibility of heterologous hybridizations (i.e. between strands of different chromosomes) of C.albicans DNA. Approximately 15% of the C.albicans genome i s repetitive [184]. Optimum renaturation of recombinant probe with f i l t e r bound C.albicans whole c e l l DNA, required a time period (5 days) intermediate between that of C.albicans and recombinant plasmid "self" hybridizations. In the recombinant plasmid and C.albicans reaction, 1/24 strand collisions would form hybrids (identical to that of the C.albicans "self" hybridizations). Since this system i s flooded with a large excess of one type of sequence (the insert of recombinant #66), saturation occurs earlier in the recombinant probe and membrane bound C.albicans hybridization reaction than in the C.albicans "self" hybridization reaction. However, the kinetics of the three different types of reactions are not directly proportional to the frequencies of hybrid formation suggested here. Other factors such as sheared C.albicans DNA (more fragments) and sequence repetitiveness of the insert (i.e. not single copy within genome), could influence reaction rates. The lowest self hybridization efficiency of a l l the yeasts studied was for C.albicans. This may be indicative of a more complex genome in C.albicans. This explanation also implies that the complexity of the S.cerevisiae genome i s similar, but less than that of C.albicans and more than that of other Candida species (as determined by the results presented here). Whelan et al [239] have shown that the mean DNA content of C.albicans isolates i s close to that of a diploid S.cerevisiae reference -121-strain. While i t may be possible that these two organisms have- comparable DNA contents, one would expect (from homology values), a closer relationship (in terms of degree of complexity), between C.albicans and other Candida species, than between C.albicans and S.cerevisiae. Hybridization appeared to proceed more ef f i c i e n t l y at 42° C. than at 20° C. The thermal s t a b i l i t y of duplexes formed between labeled DNA and bound DNA i s dependent upon the base composition, degree of base pairing and length of the pair structures [144]. Since this study did not include controls for the various parameters, one cannot confidently comment on the properties of the DNA's involved. 32 The lower sensitivity of the J P-labeled C.albicans for "self" DNA 32 (200 pg) compared to that of the P-labeled recombinants (30 pg), may be partly due to incomplete hybridization. The renaturation rate of C.albicans DNA i s lower than that of the plasmid DNA. As mentioned previously, optimum renaturation would not have occurred un t i l after at least 1 days. In this experiment, hybridization was allowed to proceed for only two days. An earlier experiment which involved a hybridization period of only 21 hours, indicated that under comparable conditions, "self" hybridizations were approximately 160 times more efficient than C.albicans "self" hybridizations. 32 C.albicans whole c e l l DNA probed with P-labeled recombinant #66 was undetectable u n t i l the 8 ng spot. Assuming that the insert in recombinant #66 i s approximately 5 kbp and i s a single copy sequence, then the insert would only represent 8.71 X 10~2 % (3-3 X 10~17 g) of the total genome (3.79 X lO - 1** g). Therefore, in a 8.0 ng spot of C.albicans whole c e l l -122-DNA (which corresponds to 2.11 X 10 c e l l s ) , approximately only 7 Pg of target DNA would be present. If the sequence of the insert in recombinant #66 i s present only once per C.albicans genome (as previously assumed for these calculations), then this probe was able to detect 2.11 X 10 32 C.albicans c e l l s . The C.albicans whole c e l l DNA probe ( P) was able to detect as l i t t l e as 0.2 ng of C.albicans DNA. The higher sensitivity of the C.albicans whole c e l l DNA probe (cf. #66 - 8.0 ng) i s expected since there are different types of target DNAs in the f i l t e r bound DNA. Radioactive probes for routine use in c l i n i c a l laboratories are somewhat cumbersome. Ideally, diagnostic probes should be non-radioactive, easy to use and rapidly detectable. Hence, investigations were made into the use of a non-radioactive reporter molecule such as biotin. Biotin i s a water-soluble vitamin which binds well with avidin (68 kd protein), to form large complexes that can be detected biochemically. Researchers at Yale University were the f i r s t to develop a two step enzymatic technique for detection of incorporated biotin-labeled analogs of TTP and UTP [117]. By polymerizing the enzyme involved in detection of biotin, the sensitivity of the biotinylated probes was reported to be at 1-10 pg of target DNA after enzyme incubations of less than one hour [117]. Subsequently, the sensitivity of these probes was increased ( < 1 pg target DNA detectable ) by modifying the biotin detection system. Comparison of three detection systems (the original BRL, the BRL Blugene, and the method described by Chan et a l . ) , indicated that under the conditions employed in this study, the original BRL system appeared superior to the others. The background signals of the Blugene system were greater than the others. It i s possible that the -123-Blugene system may have detected lower amounts of target DNA, but these signals were masked by the high background. Since other investigators have had success with the Blugene system, i t is possible that the background problem could be solved with slight changes in methodology. The biotin-labeled recombinant probes were almost one half as sensitive 32 as the P-labeled recombinant probes (i.e. biotin - 12.2 pg target DNA 32 cf P - 7 Pg target DNA). Although the biotin system required less time to complete, i t involved more manipulations than the radioisotope system. Unless the sensitivity of the biotin-labeled recombinant probes can be 32 increased, P-labeled recombinant probes with higher specific activities (to decrease autoradiography time), may have to be used for optimal sensitivity. Another p o s s i b l i l i t y i s to find an alternative nonradioactive reporter molecule with comparable or greater sensitivity. Probes of high sensitivity are required to diagnose systemic candidiasis, especially during the i n i t i a l stages of the disease. This assumption i s based on the d i f f i c u l t y in culturing C.albicans from blood. The inference i s that there are only low levels i f any, of circulating C.albicans. Hence optimal c e l l DNA recovery i s c r i t i c a l to the efficiency of the probes. The low c e l l l y s i s efficiencies of the methods used in this study may have been due to several factors, one of which was inaccurate c e l l counts. I n i t i a l c e l l counts were performed with a hematocytometer as colony counting would have involved a delay of approximately 16 hours . This delay was avoided since enzyme treatments for protoplast formation were dependent upon c e l l numbers. Subsequent comparisons of hematocytometer counts with colony -124-counts, indicated 2-10 fold differences. Thereafter, a l l c e l l number quantitations were accomplished with colony counting. Incorrect c e l l counts could also have occurred as a result of the growth phase of the C.albicans culture. According to the growth curve, the early stationary phase of C.albicans began at approximately 11 hours after inoculation. Cell cultures used in the c e l l l y s i s experiments were similarly inoculated and were allowed to grow for 15-20 hours. Presumably, these cultures would have been in mid to late stationary phase. Bernander and Edebo [11] have observed that stationary phase C.albicans blastoconidia were more l i k e l y to form germ tubes than blastoconidia in the exponential phase of growth. It has also been reported that the proportion of blastoconidia forming germ tubes was higher for cells grown in Sabouraud's broth than in a synthetic medium [37]- Thus i t i s quite probable that the C.albicans cultures in the present study contained a large percentage of germ tubes or mycelia. These morphological forms would have caused clumping (autoagglutination) of the c e l l s , leading to inaccurate c e l l counts. An alternative explanation for low c e l l l y s i s efficiencies i s the resistance to protoplast forming enzymes. The sensitivity of yeast cells to protoplast formation varies from strain to strain [169,203]. Torres-Bauza and Riggsby [226] have reported that yields of protoplast depended mainly on the phase of growth of the yeast culture. Starved cells and late-stationary phase cells gave the lowest recovery of protoplasts. Elevated levels of chitin in germ tubes [32] may be one explanation for this phenomenon. Both C.albicans and S.cerevisiae c e l l walls were more susceptible to helicase (enzyme mixture from the gut of Helix pomatia), during the exponential phase of growth [21]. Decreased susceptibility to helicase corresponded to the -125-deceleration of yeast growth. Increased resistance to helicase may be related to an increase in the production of phosphomannans or may involve alterations in existing wall polymer (i.e. formation of additional glycosidic linkages), in the c e l l walls of stationary phase cells [21]. Since the c e l l cultures used here were most l i k e l y in stationary phase, resistance to protoplast formation may be a contributing factor in the observed results. This explanation complies with the progressive decrease in the percentage of DNA recovered, beginning from late exponential or early stationary phase of growth. The absence of a significant drop in c e l l numbers during the stationary phase argues against the probability of c e l l autolysis being solely responsible for the dramatic decrease in the DNA recovered. However, c e l l autolysis may be partially responsible, as c e l l death results in the release of nucleases into the extracellular environment. C.albicans DNA is probably very susceptible to enyzme digestion. Digestion of less than five seconds with Mbo I (restriction enzyme) resulted in DNA fragments of less than 4 kbp. The lyticase and SDS treatment for C.albicans DNA extraction appeared to be the most rapid and efficient method investigated. The use of this treatment,and other procedures established for optimum probe efficiency, did not result in the detection of C.albicans DNA from any of the leukemic patients' sera specimens with either the C.albicans or the recombinant DNA probes. This implied that there were less than 0.2 ng of C.albicans DNA in the serum samples, as determined by the lower limit of sensitivity of the C.albicans whole c e l l DNA probe. These results do not give a clear -126-indication of the sensitivity of the probes as a diagnostic tool, since none of the patients screened were known to be positive for C.albicans. Further testing using sera from known positives would help to c l a r i f y the probe parameters. The highest percentage of DNA recovered using the lyticase and SDS method was less than 16%. This suggests that the recombinant probes would not be effective unless a large number of C.albicans cells were present in patient specimens. 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