Isolation and characterization of allergens from curvularia lunata 1

Chapter CHAPTER 1: BACKGROUND AND INTRODUCTION 19 Chapter 1.1 HYPERSENSITIVITY REACTIONS AND ALLERGY 1.1.1 Hypersensitivity Reactions A normal immune system is beneficial to the human body in order to differentiate self from non-self and to neutralize potentially pathogenic organisms or substances. Hypersensitivity refers to undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system. In other words, hypersensitivity refers to a pre-sensitized state of an individual being abnormally sensitive to the foreign substances causing inflammation and cellular damage. Hypersensitivity reactions were classified into four types: Type I, II, III and IV, based on the mechanisms involved (Gel and Coombs, 1975). Later Type V and VI reactions were added (Rajan, 2003) to the above classification scheme. Details of various hypersensitivity reactions are highlighted in Table 1.1. Table 1.1: Various types of hypersensitivity reactions (modified from Gel and Coombs, 1975). Mechanism Example/s I IgE-mediated immediate hypersensitivity Systemic anaphylaxis, Asthma, Eczema, Hay fever II Antibody-mediated cytotoxic hypersensitivity Haemolytic disease of newborn, Goodpasture`s syndrome III Immune-complex mediated hypersensitivity Systemic lupus erythematosus, arthritis, glomerulo nephritis Type V Cell-mediated delayed hypersensitivity Stimulated antibody mediated hypersensitivity VI Antibody dependent cell mediated cytotoxic hypersensitivity IV 20 Contact dermatitis, Tubercular lesions Graves disease Parasitic helminthes infections Chapter 1.1.2 Allergy The term as well as the concept of “allergy” was first introduced by a Viennese pediatrician, von Pirquet in 1906 (Bendiner, 1981). Allergy is used to refer to a Type I hypersensitivity reaction. Out of the four major hypersensitivity reactions, allergy has the most clearly defined and unambiguous immunological as well as pathological correlation. Allergy is characterized as a hyper response of IgE antibody to environmental substances like pollen, dust mites, animal dander, fungal spores, insect venom and food. Allergic conditions include allergic rhinitis, conjunctivitis, asthma, etc., causing clinical symptoms like sneezing, coughing, wheezing and breathlessness with reversible airway obstruction, urticaria and anaphylaxis (Stewart and Thompson, 1996). The initiation of the process is brought up by presentation of processed environmental antigens to naïve Th precursor cells (ThP) by antigen presenting cells (APCs) bringing selective proliferation of Th2-polarised memory cells (Th2M), eventually causing production of antigen specific IgE by the B cells. Re-exposure to this particular antigen elicits acute phase response brought by cross-linking of IgE receptors (FcεRI) on mast cells or basophils causing them to degranulate. This results in release of pro-inflammatory mediators like histamine, leukotrienes and prostaglandins. These in turn cause symptoms of immediate allergic reactions as mentioned above. The mast cells can also cause delayed type reactions, -8 hours after the immediate responses (Holt et al., Holgate, 1999). The mediators released by mast cells induce release of cytokines and proteases causing tissue damage. The late 21 Chapter phase or delayed type of reaction brings about nasal congestion in allergic rhinitis and bronchial obstruction in asthma which may lead to airway hyperresponsiveness (AHR) in future. The major cell types, molecules implicated in allergic reaction and the overall mechanism underlying allergic reaction is described in Figure 1.1. Allergy is often explained in terms of “atopy”. The term atopy refers to a hereditary disorder marked by the tendency to develop immediate hypersensitivity reactions to specific antigens. Hence it is also referred as “atopic allergy”. As atopy is a hereditary disorder, the atopic individual shows a predisposition for a Th2-polarised response which is further enhanced by factors like lack of pathogens in environment, vaccination, industrialization, clean housing and bedding (Figure 1.2). The development of atopy is a two-step process. As shown in Figure 1.3, phase of atopic asthma involves antigen specific immunological memory. This occurs normally in childhood and results in Th0/Th2-polarized memory, increasing risk for respiratory disease. The first phase is not sufficient for the disease presentation. The second phase occurs only in the individuals with persistent inflammation (Holt et al., 1999). 1.2 ALLERGENS 1.2.1 Definition and various allergen types The word “Allergen” is defined as an antigen that induces IgE antibody synthesis in atopic patients in response to the allergen, leading to release of histamine and other pharmacological mediators of immediate hypersensitivity from mast cells and basophils (Kurup and Banerjee, 2000). Commonly, the allergens are classified into two 22 Chapter Figure 1.1: Molecular and Cellular mechanism of allergy (Adapted from Holt et al.,1999) 23 Chapter Figure 1.2: Factors responsible for atopy (adapted from Umetsu et al., 2002) Figure 1.3: Progression of allergic sensitization from early childhood to atopy in adulthood (adapted from Holt et al., 1999) 24 Chapter types: Indoor and outdoor allergens (Boulet et al, 1997; Kerkhoff et al., 2003). Plant pollens and fungi are the two major groups of outdoor allergens (Burge, 2000; Kerkhoff et al., 2003). Indoor allergens are from house dust mites, dander of pets (cats and dogs), cockroaches and fungi (Burge, 2000; Kerkhoff et al., 2003). In industrialized nations, atopic diseases affect up to 20% of the population (Kurup and Banerjee, 2000). Biochemically, allergens are proteins, carbohydrates or glycoproteins which stimulate the immune system of the atopic individual and bind specifically to IgE produced in response to stimulation. To date there are over 300 reported allergens which comprise molecules of various physiological and biochemical functions (Scheiner, 1995). Various allergens from pollens, house dust mites and cockroaches have been well studied, but the same is not true for fungal allergens (Scheiner, 1995). Although fungal allergens are important (as they are found both indoors and outdoors), very few fungal species and fungal allergens have been studied in detail for possible allergenicity. 1.2.2 Recombinant allergens in allergy Classically, allergologists used natural products such as total protein extracts for the diagnosis and treatment of allergies. However, allergens prepared this way were highly heterogeneous in the mixture due to the varying amounts of allergenic and nonallergenic proteins. Moreover, natural extracts had various drawbacks such as chances of contamination from other allergen sources being prone to proteolysis, degradation and at times containing various lipopolysaccharides and endotoxins (Linhart and Valenta, 2005). With the development of molecular biology and recombinant DNA 25 Chapter technology, several recombinant allergens were cloned, expressed, purified and tested. More than 300 allergen (nucleotide/protein) sequences are now available in Genbank (www.ncbi.nlm.nih.gov) and various databases. These recombinant allergens would soon be used in various diagnosis and treatments of allergy (Chapman et al., 2000). These recombinant allergens may also be used to improve various forms of specific immunotherapy – SIT (Norman, 1993). 1.3 FUNGAL ALLERGY AND FUNGAL ALLERGENS 1.3.1 Fungi as environmental allergens Fungi are eukaryotic, achlorophyllus, chitinous cell walled, unicellular/multicellular organisms which form a separate kingdom in classification (Whittaker, 1969). Fungi form a large group of organisms found in every ecological niche (Hawksworth, 2001). Around 1.5 million species of fungi are present worldwide (Alexopoulos et al., 1996). Based on the spore type produced, the life cycle of a typical fungus is divided into perfect (sexual) and imperfect (asexual) phases. In modern terms, these states are referred as the teleomorph and anamorph, respectively and the fungus showing both states, known as holomorph. Conidium is a term used for asexual spores produced by anamorphs of filamentous fungi. Most fungi reproduce sexually by meiosis, producing spores on specialized structures such as basidia or in a specialized structure called the ascus. These types of fungi are referred as Fungi Perfecti. Fungi liberate spores and respirable mycelial fragments in large numbers. Fungal species that produce airborne 26 Chapter spores are found under the phyla Dikaryomycota, Zygomycota and Oomycota (Horner et al., 1995). Details of the classification of the fungal species under these phyla are shown in Table 1.2. Fungi cause a number of infectious diseases. Many fungi produce toxins (Kendrick, 1985), some of which are potent carcinogens, e.g., Aflatoxins produced by Aspergillus flavus. Fungal spores have been identified as one of the sources of indoor and outdoor allergies (Platts-Mills et al., 1996). Given their smaller size (>10µm), fungal spores can penetrate the lower respiratory tract causing allergies (Pepys, 1965; Dankaart et al., 1991; Reponen et al., 2001). The immunological manifestations of fungal allergies range from dermatitis, sinusitis and asthma, to bronchopulmonary mycoses, pneumonitis and allergic alveolitis (Lehrer et al., 1983; Fink, 1998). The immune responses in fungal allergies follow the same pattern as that of other inhalant allergens such as pollens or house dust mites (Kauffman et al., 1995). The most commonly found allergic fungi are Alternaria spp., Cladosporium spp., Epicoccum nigrum, Fusarium spp., Ganoderma spp., Penicillium spp., Aspergillus spp., etc., (Beaumont et al., 1985; Solomon and Matthews, 1988). Many yeasts and mushrooms capable of producing allergic reactions have also been reported (Horner et al., 1995 and 1998). Generally, Aspergillus spp. and Penicillium spp. are considered as indoor fungi and are less commonly seen outdoors (Beaumont et al., 1985; Licorish et al., 1985). Outdoor fungal spore counts are seen to be correlated with clinical symptoms (Malling, 1986). Most of the allergenic fungal genera belong to the class Ascomycetes. 27 Chapter Table 1.2: Taxonomic distribution of various airborne spores-producing fungal genera (adapted from Horner et al., 1995) Phylum Zygomycota Class Zygomycetes Order Mucorales………………………………Mucor, Rhizopus Phylum Dikaryomycota Subphylum Ascomycotina Class Ascomycetes (including imperfect forms) Order Dothidiales .Alternaria, Cladosporium, Epicoccum Order Eurotiales………………………….……Aspergillus, Penicillium Order Pleosporales…………………………….Curvularia(Cochliobolus) Order Helotiales……………………………….Botrytis Order Hypocreales…………………………….Fusarium Order Onyngeales…………………………… Trichophyton Class Saccharomycetes……………………… …….Saccharomyces, Candida Subphylum Basidiomycotina Class Holobasidiomycetes Order Agaricales………………………………Coprinus, Pleurotus, Psilocybe Order Aphyllophorales……………………… Ganoderma, Merulius Order Lycoperdales………………………… Calvatia, Geaster Class Teliomycetes Order Uredinales………………………….……Rusts Order Ustilaginales…………………………….Smuts, red yeasts (Sporobolomyces) Phylum Oomycota Class Oomycetes Order Peronosporales…………………… ……Phytophthora, Plasmopara (mildews) 28 Chapter 1.3.2 Recombinant fungal allergens As explained earlier, recombinant allergens are thought to offer towards allergy diagnosis as well as therapeutics. Although, the breakthrough in recombinant allergens is promising, compared to other allergens (like dust mites, pollen and foods), recombinant fungal allergens are less documented and are less studied. To date, there are only around 90 recombinant fungal allergens submitted to the International Union of Immunological Societies (IUIS): Allergen nomenclature sub-committee which maintains the list of available recombinant allergens (Table 1.3). Taking into account the importance of fungi as environmental allergens and the uniqueness of fungal airspora, it is of great importance to identify and study these recombinant fungal allergens in detail. 1.3.3 Global prevalence of fungal allergy Fungal spores are present worldwide and many species can be observed at most times of the year (Horner et al., 1995; Chou et al., 2003). Worldwide, more than 80 genera of the major fungal groups have been associated with symptoms of respiratory tract allergy (Horner et al., 1995). Fungal spores are usually present in outdoor air throughout the year in high numbers and frequently exceed pollen concentrations by 100 to 1,000-fold (Lehrer et al., 1983). Globally, fungal allergy is prevalent at 20 to 30% among atopic individuals and up to 6% in the general population (Portnoy et al., 1987). Epidemiological study on 16,204 civilians in the U.S.A. showed that 3.6% of the population was sensitized to the fungus Alternaria alternata (Gergen et al., 1987). Generally, the fungal allergic subjects are seen to have IgEs to various fungal species. 29 Chapter Table 1.3: Fungal allergens as approved by the allergen nomenclature committee (adapted from www.allergen.org/List.htm) Fungal allergen name Biochemical type Mol.wt. (kDa) Accession Alternaria alternata Alt a 28 U82633 U87807, U87808 Alt a heat shock prot. 70 Alt a prot. disulfideisomerase 57 Alt a acid ribosomal prot. P2 11 X84217 X78222, U87806 Alt a enolase 45 U82437 Alt a YCP4 protein 22 X78225 Alt a mannitol dehydrogenase 29 Alt a 10 aldehyde dehydrogenase 53 AY191815 X78227, P42041 Alt a 12 acid ribosomal prot. P1 11 X84216 Alt a 13 glutathione-S-transferase 26 AY514673 Cla h Ag54 23 Cla h acid ribosomal prot. P2 11 X78223 Cla h enolase 46 X78226 Cla h YCP4 protein 22 X78224 Cla h mannitol dehydrogenase Cla h vacuolar serine protease 55 AY787775 Cla h 10 aldehyde dehydrogenase 53 X78228 Cla h 12 acid ribosomal prot. P1 11 X85180 alkaline serine protease 34 Cladosporium herbarum AY191816 Aspergillus flavus Asp fl 13 Aspergillus fumigatus Asp f 18 M83781, S39330 Asp f 37 U56938 19 U20722 30 AJ001732 40 Z30424 26.5 U53561 Asp f peroxisomal protein Asp f Asp f metalloprotease Asp f Mn superoxide dismut. Asp f Asp f ribosomal prot. P2 Asp f Asp f 10 aspartic protease 30 12 AJ223315 11 AJ224333 34 AJ223327 34 X85092 Chapter Asp f 11 peptidyl-prolyl isomeras 24 Asp f 12 heat shock prot. P90 90 Asp f 13 alkaline serine protease 34 Asp f 15 16 AJ002026 Asp f 16 43 g3643813 Asp f 17 AJ224865 Asp f 18 vacuolar serine protease 34 Asp f 22w enolase 46 AF284645 Asp f 23 L3 ribosomal protein 44 AF464911 Asp f 27 cyclophilin 18 Asp f 28 thioredoxin 12 Asp f 29 thioredoxin 12 Asp n 14 beta-xylosidase 105 Asp n 18 vacuolar serine protease 34 Asp n 25 3-phytase B Aspergillus niger AF108944 66-100 P34754 X17561 D00434, M33218 Aspergillus oryzae Asp o 13 alkaline serine protease 34 Asp o 21 Penicillium brevicompactum TAKA-amylase A 53 Pen b 13 alkaline serine protease 33 Pen b 26 acidic ribosomal prot. P1 11 Pen ch 13 alkaline serine protease 34 Pen ch 18 vacuolar serine protease 32 Pen ch 20 N-acetyl glucosaminidas 68 AY786077 Penicillium chrysogenum (formerly P.notatum) Penicillium citrinum Pen c peroxisomal mem. prot. 18 Pen c 13 alkaline serine protease 33 Pen c 19 heat shock prot. P70 70 U64207 Pen c 22w enolase 46 AF254643 Pen c 24 elongation factor beta AY363911 Penicillium oxalicum Pen o 18 vacuolar serine protease 34 Fus c ribosomal prot. P2 11 AY077706 Fus c thioredoxin-like prot. 13 AY077707 Fusarium culmorum Trichophyton rubrum Tri r Tri r serine protease 31 Chapter Trichophyton tonsurans Tri t 30 Tri t serine protease 83 Candida albicans Cand a Cand a 40 peroxisomal protein 29 AY136739 20 J04984, J04985 Candida boidinii Cand b Psilocybe cubensis Psi c Psi c cyclophilin 16 Cop c leucine zipper protein 11 Cop c thioredoxin Coprinus comatus AJ132235 AJ242791 Cop c AJ242792 Cop c AJ242793 Cop c AJ242794 Rhodotorula mucilaginosa Rho m enolase 47 Rho m vacuolar serine protease 31 AY547285 MF1, peroxisomal 21 AB011804 20 AB011805 35 AF084828 Malassezia furfur Mala f membrane protein Mala f MF2, peroxisomal membrane protein Mala f mitochondrial malate dehydrogenase Malassezia sympodialis Mala s X96486 Mala s 18 AJ011955 Mala s 17 AJ011956 Mala s AJ011957 Mala s 19 AJ011958 Mala s 37 AJ011959 Mala s 10 heat shock prot. 70 86 AJ428052 Mala s 11 Mn superoxide dismut. 23 AJ548421 Mala s 12 glucose-methanol-choline oxidoreductase 67 AJ871960 Mala s 13 thioredoxin 12 serine protease 30 Epicoccum purpurascens (formerly E.nigrum) Epi p 32 P83340 Chapter 1.3.4 Prevalence of fungal spores in Singapore environment In line with the global prevalence of fungi, an aerobiology survey conducted in Singapore showed abundant presence of fungal spores (Tan et al., 1992). Fungal spores were found to occur perennially in the Singapore air. Numerically, the fungal spores dominated around 86-89% of the total airspora, exceeding fern and pollen spore counts (Lim et al., 1998). Cladosporium (48%) was the most abundant spore type, followed by Didymosphaeria (31%) and Pithomyces (12%), Curvularia (5%) and Drechslera (2%) (Lim et al., 1998). The abundance of Cladosporium and Curvularia was consistent with that of the surveys carried out in different parts of the world but the presence of Didymosphaeria and Pithomyces was unique as it had not been reported elsewhere (Lim et al., 1998). The abundance of Pithomyces was different from the fungal profile reported in the neighboring country and some other parts of the world where it constituted less than 1% (Lim et al., 1998). This suggests that the fungal airflora in Singapore was distinct and different on some aspects. A five year survey (June 1990-June95) was also conducted to study the indoor as well as to follow the sporulation patterns of various fungal spores. It was observed that spores of Didymosphaeria, Pithomyces and Curvularia were present in the environment for more than 80% of the days sampled (Figure 1.4). This data suggests that the climatic conditions of Singapore favor growth of these fungi almost all year round. Distinct seasonal variations in the spore densities were observed despite the absence of climatic seasons in Singapore (Lim et al., 1998). An average spore count of 1688 spores m-3day-1 was found while the maximum spore load was found around 19,000 spores m-3day-1 (Lim et al., 1998). 33 Chapter Figure 1.4: Five years` survey (June1990-June95) highlighting the occurrence of various fungal spore types (%number of days of occurrence / total number of surveyed days) in Singapore environment Pithomyces Didymosphaeria Curvularia Drechslera Cladosporium Fungal spore type Tetraploa Torula Pleospora Didymopleela Hiospira Smut fungus Alternaria Pringsheimia Grallomyces Beltrania Unknown fungi 20 40 60 80 100 Occurence (% number of days) 34 Chapter Lim et al. (1998) also observed two periods of high spore density each year, from February to March and from October to November. 1.3.5 Studies on airborne fungal allergy in Singapore Annually, an estimated 140,000 individuals suffer from asthma and more than 100 individuals die of this disease resulting in an estimated medical cost of US $33.93 million per annum in Singapore (Chew et al., 1999). As mentioned earlier, airspora studies conducted in Singapore showed the presence of fungal spores, a number of which are unique to this region. The high amount of fungal spore load in the environment raises the question of possible sensitization in atopic patients to these fungi. Hence a study (as part of an international effort to evaluate the effect of asthma and allergy around the world) was carried out in school-going children in Singapore (Goh et al., 1996). As part of the study, about 6000 school-going children (aged between 6-7 years) and about 4000 children (aged between 12-15 years) were provided with the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. The results showed that allergic disorders are common to Singaporean children and the prevalence was comparable to some populations in west countries. The overall cumulative prevalence of wheezing in children was found to be about 22% in children of age group of 6-7 years, and 12% in those of age 12-15 years. In the above study, demographics and socioeconomic factors influenced the prevalence and severity of allergic disorders with a higher prevalence of rhinitis and wheezing in male subjects with higher socio-economic status. It was also observed from the questionnaire survey that there was under-recognition of childhood asthma in these 35 Chapter school-going children, about 49% of 1856 children with asthma-like symptoms had not been previously diagnosed with asthma (Chew et al., 1999). In 2000, Chew et al. reported on the allergenicity of locally important fungal spore types that were frequently found in Singapore’s environment as previously reported by Lim et al., (1998). It was found that fungal spores induced allergic reactions in about 30% of the atopic Singaporean population on skin prick test (SPT) reactions. Curvularia spp. which induced about 26-32% of the population showed the highest response, followed by Drechslera-like spores accounting for around 30% response. Overall, about 80% of the patients showed skin reactivity to at least one of the fungal allergens tested (Table 1.4). The association of prevalent fungal spores with atopy suggested that these fungi play a major role in allergic diseases in the tropics (Chew et al., 2000). Curvularia was found to be the fungus of great importance locally, and a better understanding of allergens from this genus would assist in better understanding the allergic reactions and would also aid towards developing SIT against fungal allergens. 1.4 Curvularia Curvularia is a dematiaceous fungus. Most of the Curvularia species are facultative pathogens, plants and cereals in tropical/subtropical areas and common in the soil. They are commonly found as saprophytes on cereals (Domsch et al., 1980). The conidia are multicellular, colored and develop in acropetal manner (youngest at the tip 36 Chapter Table 1.4: Frequency of skin test sensitivity to fungi in Singaporean atopic population. The positive counts were evaluated via Fisher`s exact test comparing the patient groups with the healthy controls. * p[...]... protease 31 AY547285 MF1, peroxisomal 21 AB 011 804 20 AB 011 805 35 AF084828 Malassezia furfur Mala f 2 membrane protein Mala f 3 MF2, peroxisomal membrane protein Mala f 4 mitochondrial malate dehydrogenase Malassezia sympodialis Mala s 1 X96486 Mala s 5 18 AJ 011 955 Mala s 6 17 AJ 011 956 Mala s 7 AJ 011 957 Mala s 8 19 AJ 011 958 Mala s 9 37 AJ 011 959 Mala s 10 heat shock prot 70 86 AJ428052 Mala s 11 Mn superoxide... AJ223 315 X85092 Chapter 1 Asp f 11 peptidyl-prolyl isomeras 24 Asp f 12 heat shock prot P90 90 Asp f 13 alkaline serine protease 34 Asp f 15 16 AJ002026 Asp f 16 43 g3643 813 Asp f 17 AJ224865 Asp f 18 vacuolar serine protease 34 Asp f 22w enolase 46 AF284645 Asp f 23 L3 ribosomal protein 44 AF464 911 Asp f 27 cyclophilin 18 Asp f 28 thioredoxin 12 Asp f 29 thioredoxin 12 Asp n 14 beta-xylosidase 10 5 Asp... X78228 Cla h 12 acid ribosomal prot P1 11 X8 518 0 alkaline serine protease 34 Cladosporium herbarum AY1 918 16 Aspergillus flavus Asp fl 13 Aspergillus fumigatus Asp f 1 18 M837 81, S39330 Asp f 2 37 U56938 19 U20722 30 AJ0 017 32 40 Z30424 26.5 U535 61 Asp f 3 peroxisomal protein Asp f 4 Asp f 5 metalloprotease Asp f 6 Mn superoxide dismut Asp f 7 Asp f 8 12 11 Asp f 10 aspartic protease 30 AJ223327 34 Asp... proteins In view of these, identification of various allergenic components of Curvularia will contribute to a better understanding of the repertoire of Curvularia allergens, and of the allergies caused by Curvularia This would then be useful in targeting various diagnostics as well as immunotherapy tools towards Curvularia allergies 40 Chapter 1 1.5 Objectives of the present study The purpose of this study... study is to identify and characterize the allergens from Curvularia lunata and to characterize these allergens and cross-compare it with other putative as well as known allergen homologs The obtained data will help in understanding the allergens and allergenicity of Curvularia lunata as well as fungi in general and will assist in development of improved fungal allergy diagnosis and will provide with... (Chew et al., 2000) Curvularia was found to be the fungus of great importance locally, and a better understanding of allergens from this genus would assist in better understanding the allergic reactions and would also aid towards developing SIT against fungal allergens 1. 4 Curvularia Curvularia is a dematiaceous fungus Most of the Curvularia species are facultative pathogens, plants and cereals in tropical/subtropical... (27.3) * * * 46 (19 .9) 37 (16 .0) 8 (10 .5) 6 (7.9) 0 (0.0) 5 (6.6) 2 (2.6) 64 (27.7) * * 54 (23.4) * * 60 (26.0) * * * 60 (26.0) * * * 73 ( 31. 6) * * * 5 (6.6) 6 (7.9) 6 (7.9) 71 (30.7) * * * 71 (30.7) * * * 41 (17 .1) * Single species spore types Cladosporium Didymosphaeria Pithomyces Tetraploa Curvularia spp Spores Curvularia brachyspora Curvularia fallax Curvularia inequalis Curvularia lunata Curvularia. .. Trichophyton tonsurans Tri t 1 30 Tri t 4 serine protease 83 Candida albicans Cand a 1 Cand a 3 40 peroxisomal protein 29 AY136739 20 J04984, J04985 Candida boidinii Cand b 2 Psilocybe cubensis Psi c 1 Psi c 2 cyclophilin 16 Cop c 1 leucine zipper protein 11 Cop c 2 thioredoxin Coprinus comatus AJ132235 AJ2427 91 Cop c 3 AJ242792 Cop c 5 AJ242793 Cop c 7 AJ242794 Rhodotorula mucilaginosa Rho m 1 enolase 47 Rho... dehydrogenase 29 Alt a 10 aldehyde dehydrogenase 53 AY1 918 15 X78227, P420 41 Alt a 12 acid ribosomal prot P1 11 X84 216 Alt a 13 glutathione-S-transferase 26 AY 514 673 Cla h 2 Ag54 23 Cla h 5 acid ribosomal prot P2 11 X78223 Cla h 6 enolase 46 X78226 Cla h 7 YCP4 protein 22 X78224 Cla h 8 mannitol dehydrogenase Cla h 9 vacuolar serine protease 55 AY787775 Cla h 10 aldehyde dehydrogenase 53 X78228 Cla h 12 acid ribosomal... et al., 19 98) An average spore count of 16 88 spores m-3day -1 was found while the maximum spore load was found around 19 ,000 spores m-3day -1 (Lim et al., 19 98) 33 Chapter 1 Figure 1. 4: Five years` survey (June1990-June95) highlighting the occurrence of various fungal spore types (%number of days of occurrence / total number of surveyed days) in Singapore environment Pithomyces Didymosphaeria Curvularia . sympodialis Mala s 1 X96486 Mala s 5 18 AJ 011 955 Mala s 6 17 AJ 011 956 Mala s 7 AJ 011 957 Mala s 8 19 AJ 011 958 Mala s 9 37 AJ 011 959 Mala s 10 heat shock prot. 70 86 AJ428052 Mala s 11 Mn superoxide. mannitol dehydrogenase 29 AY1 918 15 Alt a 10 aldehyde dehydrogenase 53 X78227, P420 41 Alt a 12 acid ribosomal prot. P1 11 X84 216 Alt a 13 glutathione-S-transferase 26 AY 514 673 Cladosporium herbarum. Chapter 1 19 CHAPTER 1: BACKGROUND AND INTRODUCTION Chapter 1 20 1. 1 HYPERSENSITIVITY REACTIONS AND ALLERGY 1. 1 .1 Hypersensitivity