Produkce a Charakterizace Lektinu MCL Z Patogenní Plísně

PŘÍRODOVĚDECKÁ FAKULTA

Produkce a charakterizace lektinu MCL z patogenní plísně

Diplomová práce

MARTINA RIEVAJOVÁ

Vedoucí práce: prof. RNDr. Michela Wimmerová, Ph.D. Mgr. Lenka Malinovská, Ph.D.

Ústav biochemie

Brno 2021

Bibliografický záznam

Autor: Bc. Martina Rievajová Přírodovědecká fakulta Masarykova univerzita Ústav biochemie Název práce: Produkce a charakterizace lektinu MCL z patogenní plísně Studijní program: Biochemie Specializace: Analytická biochemie Vedoucí práce: prof. RNDr. Michela Wimmerová, Ph.D. Mgr. Lenka Malinovská, Ph.D. Rok: 2021 Počet stran: 68+1 Klíčová slova: Microsporum canis; lektín; MCL; AAL rodina; purifikace proteinu; hemaglutinace

Bibliographic record

Author: Bc. Martina Rievajová Faculty of Science Masaryk University Department of Biochemistry Title of Thesis: Production and characterization of lectin MCL from pathogenic mould Degree Programme: Biochemistry Field of Study: Analytical biochemistry Supervisor: prof. RNDr. Michela Wimmerová, Ph.D. Mgr. Lenka Malinovská, Ph.D. Year: 2021 Number of Pages: 68+1 Keywords: Microsporum canis; lectin; MCL; AAL family; protein purification; hemagglutination

Abstrakt

Lektiny jsou proteiny se schopností vázat sacharidy specificky a reverzibilně. Mnoho patogenních organismů používá lektiny pro adhezi na hostitelské buňky, takže lektiny jsou často zásadní pro iniciaci infekce. Znalosti o těchto lektinech lze využít pro vývoj optimální léčby. Tato diplomová práce se zaměřuje na purifikaci a charakterizaci lektinu MCL z patogenní houby Microsporum canis. Tento dermatofyt způsobuje povrchové kožní infekce známé jako tinea. Infikuje převážně zvířata, ale napadá i člověka. Pro purifikaci MCL byla použita afinitní chromatografie a také byla provedena renaturace MCL z inkluzních tělísek. Pro charakterizaci tohoto proteinu byly použity metody CD spektroskopie, nanoDSF a testy inhibice hemaglutinace.

Abstract Lectins are ubiquitous proteins with the ability to bind saccharides specifically and reversibly. Many pathogenic organisms use lectins for adhesion to host cells. Therefore, lectins are often crucial for the initiation of infection, and knowledge about these lectins can be used for the development of optimal treatment. This diploma thesis focuses on the purification and characterization of lectin MCL from the pathogenic fungus Microsporum canis. This dermatophyte causes superficial skin infections known as tinea and predominantly infects animals, but also humans. For purification of MCL, affinity chromatography was used, and refolding of MCL from inclusion bodies was performed. For characterization of this protein were used methods CD spectroscopy, nanoDSF, and hemagglutination inhibition assay.

Declaration I declare that I wrote this diploma thesis by myself under the supervision of my supervisors, using only the literature listed in the references.

Brno May 28, 2021 ...... Martina Rievajová

Acknowledgment I would like to thank prof. RNDr. Michaela Wimmerová, Ph.D., for the opportunity to work in the Glycobiochemistry group and for all the professional advice. I am also very grateful for all the help, guidance, and incredible amount of patience I received from Mgr. Lenka Malinovská, Ph.D.. To all members of the group, thank you for the friendly environment and many helpful pieces of advice. And last but not least, I would like to thank my family, who supported me and encouraged me throughout my studies.

Table of Contents

List of abbreviations ...... 12 Introduction ...... 13 Theoretical part ...... 14 1. Lectins ...... 14 1.1. Characterization of lectins ...... 14 1.2. Animal lectins ...... 14 1.3. Plant lectins ...... 15 1.4. Microbial lectins ...... 16 1.5. Fungal lectins ...... 17 1.5.1. Lectins from pathogenic fungi ...... 17 1.6. Applications of lectins ...... 18 2. Dermatophytes ...... 19 2.1. Characterization ...... 19 2.2. Pathogenesis ...... 20 2.3. Genus Microsporum ...... 21 2.3.1. Microsporum canis ...... 21 3. Hypothetical lectin from Microsporum canis and its homologues ...... 23 3.1. Microsporum canis lectin...... 23 3.2. Aleuria aurantia lectin ...... 24 3.3. Aspergillus fumigatus lectin ...... 24 Aims of the thesis ...... 26 Experimental part ...... Error! Bookmark not defined. Material and methods ...... Error! Bookmark not defined. 1. Production of MCL ...... Error! Bookmark not defined. 1.1. Production of MCL in Tuner (DE3) cells ...... Error! Bookmark not defined. 1.2. Production of MCL in ArcticExpress (DE3) cells...... Error! Bookmark not defined. 1.2.1. Selection of the best clone for large scale production of MCL ...... Error! Bookmark not defined. 1.2.2. Large-scale production of MCL in ArcticExpress (DE3) cells ...... Error! Bookmark not defined. 1.3. Disruption of cells ...... Error! Bookmark not defined. 1.3.1. Solubility test ...... Error! Bookmark not defined. 2. SDS-PAGE ...... Error! Bookmark not defined. 2.1. Coomassie Brilliant Blue R250 staining ...... Error! Bookmark not defined. 2.2. Silver staining ...... Error! Bookmark not defined.

3. Purification of MCL ...... Error! Bookmark not defined. 3.1. Fast protein liquid chromatography ...... Error! Bookmark not defined. 3.2. Purification of MCL on gravity column ...... Error! Bookmark not defined. 4. Refolding of MCL from ArcticExpress (DE3) inclusion bodies . Error! Bookmark not defined. 5. Refolding of MCL from Tuner (DE3) inclusion bodies ...... Error! Bookmark not defined. 5.1. Optimisation of refolding from inclusion bodies Error! Bookmark not defined. 5.2. Refolding of MCL from inclusion bodies after optimization..... Error! Bookmark not defined. 6. Lyophilization ...... Error! Bookmark not defined. 7. Characterization of MCL ...... Error! Bookmark not defined. 7.1. CD spectroscopy ...... Error! Bookmark not defined. 7.2. nano Differential Scanning Fluorimetry (nanoDSF) ...... Error! Bookmark not defined. 7.3. Hemagglutination and hemagglutination inhibition assay .. Error! Bookmark not defined. Results and discussion ...... Error! Bookmark not defined. 1. Production of MCL in ArcticExpress (DE3) cells . Error! Bookmark not defined. 2. Purification of MCL using fast protein liquid chromatography Error! Bookmark not defined. 2.1. Purification of MCL on fucose-sepharose column ...... Error! Bookmark not defined. 2.2. Purification of MCL on mannose-agarose column with isocratic elution ...... Error! Bookmark not defined. 2.3. Purification of MCL on galactose-agarose column...... Error! Bookmark not defined. 3. Disruption of ArcticExpress (DE3) cells ...... Error! Bookmark not defined. 4. Purification of MCL using gravity column ...... Error! Bookmark not defined. 5. CD spectroscopy ...... Error! Bookmark not defined. 6. Nano differential scanning fluorimetry ...... Error! Bookmark not defined. 7. Refolding of MCL from inclusion bodies of ArcticExpress (DE3) cells ...... Error! Bookmark not defined. 8. Hemagglutination inhibition assay ...... Error! Bookmark not defined. 9. Optimization of refolding from inclusion bodies ... Error! Bookmark not defined. 9.1. Optimization of extraction buffer ...... Error! Bookmark not defined. 9.2. Optimization of refolding buffer and protein concentration...... Error! Bookmark not defined. 10. Refolding of MCL from inclusion bodies of Tuner (DE3) cells ...... Error! Bookmark not defined.

Summary ...... Error! Bookmark not defined. List of references ...... 27

List of abbreviations

AAL Aleuria aurantia lectin AFL Aspergillus fumigatus lectin APS ammonium persulfate BanLec lectin from banana CD circular dichroism Cpn60 chaperonin 60 DLS dynamic light scattering EDTA ethylenediaminetetraacetic acid GdmCl guanidium chloride His-tag polyhistidine-tag IgE immunoglobulin E

IPTG isopropyl β-D-1-thiogalactopyranoside LB medium Luria-Bertani medium MBL mannose-binding lectin MCL Microsporum canis lectin MES 2-(N-morpholino) ethane sulfonic acid MS mass spectrometry nanoDSF nano differential scanning fluorimetry

OD600 optical density at 600 nm OmpA outer membrane porin A OmpF outer membrane porin F PA-IIL Pseudomonas aeruginosa lectin 2 PBS phosphate-buffered saline buffer PCR polymerase chain reaction SDS sodium dodecyl sulphate SDS-PAGE sodium dodecyl sulphate–polyacrylamide gel electrophoresis Sub3 subtilisin 3 TEMED tetramethyl ethylenediamine Tris tris(hydroxymethyl)aminomethane

Introduction Lectins are proteins with the ability to specifically and reversibly bind saccharides. They are ubiquitous in nature and are involved in many physiological and pathological processes. Many pathogens use lectins for adhesion to host cells in the early stages of infection. Knowledge about lectins involved in pathogenesis can be used for the development of anti-adhesion therapy for the prevention and treatment of infections. Microsporum canis is a pathogenic dermatophytic fungus that infects the upper layers of skin. It causes a disease known as ringworm or tinea. It infects animals predominantly, but it can also infect humans. A hypothetical Microsporum canis lectin was identified in the genome of this organism. The aim of this thesis is to purify the hypothetical Microsporum canis lectin in recombinant form and then characterize the protein using various techniques. Affinity chromatography was used for the purification of the protein. Refolding from inclusion body proteins was also performed. CD spectroscopy, nanoDSF, and hemagglutination inhibition assay were used for the characterization.

Theoretical part

1. Lectins 1.1. Characterization of lectins Lectins are proteins that have the ability to specifically and reversibly bind saccharides. They are of non-immune origin, and they do not change the bound saccharides in any way, distinguishing them from antibodies and enzymes. Lectins are ubiquitous in nature, they can be found in organisms ranging from viruses to animals and humans. Based on their organism of origin, they can be classified into microbial, fungal, plant and animal lectins (1). Another type of classification of lectins is based on their monosaccharide specificity. Most lectins show an affinity for one of five monosaccharides that naturally occur on the surface of eukaryotic cells. These saccharides are L-fucose, D-galactose/N-acetyl-D-galactosamine, sialic acid, D-mannose, and N-acetyl-D-glucosamine. However, lectins with a high affinity for the same monosaccharide may have vastly different affinities for various oligosaccharides. That is because the affinity of lectins for monosaccharides is generally low. Meanwhile, their affinity to oligosaccharides can be up to 1000-fold higher. Some even bind oligosaccharides exclusively (2). In general, lectins are oligomeric proteins consisting of several subunits. Each subunit may contain two or more saccharide binding sites. Because of that, their interaction with surface saccharides of cells leads to crosslinking and subsequent precipitation of the cells. Crosslinking of erythrocytes is called hemagglutination and is a common feature of most lectins, regularly used for their detection and characterization. Another common method for the identification of new lectins is using amino acid sequence homology with known lectins (1,2).

1.2. Animal lectins Animal lectins are a structurally diverse group, at least 15 structural families are known. Additionally, many animal lectins do not belong to any of these families, thanks to their unique structure. Besides the carbohydrate-binding domain, animal lectins frequently also have other domains which mediate binding of other ligands than saccharides through protein-protein, protein- lipid, or protein-nucleic acid interactions. However, the carbohydrate-binding site may also be responsible for binding non-saccharide ligands (3,4). The functions of animal lectins are various. They may be involved in cell adhesion and cell recognition, can monitor the migration of leucocytes in blood vessels, or contribute to proliferation and metastasis of tumour cells (5). Many animal lectins have roles within the immune system. One of the best characterized is a component of innate immunity called mannose-binding lectin (MBL). This C-type lectin functions as

a pathogen-recognition molecule and can activate the complement system. Its deficiency is prevalent in all studied human populations, potentially leading to higher susceptibility to infectious diseases (6,7). Examples of lectins with non-immune function are calnexin and calreticulin. These proteins are chaperons located predominantly in the endoplasmic reticulum. Calnexin is a membrane protein, and calreticulin is its soluble sequential homologue. The primary function of calnexin is protein chaperoning. Calreticulin also controls the regulation of Ca2+ homeostasis, regulates genes, and binds RNA in addition to protein chaperoning (5,8).

1.3. Plant lectins It is generally believed that the first description of lectins dates back to 1888 when a highly toxic plant lectin called ricin was observed in seeds of castor beans. Plant lectins are located either in the whole plant or in various tissues, like the seeds, bark, leaf, or rhizome. They can be found most often in Leguminosae and Euphorbiaceae families but are also present in many others (9). Based on the structure of their carbohydrate-binding domain, all presently known and characterized plant lectins can be classified into 12 families, with the exception of a lectin from Dioscorea batatas (10). Even though plant lectins are studied the longest among all lectins, their functions still have not been fully elucidated. Lectins from leguminous plants are presumed to facilitate the establishment of symbiosis with the nitrogen-fixing bacteria. Another more general function of plant lectins is their role in defence mechanisms against the attack of microorganisms. They can inhibit the growth of several phytopathogenic and non-pathogenic fungi and are also involved in defence against oomycetes (1,11). For example, a lectin from Nicotiana benthamiana is required in the resistance reactions against Phytophthora infestans (12). As lectins can be found in many common food crops, such as wheat, rice, tomato, or bean, a lectin-free diet has become popular in recent years, claiming lectins have a detrimental effect on health. The research on dietary effects of lectins is currently minimal, so no scientific evidence supporting the claim exists (13). However, some harmful effects have been noticed. For example, in Japan, lectin poisoning occurred after the consummation of powdered toasted white kidney beans introduced by a TV broadcast. This preparation method was inadequate, as lectins did not lose activity and were very likely the reason for acute intestinal problems for a number of people (14). Food lectins have also been found to cause non-allergic food hypersensitivity through interaction with glycans of IgE antibodies on the surface of basophils and mast cells, one of these lectins being BanLec from banana fruit. (15).

1.4. Microbial lectins The first identified microbial lectin was isolated from the influenza virus. This lectin, called hemagglutinin, binds to sialic acid located on the host cell surface (Fig. 1), facilitating the release of the viral RNA into the host cell. The difference in specificity for oligosaccharides plays a role in the host specificity of various influenza strains. It is also an important antigen against which antibodies are created. Changes in this protein are partially responsible for the regular need for a new vaccine formulation (16).

Figure 1. Lectin-saccharide interactions on the cell surface. Lectins are used for the attachment of cells, bacteria, and viruses to other cells. (Taken and edited from 17).

Lectins can also be found in bacteria, where most known bacterial lectins are located on the bacterial cell surface. However, a small number of soluble bacterial lectins have also been described. The bacteria use cell surface lectins for the attachment to the host cell, a crucial step in the initiation of infection. Inhibition of these lectins can be used for anti-adhesion therapy of bacterial diseases. These lectins are usually in the form of fimbriae or pili, which are submicroscopic filamentous appendages. Some of the best-characterized bacterial lectins are type-1 fimbriae, P fimbriae, and F- 17 fimbriae found in E. coli strains. The specificity of bacterial lectins can indicate which tissues are susceptible to infection, based on the type of glycans the tissues express (2,16). Several bacterial toxins also use the binding of sugars in the process of recognition and entering their host cells. These toxins are, for example, Shiga toxin from Shigella dysenteriae, tetanus toxin from Clostridium tetani, or extensively studied cholera toxin from Vibrio cholera. Cholera toxin is a complex formed by two types of subunits, one of which contains a carbohydrate-recognizing 16

domain. This type of subunit is used for binding on the membrane. After endocytosis and transport of the toxin to the endoplasmic reticulum, the other type of subunit relocates to the cytosol, severely altering the infected cell's ion homeostasis (16). Apart from viruses and bacteria, other parasites such as protozoa use lectins for adhesion. One of them is Entamoeba histolyca, which expresses several lectins mediating the adhesion to host cells (18).

1.5. Fungal lectins

Fungi are a rich source of new lectins with unique saccharide specificities. The research of fungal lectins began with lectins from higher fungi. The first fungal lectin was isolated from fly agaric (Amanita muscaria) in 1910. Its activity was found to be linked to the toxicity of the fungus. Later, lectins were also found in edible fungi such as Boletus edulis and Lactarius deliciosus. Most lectins were identified in mushrooms. A smaller number was also found in microfungi and yeasts. They have been isolated from mycelium, conidia, basidiomes, and fruiting bodies of fungi (19,20). Based on their structure, fungal lectins can be divided into nine structural families (21). The physiological functions of fungal lectins are various and, in many cases, unknown. They may serve as storage proteins and play a role in growth and morphogenesis, defense, or molecular recognition in mycorrhization and lichens. As many fungi are pathogenic, their lectins can be involved in recognition of host glycans and early stages of infection (20).

1.5.1. Lectins from pathogenic fungi Lectins from pathogenic fungi are presumed to mediate interaction with the host leading to invasion and infection, similarly to lectins from pathogenic bacteria and viruses. Lectin from phytopathogenic fungus Magnaporthe oryzae, the rice blast fungus, is speculated to play a role in appressorium development, as it is preferentially expressed and localized in cells specialized in plant infection (22). Paracoccin, a lectin from fungus responsible for human paracoccidioidomycosis, binds to a component of the extracellular matrix called laminin during all stages of infection, leading to stimulation of release of key mediators of the infection (23). Epithelial adhesins of Candida glabrata, an opportunistic pathogen causing candidiasis, have domains with lectin activity that enable them to adhere to host cells by interactions with glycans expressed on the cell membrane of the host (24). Another example of lectins from pathogenic fungi potentially involved in infection are lectins from Trichophyton rubrum and Trichophyton mentagrophytes. Both these dermatophytic fungi express lectins on the surface of their microconidia, and in vitro experiments suggest that they may be involved in the infection (25,26).

1.6. Applications of lectins Lectins have a wide range of applications. They can be used as models for the study of protein- saccharide interactions, analysis of glycosylation, or in diagnostics and treatment of diseases (27). Protein-saccharide interactions are the basis of many critical biological processes, and their research may lead to the development of medicines and vaccines (28). They can be used to study glycosylation in proteomics and for the detection of altered glycosylation patterns typical for diseases such as cancer (27,29). They can also serve as carriers in lectin-drug conjugates, effectively delivering the drug to cells presenting specific glycans (30). Some plant lectins were even found to induce cell death and eliminate cancer cells (31).

The knowledge about the interaction of lectins with saccharides can be applied in anti-adhesion therapy. Pathogenic microbial organisms often use lectins for adhesion to host cells or for colonization of the host. Anti-adhesion therapy focuses on inhibition of lectin-saccharide interaction with the use of appropriate inhibitors (Fig. 2), which are either glycomimetic and neoglycoconjugate structures specifically synthesized based on properties of the involved lectin or also simple monosaccharides can be used (32,33). A significant advantage of anti-adhesive therapy is that it does not kill the pathogen, so the probability of development of resistance by the pathogen should be low (34). It shows a great promise, especially with the ever-rising cases of resistance to antibiotic treatment. It can be used as an alternative approach to treatment or with other agents to boost the therapeutic effect (32). A commonly available example of anti-adhesive therapy applications is ear cleansers for animals used for the prevention and treatment of bacterial and yeast infections. These drugs are ordinarily available in pharmacies. In addition to other active substances such as salicylic acid, they also contain saccharides supposed to block lectins from bacteria causing ear infections and help with the treatment of the infection (33).

Figure 2. Schematic illustration of anti-adhesive therapy mechanism. On the left, the bacterium is bound to glycans on the host cell. On the right, the binding of the bacterium was inhibited by a carbohydrate-based drug (35).

2. Dermatophytes 2.1. Characterization Dermatophytes are closely related pathogenic keratinolytic fungi, traditionally from three genera Microsporum, Trichophyton, and Epidermophyton. According to recent changes in taxonomy, three additional genera Nannizzia, Lophophyton, and Arthroderma, belong to dermatophytes (36). Based on their primary host, they are categorized as anthropophilic, zoophilic, or geophilic. They developed from keratinophilic soil organisms, but few pathogenic species remain primarily in this environment, and most have adapted to human and animal hosts. The dermatophytes that remained geophilic are saprophytes, deriving nutrients from keratinous substrates in soil, but they may infect humans and animals through contact with contaminated soil. Anthropophilic and zoophilic species are usually associated with a particular host, and infections of other species are not maintained long term. Anthropophilic species can occasionally also infect animals, and zoophilic species can infect humans (37,38). Dermatophytes degrade keratin as a source of nutrients. They invade keratinous tissues such as skin, hair, or nails. Dermatophytosis, also called ringworm or tinea, is a superficial skin infection. The spread of these pathogens stops when in contact with living cells or inflammation. The symptoms vary based on the dermatophyte species, affected tissue, and area of the body. Typical for the lesions are erythema, scaling, occasional blistering, and in haired areas brittleness of hair and alopecia. In hairless areas, the lesions are most strongly inflamed at the periphery as the pathogen moves circularly from the site of infection. The centre may heal, resulting in the lesion's typical ringworm appearance 19

(Fig. 3). In humans, lesions caused by geophilic and zoophilic species are usually more severely inflamed (37,38).

Figure 3. Ringworm in a cat and its owner caused by Microsporum canis. The cat has a lesion on the nose and front, the owner has a typical ringed lesion on the forearm (39).

2.2. Pathogenesis In the process of infection by dermatophytes, three steps can be recognized (Fig. 4). First is the adherence of arthroconidia to the stratum corneum. This step is essential for the establishment of the infection (40). Not much is known about the factors mediating the adhesion, but it is presumed to be mediated through the interactions of adhesins with host receptors. It has been found that Trichophyton rubrum and Trichophyton mentagrophytes recognize and bind mannose and galactose. These two saccharides are also able to inhibit adhesion. Treatment with trypsin and heating of the fungus reduced the adhesion, suggesting that the adhesion of these two fungi is mediated by proteins, probably lectins (25,26). It is also presumed that secreted proteases play a role in this complex process, but the mechanism of their contribution is unknown. One of these proteases is subtilisin 3 (Sub3), which was found to be necessary for the adherence of Microsporum canis and was identified on the surface of arthroconidia both in vitro and in vivo. It is presumed that Sub3 plays an important role in the colonization of the host (40,41,42). The next step in dermatophyte infection is germination. It occurs when adhered arthroconidia detect appropriate conditions for metabolic activity reactivation and start to grow into hyphae. Once grown, hyphae invade the epidermal cornified layer and start digesting keratin, which is the last step. In this step are essential secreted proteases, which digest keratin into short peptides and amino acids (42).

Figure 4. Schematic summary of epidermal infection by Trichophyton rubrum. One hour after contact begins adhesion of arthrospores to the epidermis, after four hours germination begins, after 24 hours hyphae invade stratum corneum. After four days immune system is activated when the epidermal barrier loses its integrity (40).

Overall, not much is known about the mechanism of infection. The research has been hindered by differences in results between in vivo and research models. Because of that, suitable models need to be used for the research of the mechanisms of infection, the immune response of the host, or the evaluation of treatment efficacy (40,42). 2.3. Genus Microsporum Genus Microsporum was first described by David Gruby in 1843 as a causative agent of human skin disease tinea capitis. Gruby also formulated the genus name based on the small spores produced around the hair shaft. According to older publications, the Microsporum genus contains 18 species, and several of them, for instance, M. canis, M. gypseum, and M. audounii, are clinically relevant pathogens (38). New taxonomy has been proposed in recent years, according to which genus Microsporum contains only three species (M. canis, M. audounii, and M. ferrugineum). The rest has been divided into other genera (36).

2.3.1. Microsporum canis

Microsporum canis is a zoophilic filamentous fungus, first described by E. Bodin in 1902 (36). The taxonomic classification of M. canis is listed in Table I. Primary hosts of M. canis are cats, dogs, and horses, but infection can also occur in other mammals. The most frequent hosts are cats, where M. canis is responsible for more than 90 % of cases of ringworm. The number is lower in dogs, around 21

70 to 80 %, but M. canis is still the predominant cause of ringworm (39). M. canis has also been isolated from rabbits (43), sheep (44), and elephants (45). Furthermore, M. canis can also infect humans through close contact with infected animals. The infection is most common in children and the elderly (38).

Table I. Taxonomic classification of M. canis (46).

Superkingdom Eukaryota Kingdom Fungi Phylum Ascomycota Class Eurotiomycetes Order Onygenales Family Arthrodermataceae Genus Microsporum Species M. canis

M. canis forms septate hyphae, which in living host contain arthroconidia. Arthroconidia are asexual spores developed inside the hyphae. In laboratory culture, the hyphae contain numerous macroconidia and less abundant microconidia (Fig. 5). The microconidia and macroconidia are asexual spores developed outside of hyphae (37). Macroconidia are long and spindle-shaped, have rough, thick walls, and their ends are beaklike. Their size varies between 10-25 × 35-110 µm. The surface of macroconidia is rough, especially at the ends. On the inside, they are typically divided into six or more compartments. Microconidia forming along the hyphae are club-shaped and have smooth walls (47). The macroscopic appearance of M. canis (Fig. 6) is fuzzy colonies white on one side, and the other side is bright yellow (48).

Figure 5. Microscopical structure of Microsporum canis. a) Structure of hyphae, microconidia, and macroconidia (49); b) Spindle-shaped macroconidia under the microscope (38); c) Macroconidia observed with cryo scanning electron microscopy (50).

Infections caused by M. canis are highly contagious, and treatment is necessary to prevent transmission. However, the symptoms are highly similar to those of other dermatophytes and other skin diseases, so correct diagnosis is essential for successful treatment. The quickest way to test for M. canis infection is to use Wood's lamp. M. canis fluoresces under UV light along with few other dermatophytes, so it is not a method for a specific diagnosis. However, it can be used to choose the site for the collection of samples for analysis by other methods. Fungal culture combined with direct examination is a more reliable method, and it is considered the golden standard for diagnosing dermatophytosis. This method is usually time-consuming, and there may be some problems, such as contamination by normal fungal flora. For a more quick, sensitive, and specific diagnosis, PCR and its variation are used. For treatment are used oral and topical antifungal drugs, such as griseofulvin, terbinafine, or itraconazole. M. canis infections are often recurring, and treatment failure is relatively high, possibly because of irresponsible pet owners, poor drug penetration, or resistance of pathogen (51,52).

a) b) Figure 6. Macroscopic appearance of Microsporum canis. a) Top side of the culture; b) bottom side of the culture showing bright yellow pigment (48).

3. Hypothetical lectin from Microsporum canis and its homologues 3.1. Microsporum canis lectin Hypothetical protein Microsporum canis lectin (MCL) is potentially the first lectin discovered in M. canis. It is presumed that this lectin could be involved in the pathogen's adhesion and may serve as a potential target for the prevention and treatment of M. canis infections. The molecular weight of this protein is 34,435 Da, and it consists of 313 amino acids. The sequence of this protein also contains signal peptide. MCL has been identified as a sequence homologue of the lectin AAL from Aleuria aurantia with 28% sequence identity. The high similarity is also shown with lectin AFL from Aspergillus fumigatus with the sequence identity 42 %. These lectins belong to the AAL family,

comprised of L-fucose specific lectins with the six-bladed beta-propeller fold. MCL was also predicted to have this fold using bioinformatics research (53). Earlier research of this protein exposed a mistake in the prediction of introns in the sequence of MCL from the database, where one exon is predicted shorter than it should be and another one has been entirely predicted as intron (54). However, even after correction of the sequence, this protein has not been successfully obtained in purified recombinant form thus far. As for saccharide specificity, experiments with a cell lysate containing MCL show that this protein prefers L-fucose, like the rest of the AAL family (53). 3.2. Aleuria aurantia lectin A close characterized homologue of MCL is Aleuria aurantia lectin (AAL). Aleuria aurantia, also known as orange peel fungus, is an edible saprophytic fungus belonging to the group Ascomycota. It has a bright orange colour and is shaped like a cup; its regular form becomes irregular and contorted by age. A. aurantia grows predominantly in North America and Europe on sandy or clay soil near forest roads (58,59). Aleuria aurantia lectin (AAL) was purified by affinity chromatography from fruiting bodies of A. aurantia as a 36 kDa L-fucose specific lectin. This lectin is a dimer in solution and crystal structure. The subunits are identical, and each consists of 312 amino acids. The monomer is made of 6 tandem repeats. The individual repeats are made of twisted four- stranded antiparallel beta-sheets. The overall fold of the monomer is a six-bladed beta- propeller (Fig. 7). Each monomer contains five binding sites for L-fucose located between consecutive blades (60). 3.3. Aspergillus fumigatus lectin Another close characterized homologue of MCL is Aspergillus fumigatus lectin (AFL). Aspergillus fumigatus is a saprophytic fungus living in the soil, and it has an important role in the recycling of environmental carbon and nitrogen. Conidia of A. fumigatus are commonly found in the air, and humans typically inhale several hundred conidia a day. The immune system of healthy individuals effectively eliminates these spores. However, they are dangerous for immunocompromised patients, who can develop severe infectious diseases such as invasive aspergillosis or allergic bronchopulmonary aspergillosis (55). Aspergillus fumigatus lectin was identified in the genome of A. fumigatus using a homology search of AAL lectin. This 34 kDa lectin is specific for L-fucose and is a dimer in solution and crystal structure. The monomer structure is a six-bladed beta-propeller. Each blade is formed by four antiparallel beta-sheets. The monomer has six binding sites for L-fucose located between consecutive blades (56). AFL is located on the surface of conidia and has a strong pro-inflammatory effect. This 24

lectin was considered a virulence factor, but new findings lead to a hypothesis that recognition of this lectin by the immune system actually prevents the infection (57).

a) b) Figure 7. Structure of lectins AAL and AFL. a) Six-bladed beta-propeller fold of AAL monomer (1OFZ); b) six-bladed beta-propeller fold of AFL monomer (4AGI).

Aims of the thesis

• Purification of the recombinant Microsporum canis lectin • Characterization of structural and binding properties of the recombinant Microsporum canis lectin

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