Fungal infections

Esther Keizer; Ivan Valdes; Adriana Celis Ramirez
Han Wösten
Hans de Cock

Pathogenic fungi have developed strategies to live and survive in human hosts. The immune system of the host is involved in clearing fungi thereby preventing the development of a mycosis. Candida spp, Cryptococcus spp and Aspergillus spp belong to the most common fungal species that give fungal infections in human. In my lab we focus on Aspergillus spp, a common fungus found in our surroundings. Daily we come into contact with this fungus, mostly by inhalation of Aspergillus spores without being affected. Aspergillosis is developing especially in humans with a weakened immune system or with certain lung diseases. Aspergillus fumigatus is frequently the causative agent for lung infections whereas Aspergillus niger is associated with lung or ear infections. Due to the increased use of immunosuppressive drugs the number of fungal infections is increasing in the western world. 

Our immune systems is composed of the innate and adaptive immune system which is in general efficient to eliminate pathogens. Recognition of pathogens via their PAMPs (Pathogen-Associated Molecular Pattern) of MAMPs (Microbe-Associated Molecular Pattern) by the Pattern Recognition Receptors (PPRs) present on cells of the innate immune system helps to eliminate most pathogens. Fungi are often recognized cell wall components like α or β glucans, chitin or O- or N-linked mannan. 

We are interested in components released or actively secreted by Aspergillus spp that interact with components of the innate or adaptive immune system. These components include (but are not limited by) cellular components (neutrophils, monocytes), complement system, clotting cascade, endothelium and lung epithelium. 
 

Project 1 “Aspergillus infections and immune evasion”

Expression profiles of Aspergillus is highly dependent on culture conditions, like media, temperature, growth phase and possibly varies between different strains. Therefore, different culture conditions and Aspergillus strains will be used to obtain an Aspergillus supernatant. This supernatant will be screened for components that interact with the human immune system and components which will be purified and characterized. Interactions with various components of the immune system will be studied using various binding and (semi)functional essays. There is an extensive collaboration within the university of Utrecht on protein-protein interactions and protein structure. 

In order to study the importance of identified proteins for Aspergillus growth and pathogenicity, gene knock outs will be constructed. These gene knock-outs will be tested in animal models of invasive aspergillosis. These include mouse models of invasive pulmonary and cutaneous aspergillosis. 

Interactions between fungi and lung epithelium will be studied in newly developed in vitro systems in which interaction, binding and growth of A. niger will be compared to clinically relevant strains. 
 

Project 2 “Gene expression during fungal infections”

In order to obtain more insight into the infections process of Aspergillus spp, and especially gene expression during fungal infections in vivo in patients, we have initiated studies on fungal infections in dogs in collaboration with the Veterinary Medicine at Utrecht University. The form of human fungal sinusitis that most closely approximates the disease occurring in the dog is chronic erosive non-invasive fungal sinusitis. This disease is characterized by destruction of bone in the absence of tissue invasion by the fungus and requires both removal of necrotic tissue and medical therapy with antifungals. Aspergillus fumigatus has been reported to be the most common species in these infections and typing of the strain is based on morphology and culturing. 

Mucosal immune response in SNA infections has been studied in more details in material obtained via biopsy and cytokine profiling as well as transcriptomic analysis of the tissue. Results indicated an upregulation of the Th-1 response (cellular immunity). The unsuccessful clearance of the infection might be due to increased expression of factors that dampen the immune response (like IL-10 and Transforming Growth factor beta). Tissue damage might be due to the excessive immune response as well as expression of necrotic toxins by the fungus (like gliotoxin). 

After removal of fungal plaque the material can be used to purify RNA and to perform transcription analysis by RNA sequencing. Strains can be identified and typed via molecular tools (in collaboration with KNAW-CBS, group Samson/Houbraken). Such novel studies will provide for the first time insight into the genes expressed during infection in vivo in a patient and is expected to provide novel information on the molecules produced during pathogenesis and initiate further studies to the role they might have during infection(e.g. involvement in immune modulation). 

We will compare gene expression between in vitro (lab culture) and in vivo (in patient) grown Aspergillus and focus on genes up regulated during in vivo growth. We hope to identify regulators required to up regulate gene expression under conditions of growth in vivo and which products are required for virulence. 
 

Project 3 “Understanding the pathogenic properties of Malassezia spp”

These yeasts are part of the normal microbiota of humans and some warm-blooded animals, mostly present in regions rich in sebaceous glands. They are characterized by lipid-dependency, due to the lack of a FAS which results in a defect to synthesis of fatty acid palmitate. Malassezia species have been associated with different dermatologic conditions of high prevalence including dandruff, seborrheic dermatitis and pityriasis versicolor, among others. Moreover, they have been related to systemic infections in neonates and thus recognized as opportunistic pathogens. However their pathogenic role has not been fully elucidated. 

The pathogenic role of Malassezia spp. has been related to several factors, including the ability to produce esterases, lipases, lipoxygenases and proteases. On the other hand, the final products of lipids metabolism appear to be associated to the production of inflammation, irritation and scaling in susceptible individuals. 

We are interested in the genomics and transcriptomics of virulence factors in vitro and in vivo and will initiate studies to genetically modify Malassezia spp. 

Collaborations 

Project 1 is a collaboration with the medical microbiology group at UMCU (van Strijp) at Utrecht University and the Molecular Host Defence group at Veterinary Medicine (Prof. dr. H Haagsman). With the veterinary hospital (Small Animal Surgery, Department of Clinical Sciences of Companion Animals Faculty of Veterinary Medicine, Utrecht University) we will investigate the SNA in dogs (project 2). 

Project 3 is a collaboration with Adriana Celis (grant from Netherlands Fellowship Programme (NFP), Universidad de Los Andes, Faculty of Science, Mycology and plant pathology lab (LAMFU), Bogota, Colombia.