Candida Albicans Adaption to Host Microenvironments Drives Immune Evasion

Candida albicans adaption to host microenvironments drives immune evasion

José Pedro Lopes

Department Clinical Microbiology Umeå 2018

This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7601-919-1 ISSN: 0346-6612 Cover “Pink Peacock door stopper” by José Pedro Lopes Electronic version available at: http://umu.diva-portal.org/ Printed by: Umu Print & Media Umeå, Sweden 2018

Para Kristina, Matias e Klara

Table of Contents

Abstract ...... iii Populärvetenskaplig sammanfattning ...... vi Publications ...... ix Included in the thesis ...... ix Not included in the thesis ...... x Abbreviations ...... xi Introduction ...... 1 Fungal kingdom ...... 1 Human fungal pathogens...... 3 Fungal host adaptation mechanisms...... 7 Host immunity ...... 10 Innate immune system...... 10 Adaptive immune system ...... 18 Final remarks to host immunity ...... 21 Treatment strategies ...... 22 Aim of the thesis ...... 25 Methodological considerations ...... 26 Results and Discussion ...... 32 Paper I - Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells ...... 32 Paper II - Evasion of immune surveillance under low oxygen enhances Candida albicans survival ...... 36 Paper III - Stable redox-cycling nitroxide tempol has antifungal and immune- modulatory properties ...... 41 Paper IV - Antifungal application of non-antifungal drugs ...... 45 Outlook ...... 50 Acknowledgment ...... 51 References ...... 53

Abstract

Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen.

Mast cells are present in tissues prone to fungal colonization being among the first immune cells to get into contact with C. albicans. Here, we describe how mast cell responses initially reduced C. albicans viability. During the encounter, mast cells released granular proteins, cytokines, and extracellular DNA traps. A major cytokine released was chemokine IL-8 resulting in neutrophil recruitment to the site of infection. Despite being transiently attenuated, C. albicans eventually recommenced growth and induced mast cell killing. Therefore, we propose that mast cells act as tissue sentinels modulating initial antifungal immune responses and recruiting immune cells to the site of infection.

Upon chemokine sensing circulating neutrophils rapidly infiltrated tissues to fight infection in a murine abscess model of subdermal C. albicans infection. A high number of infiltrating cells coupled with the formation of multicellular, microbial structures, such as biofilms, led to the induction of hypoxic and anoxic niches. The abscesses were formed by high numbers of neutrophils. Thus, we decided to characterize the effect of anoxia on the responses of neutrophil encountering C. albicans. We found that persistent anoxia did not affect neutrophil viability nor metabolism. However, neutrophil responses against C. albicans were hampered affecting PAMP sensing and consecutive activities (phagocytosis and NET formation). On the other hand, anoxia contributed to increased fungal survival of C. albicans. Increased fungal survival was combined with higher infectivity of C. albicans which we confirmed in a worm model of

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infection. Hence, we conclude that adaption to low oxygen by C. albicans is a pre- requisite for successful infection.

Hypoxic and anoxic environments did not allow neutrophils to efficiently produce reactive oxygen species. Neutrophil oxidative burst is essential for antifungal activity and many fungal pathogens evolved antioxidative factors to mediate survival during infection. We reasoned that targeting of fungal redox balances could be a promising therapy approach. We have tested tempol, a redox-cycling nitroxide, as a new antifungal drug. Tempol is as an anti-cancer and anti- inflammatory agent. Here, we demonstrated tempol’s antifungal activity, by determining IC values in response to C. albicans. In addition, we have characterized the antifungal activity of tempol using RNA-Seq and metabolomics and found several genes and metabolites affected by tempol. Further bioinformatic analysis showed that affected genes and metabolites were part of similar signaling pathways in C. albicans. Tempol affected fundamental pathways for fungal homeostases, such as glycolysis and steroid biosynthesis. When tested in a murine model for systemic candidiasis, tempol reduced fungal burden compared to untreated controls during the initial phase of the infection. Finally, we infected whole blood from healthy volunteers and measured the cytokine response in treated and untreated blood. In summary, tempol induced an enhanced cytokine response upon C. albicans infection. Tempol may serve as the basis for the development of new antifungal compounds and open new opportunities for future therapies.

Efficient antifungal therapies are urgently required since currently, only 6 classes of antimycotics exist of which all aim at similar fungal targets. Since fungal infections primarily affect patients with immuno-suppressive conditions caused either by a disease directly or by the treatment of an underlying disease, we reasoned that repurposing drugs could offer clinical benefits. We performed a screen of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library identifying anti-Candida activity of compounds based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. From 844 drugs, 26 agents showed

activity against C. albicans. We identified 7 off-target drugs with previously unknown antifungal activity. The use of these old drugs for new purposes could provide fast therapy options by bypassing tedious and time-intensive development of de novo antifungal agents.

Overall, in this thesis work, we focused on the immune recognition and clearance of fungal pathogens and how we can harness lessons learned from our immune system to efficiently eradicate fungal pathogens. The findings open new doors for a better understanding of fungal pathogenicity and pave the way for the development of future therapies.

Populärvetenskaplig sammanfattning

Den mänskliga kroppen är ständigt utsatt för angrepp från mikrober i vår miljö. Varje dag träffar vår kropp på organismer som potentiellt kan infektera oss, så kallade patogener så som bakterier, virus, parasiter och svampar. Det är immunsystemets uppgift att befria oss från oönskade mikroorganismer. Vårt immunförsvar är mycket effektivt vilket framgår av att trots den ständiga närvaron av mikroorganismer så blir vi sällan allvarligt sjuka. Men, människor med nedsatt immunförsvar kan utveckla allvarliga infektioner från opportunister patogener så som svampen Candida albicans. C. albicans är en viktig orsak till sjukdom och död över hela världen.

Syftet med denna avhandling är att bättre förstå samspelet mellan dessa svampar och vårt immunförsvar. Vi fokuserar på det medfödda immunförsvaret och försöker identifiera de delar som är nödvändiga för kontroll under en infektion. Vi studerar också hur svamparna utvecklar mekanismer för att möta värdorganismens försvar och vad som reglerar dessa mekanismer.

Det medfödda immunförsvaret finns i hela kroppen och består bland annat av vävnader - huden och slemhinnorna - och en mängd olika celltyper. Mastcellerna är en del av det medfödda immunförsvaret och finns utspridda på kroppens ytor mot omvärlden. Främst finns de i huden, tarmens slemhinnor och luftvägarna och det är också här som patogena svampar förekommer i kroppen. Mastcellerna är därför strategiskt placerade för att snabbt kunna möta infekterande mikroorganismer. Här har vi visat att mastceller är kroppens vaktposter och mycket viktiga vid svampinfektioner, men de kan bara tillfälligt kontrollera C. albicans. Mastcellerna aktiveras när de möter C. albicans och släpper omedelbart ut inflammatoriska mediatorer som är lagrade i cellernas granula. Denna degranulering kan också rekrytera fler immunceller så som neutrofiler. De kan även släppa ut strukturer som kallas extracellulära fällor (MCETs), som fångar in inkräktarna för nedbrytning. Med dessa metoder kan Mastcellerna ta upp och

oskadliggöra invaderande mikroorganismer, men här har även C. albicans utvecklat ett eget försvar mot Mastcellerna. När C. albicans tas upp av mastcellerna kan de undkomma avdödning och utnyttjar då istället den skyddade intracellulära miljön för döda mastcellerna inifrån.

Neutrofiler är också en del av det medfödda immunförsvaret och de är den vanligaste typen av vita blodkroppar. Dessa celler cirkulerar i blodet men reagerar snabbt på hot och migrerar då från blodet in i vävnaden där bekämpandet av svamparna sker. De är oftast först på plats vid en infektion.

I det andra projektet har vi tittat närmare på neutrofilers försvar mot C. albicans infektioner och hur detta skiljer sig mellan syrerika och syrefattiga miljöer. Vi fann att neutrofilernas försvarsförmåga är kraftigt minskad under syrefattiga förhållanden. Både formationen av neutrofila extracellulära fällor (NETs) och även fagocytering, eller förtäring är kraftigt nedsatta under dessa förhållanden och detta kan ha en stor betydelse för immunförsvarets förmåga att bekämpa C. albicans infektioner. Bekämpandet av mikroorganismer är en aktiv process, driven av neutrofilernas snabba migration från blodet ut till vävnaden där mycket av syret förbrukas. I syrefattiga miljöer kan C. albicans också ändra cellväggens komposition och det ändrar i sin tur hur immunförsvaret reagerar mot dem.

Produktion av reaktiva syreradikaler är en av de mest påverkade försvarsmekanismerna i syrefattig miljö. Neutrofilerna använder reaktiva syreradikaler som en robust och effektiv kontroll-mekanism mot svamppatogener. I det tredje projektet undersöker vi om vi kan förhindra spridning av svampceller genom att påverka redoxpotentialen. Vi använder Tempol som är ett ämne med bevisad påverkan på metabolism och cellväggens komposition och har även inflammatorisk effekt. Behandling av svampcellerna med Tempol gav en framgångsrik anti-Candida effekt som karakteriserades med hjälp av RNA-sekvensering och metaboliska tester.

Idag finns det ett mycket stort behov av nya och bättre läkemedel mot systemisk svampinfektion. Opportunistiska svampinfektioner är ett stort hot mot patienter med nedsatt immunförsvar e.g patienter med leukemi eller som fått

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organtransplantationer. I projekt nummer fyra tänker vi oss att dessa patienter kanske skulle kunna får endast ett läkemedel mot både sjukdomarna. Därför har vi undersökt 844 godkänt-FDA läkemedlen för effekt mot C. albicans. Vi identifierat 7 nya molekyler med god effekt mot C. albicans. Vi har karakteriserad dessa läkemedlen och visat att de är lika effektiva som kommersiellt tillgängliga läkemedel.

Sammanfattningsvis visar våra resultat nödvändigheten till en fördjupad förståelse om immunceller och svampars samspel i mikromiljöer, så som de syrefattiga. En mer detaljerad förståelse kan därmed bidra till att utveckla bättre diagnostiska och terapeutiska verktyg för svampinfektioner.

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Publications

Included in the thesis

Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells

Lopes J.P*, Stylianou M*, Nilsson G, Urban CF

(2015) Scientific reports; 5:12287.

*equal contribution

Evasion of immune surveillance under low oxygen enhances Candida albicans survival

Lopes J.P. Stylianou M, Backman E., Holmberg S., Jass J., Claesson R. and Urban, C.F

Manuscript under revision

Stable redox-cycling nitroxide has antifungal and immune-modulatory properties

Hosseinzadeh A.*, Stylianou M*, Lopes J.P*, Müller D., Holmberg S., Grumaz C., Johansson A., Sohn K, Dieterich C and Urban C.F –

Manuscript

*equal contribution

Antifungal application of nonantifungal drugs.

Stylianou M., Kulesskiy E., Lopes J.P, Granlund M., Wennerberg K., and Urban C.F.

(2014) Antimicrob Agents Chemother 58, 1055-1062.

Not included in the thesis

Novel high-throughput screening method for identification of fungal dimorphism blockers.

Stylianou M., Uvell H., Lopes J.P., Enquist,P.A., Elofsson M., and Urban C.F.

(2015). J Biomol Screen 20, 285-291.

Dual transcriptome of the immediate neutrophil and Candida albicans interplay

Niemiec M.J., Grumaz C., Ermert D., Desel C., Shankar M., Lopes J.P., Mills I.G., Stevens P., Sohn K., and Urban C.F.

(2017) BMC Genomics 18, 696.

Soluble factor of Cryptococcus neoformans capsule prolongs mast cell lifespan

Lopes J.P. Stylianou M, Backman E., Holmberg S., Nilsson. G.. and Urban, C.F

Manuscript in preparation

Abbreviations

ASC Apoptosis-associated speck-like protein CBMC Cord blood-derived mast cells CLR C-type lectin receptor DCs Dendritic cells DC-SIGN ICAM3-grabbing non-integrin ELISA Enzyme-linked immunosorbent assays ETs Extracellular traps EUCAST European Committee on Antimicrobial Susceptibility Testing FcγR Fcγ receptor FDA US Food and Drug Administration IFNγ Interferon-γ IL Interleukin ILCs Innate-like lymphocytes MBL Mannose-binding lectin MCETs Mast cells extracellular traps MCP-1 Monocyte Chemoattractant Protein MIC Minimum inhibitory concentration MIF Migration inhibitory factor MMR1 Macrophage mannose receptor 1 MPO Myeloperoxidase MyD88 Myeloid differentiation primary response 88 NETs Neutrophil extracellular traps NF-κB Transcription factor nuclear factor-κB NK cells Natural Killer cells NLR NOD-like receptor pDCs Plasmacytoid dendritic cells RANTES Regulated on activation, normal T cell expressed and secreted ROS Reactive oxygen species SADs Standard antifungal drugs SEM Scanning electron microscope SYK Spleen tyrosine kinase TGF-β Transforming growth factor-β Th T-helper TLR Toll-Like Receptor TNF Tumor necrosis factor Tregs Regulatory T cells

TRIF TIR-domain-containing adapter-inducing interferon-β Type I IFNs Type I interferons response VEGF Vascular endothelial growth factor

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Introduction

Fungal kingdom

Fungi constitute one of the largest eukaryotic kingdoms and have a variety of life cycles with adaptations in metabolism and morphogenesis to fit their changing ecologies1. As a diverse group Fungi comprise several life forms such as molds, mushrooms or yeasts. Fungi break down dead organic material contributing to recycling the nutrients in various ecosystems. By synthesizing a plethora of biomolecules, fungal metabolism is essential for other eukaryotes as food. Humans widely use fungal metabolism as well, for instance by applying fungus- synthetized antibiotics. Fungi are ubiquitous in all zones of the planet and have a profound impact on the Earth’s ecosystem2.

The architecture of fungal cells is comparable to other eukaryotic cells with the exception that fungal cells are surrounded and shielded by a cell wall structure (Figure 1). The cell wall of fungi contains carbohydrates and proteins in a distinct composition which cannot be found in humans. Fungal cell walls are composed of two layers: The outer layer is mainly composed of glycoproteins and the inner layer of carbohydrate polymers, such as glucan and chitin. The polymer chains are interlinked and form a robust three-dimensional network. The fine tuning of the cell wall composition varies depending on the fungal species, morphotype, and growth stage. The expression of cell wall proteins is highly regulated and vary depending on the life cycle and morphotype of the fungi.

Figure 1. Schematic representation of a fungal cell and its organelles.

The subkingdom Dikarya contains the two major fungal phyla Ascomycota and Basidiomycota, home to approximately 98 % of all describe fungi. One of the reasons for their high dominance within the fungal kingdom is the Dikarya’s mating feature. During cell division, the daughter cell contains both nuclei from the two compatible mating types with a common cytoplasm, a so-called dikaryon. Dikaryotic cells have two sets of homologous chromosomes, even though each set locates to a separate nucleus. Together, the two nuclei control and regulate homeostasis.

Several groups of fungal species can change their morphologic appearance as an adaption to environmental changes. Dimorphic transition is a tightly regulated process crucial for the response against environmental changes and commonly confers essential virulence traits to pathogenic members of this group of fungi3. Dimorphic fungi can reversibly switch from yeast growth to filamentous, hyphal growth. Growing as yeast, fungal cells separate budding daughter cells from mother cells leading to a logarithmic increase in cell number. Growing as hyphae,

germ tubes grow out of a mother cell and growth continues at the tip of the hypha. The individual cells composing a hypha do not separate from each other resulting in an increasingly long filament that branches occasionally. The frequency of branching depends on the fungal species. As the dimorphic transition is frequently associated with virulence of pathogenic species, this process has been a topic of intense research during the past decades.

Human fungal pathogens

The omnipresence of fungi as part of the soil, water, and air, as well as our normal flora, forced humans to be able to cope with fungal infection4. However, human social evolution and the emergence of diseases and treatments that affect immune function have modulated our lifespan and rendered increasing amounts of the population more susceptible to mycoses5. Medically important fungal infections can broadly be classified into two types: superficial and invasive6. Superficial mycoses affect the skin and the mucosa. Specific examples include oropharyngeal candidiasis (thrush), and dermatophyte infections. Superficial mycoses occur in people with intact immune functions, although immunocompromised patients are at risk for increased severity and rate of disease. Despite being not life- threatening mucosal infections such as vulvovaginal candidiasis, have an impact on the life quality of affected women7. Invasive fungal infections target sterile body sites such as the bloodstream, central nervous system, liver, and kidneys and are associated with a high rate of mortality regardless of available antifungal therapy8 (Figure 2).

Figure 2. Pathogenesis of invasive candidiasis. Invasive candidiasis is one of the most common hospital-acquired infections in the developed world. This form of candidiasis arises from either hematogenous dissemination or inoculation of fungi to a sterile site. Mortality among patients with invasive candidiasis exceeds 40%. [Image reproduced with permission from Kullberg et al.9 Copyright Massachusetts Medical Society].

Fungi infecting humans can be divided into primary and opportunistic pathogens. Primary pathogens are capable of infecting immunocompetent hosts and are more common in the African, Asian, and American continent. These fungi

causing primary mycoses in humans belong to three major phyla: Entomophthoromycota, Ascomycota, and Basidiomycota.

The phylum Entomophthoromycota includes pathogens of insects which by accidental contact with contaminated soil or plant debris lead to human infection10. The phylum Ascomycota encompasses soil-inhabiting fungi that cause disease in mammals. Main representatives are Histoplasma capsulatum, Paracoccidiodes, and Coccidiodes spp. These pathogens typically enter the lung via inhalation of spores. In lung tissues, vegetative cells formed by spores trigger influenza-like symptoms. Subsequently, the fungal cells can disseminate from the lungs and persist in different organs11,12. Basidiomycota yeasts include Cryptococcus gattii, the responsible microbial agent for the cryptococcosis outbreak on Vancouver Island. This outbreak was unprecedented and led to an exceptionally high mortality among infected individuals. C. gattii benefits from an extraordinary ability to replicate within mammalian organisms after inhalation of spores13,14.

In contrast to primary pathogens, opportunistic pathogens are dependent on hosts with a compromised immune system to cause infection. These pathogenic fungi belong to three major divisions: Basidiomycota, Mucoromycota, and Ascomycota. Within the division of Basidiomycota, relevant pathogens are Malassezia spp and Cryptococcus neoformans. Malassezia spp. are lipophilic commensal fungi that colonize the human skin and thus constitute the dominant fungal commensal of the human body15. In case the immune balance of the skin is disturbed Malassezia spp may cause skin or hair root infections acumulating in different diseases, such as seborrheic dermatitis, dandruff, folliculitis, and onychomycosis16. C. neoformans is a recent human pathogen emerging in conjugation with the worldwide HIV/AIDS epidemic17. Similarly to C. gattii, it possesses a thick polysaccharide capsule composed of glucurono- and galactoxylomannan18. Both fungi have tropisms for the lung and central nervous system frequently causing meningoencephalitis as a life-threatening complication.

Diabetic patients with ketoacidosis due to bad glucose management are highly susceptible to infections caused by species from the order Mucorales, which belong to the division of Mucoromycota19. Species from this order typically enter the human body via inhalation of sporangiospores and invade blood vessels disseminating in the blood leading to vessel thrombosis, and tissue necrosis20.

Species belonging to the Ascomycota are responsible for most of the opportunistic infections by fungal pathogens. Three genera dominate: Fusarium, Aspergillus, and Candida. Despite being common plant pathogens, Fusarium spp occasionally infect humans, causing disease with varying severity grades, ranging from superficial, locally invasive, to disseminated infection depending on the host status21. From the genus Aspergillus, the most prevalent human mold pathogen is A. fumigatus, which is responsible for approximately 90 % of the cases of invasive aspergillosis22. Neutropenic patients or individuals suffering from diseases impairing neutrophil function, such as chronic granulomatous disease, are especially at risk for infection23. To prevent Aspergillus infections, lung neutrophils of immunocompetent persons trigger programmed cell death with apoptosis-like features in A. fumigatus conidia24. A. fumigatus spores cause allergic bronchopulmonary aspergillosis, a disease where spores get trapped in the sputum of asthmatic subjects triggering inflammatory reactions resulting in fungal-induced asthma 25.

Candida spp are the most common opportunistic human fungal pathogens, even though only a limited number of species from this family causes disease. Despite the emergence of multi-drug resistant Candida glabrata and the Candida auris global health threat, Candida albicans remains the most frequent cause of infections26,27. In a large proportion of the human population, C. albicans colonizes the oral, gastrointestinal and genital tract asymptomatically as an ubiquitous commensal28. However, the fungus can transmigrate through the epithelium and access deep-seeded areas causing infection. The ability to reach deep-seeded, non-commensal niches under immunosuppressive conditions of the host allows C. albicans to cause invasive infection, the most severe form of candidiasis. Despite improved diagnostics and therapy options, systemic

candidiasis remains responsible for high morbidity and mortality rates among susceptible individuals29.

Fungal host adaptation mechanisms

The total number of fungal species is estimated to be 1.5-5 million of which only a few hundred cause disease in humans30-32. The remarkable resistance of mammals against fungal pathogens has evolved in fungal species special characteristics to successfully infect these hosts. To parasitize humans, the fungus (i) must be able to grow at temperatures above 37 °C, a trait which most fungal species fail to accomplish. Hence, mammalian hosts’ endothermy creates a zone of thermal exclusion that protects against fungal pathogens33. Effective fungal pathogens, such as C. albicans, can tolerate temperatures above 40 °C 34. (ii) The fungus must be able to invade barriers to colonize underlying tissues and to establish invasive infection. Dimorphic C. albicans uses its ability to change morphology from yeast to hyphae to breach mucosal barriers. The yeast form is defined as the commensal form allowing colonization of superficial commensal niches. In contrast, the hyphal form is defined as the invasive form allowing C. albicans to penetrate host tissue barriers and to grant access to new tissues35 (Figure 3). Host temperature is one of the cues to initiate yeast-to-hypha conversion36 and the necessity of morphogenic transition for C. albicans’ virulence is reflected by that fact that nonfilamentous strains are avirulent37. Other cues include quorum sensing molecule farnesol38. C. albicans secretes quorum sensing molecules to communicate with other Candida cells and bacteria39. C. albicans secretes farnesol to inhibit filamentous growth in planktonic cultures or to block biofilm formation on adhesive surfaces40.

Figure 3. Steps in tissue invasion by C. albicans. Yeast cells adhere to the epithelium and trigger hyphal elongation and subsequent penetration of host cells. Hyphal cells can then access blood vessels and take advantage of the bloodstream to reach distant body sites. Thereafter, endothelial penetration initiates hazardous colonization and disseminated disease. [Image adapted with permission from Gow et al.35]

(iii) The fungus must also be able to absorb and digest nutrients gained from human tissues. The fight for nutrients in any given habitat led to the evolution of transporters of nitrogen and carbon sources as well as metal ion sequestering

strategies41,42. Iron acquisition is a central factor during infections since iron is an essential micronutrient for most living organisms being scarcely available throughout most habitats. Under iron starvation, C. albicans transcription factor Hap43p is essential to coordinate the iron starvation response 43,44. Interestingly, HAP43-deficient strains are more susceptible to neutrophil killing than wildtype strains45 reflecting the importance of sequestering trace metals by immune cells, such as neutrophils, to disturb microbial access to these trace elements. This part of innate immunity is referred to as nutritional immunity and has a fundamental role in preventing fungal growth within the host 46. As iron, zinc ions are an additional example of an essential micronutrient. C. albicans produces a zinc- binding protein, encoded by pH-regulated protein-1 -PRA1, which scavenges zinc from host tissues47,48. Interestingly, under zinc limitation, C. albicans induces a hyper-adherent phenotype termed Goliath cells49. (iv) Ultimately, all of these strategies are designed to be able to withstand the human immune system. Pathogenic fungi developed several strategies to circumvent the activation of an immune response. C. albicans releases a fungal peptide toxin, candidalysin, which can lyse mucosal cells. To escape from immune surveillance C. albicans can induce cell wall rearrangements upon environmental cues leading to masking of cell wall components which immune cell receptors fail to recognize 50. For instance, cell wall mannosylation impairs immune responses and contribute to virulence in vivo51. Moreover, C. albicans binds to complement regulators and detoxifies reactive oxygen species (ROS) to escape from immune attacks 52-55.

Taken together, fungal species require temperature adaption, invading mechanisms, sophisticated nutrient acquisition systems, and immune evasion strategies to become successful human pathogens. Compared to the bacterial world, only a few fungal species hold the aforementioned traits. However, due to the narrow evolutionary distance to animals and the high frequency of strains that are naturally resistant against common antifungal drugs, mycoses remain difficult to treat and emerge as a clinical concern.

Host immunity

Daily, humans are in contact with thousands of fungal cells, but during most of these encounters, healthy individuals do not acquire any form of fungal disease24,56,57. This is mainly due to the asymptomatic clearance of fungal cells by our effective immune system. The human immune system can be divided into the innate and adaptive branch.

Innate immune system Innate immune responses confer the first line of defense against fungal pathogens. The first line of protection in the human body are physical/mechanical (skin, hair, nails, and mucosal tissues), chemical (tissue pH, antimicrobial peptides, tear enzymes), and microbial in form of the resident microbiota.

Innate recognition When tissue barriers are breached, the immune system and its effector cells use receptors (Pattern Recognition Receptors-PRR) which have evolved to recognize molecular structures of fungal pathogens (Pathogens Associated Molecular Patterns-PAMPs) and to trigger cellular responses58 (Figure 4). Mutations in PRRs or downstream signaling molecules may result in high susceptibility to infections with fungal pathogens and severe outcomes underlining the importance of proper detection of fungal microbes by the immune system59,60. There are several families of PRRs which are grouped according to function, structure, and phylogeny. The key PRRs involved in antifungal immunity are Toll- like receptors (TLR), C-type lectin receptors (CLR), and the NOD-like receptors (NLR). PRRs can work alone or be co-activated simultaneously due to pleiotropy for ligands or redundant receptor functions. PRR activation leads to a multitude

of responses including phagocytosis61, cytokine production62, and inflammasome activation63.

Upon fungal infection, epithelial cells and innate immune cells are the first cell types to detect the infection. Host cells recognize several ligands on fungal pathogens and trigger immune responses. PRRs can cluster and synergistically enhance their signal intensity. TLR-4 recognizes O-linked mannans, whereas TLR2 recognize phospholipomannans or recognizes β-mannosides together with galectin 3. TLR9 has cytosolic localization and recognizes fungal DNA. Mannose-rich structures are bound by soluble lectin receptor mannose- binding lectin (MBL) and by other membrane-bound CLRs. Most well- described CLRs with fungal ligands are dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), macrophage-inducible C-type lectin (MINCLE), and macrophage mannose receptor 1 (MMR). Other CLRs include dectin-1 which binds β-glucans, and dectin-2 which, together with Fcγ receptors (FcγR), recognizes mannose polymers. Upon recognition of the fungal ligand, the membrane receptor signals downstream to adaptor molecules to eventually induce transcription of cytokines via transcription factor nuclear factor-κB (NF-κB) or type I interferons responses (Type I IFNs). C-lectin receptor responses are modulated by adaptor molecule spleen tyrosine kinase (SYK). SYK signals downstream to caspase recruitment domain-containing protein 9 (CARD-9) triggering antimicrobial responses. In contrast, TLR response activation is coordinated by myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF). Furthermore, NLRs can also sense non-microbial danger signals and form large cytoplasmic complexes called inflammasomes. Among these inflammasomes, the nucleotide-binding domain, leucine-rich-containing family, pyrin domain- containing-3 (NLRP3) inflammasome has been under intensive investigation given its involvement in several human diseases64. NLRP3 can form a complex with apoptosis-associated speck-like protein (ASC) and the caspase-1 enzyme. This complex promotes the maturation and secretion of pro-inflammatory cytokines interleukin (IL)-1β65. Other NLRs are also expressed in the cytosol, where they bind ligands from intracellular invaders.

C. neoformans C. albicans Aspergillus Malassezia

Figure 4. Recognition of human fungal pathogens. PAMP-PRR interactions depend on the fungal ligands recognized by PRRs and vary within the group of human fungal pathogens. The majority of fungal ligands is recognized by CLRs, TLRs and NLRs. [Image reproduced with modifications from Gow et al.35].

Cells of the innate immune system At the interface between environment and tissue, several immune cells reside. There, they contribute to regulation of surveillance and maintenance of commensal relationships as well as to protection from invasion. These cells express a wide range of PRRs and perform a broad range of antifungal defenses. Below, we briefly refer to a selection of these cells which are of importance for fungal clearance.

Epithelial cells Epithelial cells are the first cells in contact with microbes. They compose the lining of the most important barrier tissues such as the skin, genital-, gastrointestinal-, and respiratory tract, therefore, encountering different fungal pathogens66. Epithelial cells express TLRs67 and CLRs68 and contribute to immunity by producing cytokines and antimicrobial peptides69. Epithelial cells respond to C. albicans by activating NF-κB and a biphasic MAPK signaling pathway. Activation of NF-κB and the first MAPK phase is due to fungal cell wall recognition but independent of the recognized C. albicans morphology. Activation of the second MAPK phase is dependent on the hyphal form of C. albicans and is characterized by the induction of proinflammatory molecules. This dual response may represent a danger signaling pathway that is critical for the recognition of the pathogenic switch of C. albicans by epithelial cells. Additionally, upon C. albicans infection epithelial cells induce and secrete chemokines to recruit leukocytes, such as neutrophils 70.

Mast cells Mast cells are tissue-dwelling cells derived from hematopoietic progenitors. After migration of their immature form from blood to tissues – of skin, airways, gastrointestinal tract, and eye - the final differentiation is governed by cytokines secreted by surrounding endothelial cells71. Mast cells are known for their involvement in hypersensitivity reactions at body interfaces exposed to the external environment72. Comparably little is known about their contribution to antimicrobial defence. Nevertheless, mast cells express numerous PRRs enabling them to respond to external stimuli73. As tissue sentinels, they are capable of rapid degranulation of cytoplasmic vesicles, residual phagocytosis, and production of ROS to attack invading microbes and to enhance inflammation74,75. Fungal pathogens enter the body via regions rich in mast cells, such as the gastrointestinal or the respiratory tract.

Some insight into the role of mast cells in antifungal defence is derived from studies with A. fumigatus. During infection, mast cells induce a pro-

inflammatory response by degranulating β-hexosaminidase upon contact with A. fumigatus hyphae76. This capacity of producing an inflammatory milieu might be detrimental as described with the help of a model of cystic fibrosis where mast cells exacerbate inflammation upon A. fumigatus infection77. Mast cells participate in anti-C. albicans immunity at mucosal surfaces and as for aspergillosis, mast cells degranulate mediators to kill C. albicans78. Surprisingly, mast cells contribute to tolerance during commensal colonization of C. albicans by regulating the activity of indoleamine 2,3-dioxygenase, a modulator of inflammation, known to be involved in activation of regulatory subsets of T lymphocytes79,80.

Neutrophils Polymorphonuclear leukocytes, shortly referred to as neutrophils, belong to the family of granulocytes and are the first-line eradicators of invading microbes. Neutrophils are the most abundant leukocyte type in circulation and essential for clearance of fungal pathogens. The release of chemokines or chemotactic factors from the infected tissue can prime neutrophils to migrate from the blood towards the infection site. Priming can happen from inflamed tissue producing pro- inflammatory cytokines, such as IL-8, or from the sensing of fungal PAMPs like β-glucan81. Extravasation, the passage of neutrophils through the endothelial lining, at first requires the slowing down of circulating neutrophils through binding to P- and E-selectin expressed on endothelial cells82. This is followed by firm adhesion and diapedesis of neutrophils on endothelial cells. Transendothelial migration occurs either by a transcellular route - neutrophils penetrate through individual endothelial cells - or by a paracellular route - neutrophils squeeze through tight junctions between endothelial cells. When reaching the infection site, neutrophils prevent the growth of pathogens via four processes: launching of an oxidative burst, secretion of cytoplasmic granule contents, phagocytosis of particles, and formation of neutrophil extracellular traps (NETs)83-85. The oxidative burst refers to the induced production of ROS which contributes to the killing of engulfed microbes and be delivered to the

extracellular space or directly to the phagosome86. ROS are initiated by superoxide production through the NADPH oxidase. Upon activation, this large protein complex is assembled in the cytoplasmic membrane where it adds electrons to molecular oxygen. Generated superoxide is converted to hydrogen peroxide by superoxide dismutases. Hydrogen peroxide can react with available cations via the Fenton reaction to create hydroxyl radicals. Eventually, myeloperoxidase (MPO) further converts hydrogen peroxide to hypochlorous acid. Each of these reactive molecules contributes to damaging microbes that are in contact with the neutrophil. Patients with a defective oxidative burst in phagocytes are particularly susceptible to mold infections87.

The nonoxidative antifungal mechanisms of neutrophils include the release of enzymes, such as elastase or cathepsin-G84. These enzymes are stored in cytoplasmic vesicles, so-called granules. The neutrophil granules fuse with phagosomal or cytoplasmic membranes to release their content into the phagosome or to the extracellular space, respectively. Granule generation occurs during granulopoiesis in the bone marrow and three different granule types are produced: primary (azurophil), secondary (specific), and tertiary (gelatinase) granules. Their marker proteins are myeloperoxidase, lactoferrin, and gelatinase MMP9, respectively82.

Neutrophils can sense hyphae and, if these are too large to be phagocytosed, selectively release NETs88,89. NETs have microbicidal properties towards a variety of fungal pathogens including C. albicans90-92. The NET meshwork of DNA decorated with cytoplasmic proteins traps and kills microbes as well as exacerbating inflammatory processes85,90,93. Antifungal activity by NETs is mainly mediated by calprotectin and described to be dependent on the presence of oxygen90,94.

Macrophages Monocytes are generated in the bone marrow and, after myelopoiesis, from there released into the bloodstream. Monocytes leave the bloodstream and enter the

tissue where they differentiate into macrophages. Macrophages further develop into distinct functional phenotypes, which are determined by the cytokine milieu. Proinflammatory cytokines, mainly interferon γ, drive the classically activated phenotype, called M1, whereas anti-inflammatory cytokines, such as transforming growth factor-β (TGF-β), drive alternatively activated macrophages, called M295. However, most tissue-dwelling macrophages in adults do not arise from monocyte differentiation96. Adult tissue macrophages are derived from embryonic precursors seeding the tissues before birth and maintaining themselves by self-renewal97. During the early stages of fungal infections, infected hosts rely on tissue-resident cells, including macrophages, to act as the first line of defense. Alveolar macrophages of the lungs can produce cytokines and take up C. neoformans spores and A. fumigatus conidia98-100. In the kidney, macrophages internalize conidia and hyphae 101. Whereas in the brain specialized macrophage-related cells, termed microglia, respond to fungal invaders crossing the blood-brain barrier102-104. In all these situations, macrophages depend on their ability to phagocytose particles. Upon engulfment, pathogens are trapped in the phagosome which then fuses with endosomes and lysosomes to originate the phagolysosome. Macrophages subsequently mount an oxidative burst, similarly as neutrophils, and concurrently lower the phagolysosomal pH which contributes to the eradication of the engulfed microbe105.

In addition, macrophages increase resistance against C. albicans re-infection by maintaining a type of immunological memory, a process which has been termed trained immunity106. This process induces epigenetic reprogramming of monocytes leading to enhanced cytokine production in vivo and in vitro after secondary exposure to C. albicans β-glucans107.

Dendritic cells Dendritic cells (DCs) are a heterogeneous population of antigen- presenting cells that have crucial roles at the intersection of innate and adaptive immunity108. DC monitor the body's tissues and integrate multiple signals from the

environment in order to initiate an appropriate adaptive immune response. Inflammatory signals promote DC activation and their migration to draining lymphoid tissues where they prime effector T cell responses. However, DCs promote a homeostasis state in the tissue by inducing regulatory lymphocyte subsets108.

DC progenitors give rise to three broad DC subsets: plasmacytoid cells (pDCs), conventional DCs, and migratory DCs. The differential contribution of these subtypes to antifungal immunity remains unclear109. For pDCs, a role against A. fumigatus hyphae has been described110. Residues of α-mannan, present during hyphal stimulation of pDCs, trigger recognition via the Dectin-2 receptor. In turn, this leads to a distinct pattern of pDC gene expression resulting in cytokine expression and extracellular traps (ETs) release. ET formation seems partially mediated by fungal gliotoxin secretion111.

The unique ability of DCs to initiate adaptive responses against fungal pathogens and to directly engage in fungal recognition and eradication positions DCs as potential targets for the development of fungal vaccines. A provisional vaccine constituted by a laminarin-β-mannan-tetanus toxoid formulation was tested in bone marrow-derived DCs and enhanced removal of fungal pathogens. Uptake of the tricomponent vaccine by DCs increased cytokine production and activation of effector T-cells subsets112.

Natural killer cells Natural killer cells (NK cells) are cytotoxic lymphocytes and an important component of the innate immune response during viral infections and antitumor defence113,114. In contrast to T or B cells, NK cells do not undergo somatic rearrangement of genes coding for antigen-specific receptors. NK cell receptors are germ-line encoded proteins and comprise several inhibitory and activating molecules that regulate NK cell function 115,116. NK cells exert cytotoxic effects through the release of granule content, including perforin, granzymes, and granulysin. The antifungal activity of NK cells has been described for several

fungi117-119. For instance, NK cells recognize C. albicans using NK cell receptor NKp30120,121. Upon recognition, NK cells secrete several chemokines namely, GM- CSF, IFN-γ, and TNF-α. These cytokines enhance neutrophil activation and fungicidal activity. The direct antifungal activity of NK cells against C. albicans depends on perfornin122. Stored in secretory granules of cytotoxic lymphocytes, this protein is release into a immunological synapse, a tight binding interface present between the NK cell and the target cell. In the fungal cell membrane, perforin oligomerizes and forms large membrane-spanning pores that penetrate and damage the fungal membrane123.

Adaptive immune system

T cell Activation of the adaptive immune system is critical for resolution of infection. T cells generate in the thymus and are program to be specific for one antigen. Once they leave the thymus, T-cells express the T cell receptor and either the CD4 or the CD8 co-receptor. T cells reside in lymph nodes or circulate throughout the body until they recognize their matching antigen. Antigens are process and present to T cells by professional antigen presenting cells (APCs: DCs, macrophages and B cells) via MHC class II molecules. Normally this presentation takes place in secondary lymphoid organs. However, antigens might also be present to T cells via MHC class I molecules, which are present in all nucleated cells. MHC II presentation to T cells serves exclusively to activate T cells with matching recognition for foreign antigens. MHC class I presentation shares this function however additionally is important to regulate differentiation of self from non-self molecules. The T cell receptor, together with the CD4+ or CD8+ molecule, binds to the MHC complex on the surface of the antigen presenting cells carrying the antigen. Additionally, signals are require to fully activate the T-cell. For T- helper cells (CD4+ T-cells) proliferation only occurs after successful binding of CD28 and B7.1 or B7.2 molecules. Cytotoxic T cells (CD8+ T-cells) are less

dependent on CD28 for activation requiring signals from other co-stimulatory molecules such as CD70 and 4-1BB124.

Differentiation of T helper cells into distinct subtypes is influence not only by antigen presentation itself but also by the cytokine milieu. The cytokines IL-12 and IL-18, for instance, drive naïve T cells towards differentiation into a Th1 phenotype. The Th1 response is common against intracellular pathogens. During mycoses, Th1 cells produce interferon-γ (IFNγ), tumor necrosis factor (TNF)-α, and GM-CSF. IFNγ prompts the cellular production of nitric oxide and ROS in macrophages to enhance eradication of intracellular fungal pathogens125. TNF-α increases resistance to fungal infection whereas as GM-CSF acts through sequestration of zinc from intracellular yeasts and stimulation of ROS126-128.

The cytokines IL-4 and IL-18 govern Th2 differentiation which is favor during parasitic infection. Differentiated Th2 cells produce IL-4 and IL-13. In experimental models of fungal infection, the undesire activation of Th2 responses leads to nonprotective allergic inflammation in mice129,130.

Development of Th17 cells occurs during bacterial and fungal infection in a milieu and is govern by IL-23, IL-1β, IL-6, and TGF-β131. Th17 cells produce a distinct cytokine profile, namely IL-17 (A and F) and IL-22. Patients with chronic mucocutaneous candidiasis exhibit genetic alterations of the IL-17 pathway132. Mice deficient in IL-17A show delay healing of dermal C. albicans infection compare to wild-type mice. IL-17A injection at the site of skin infection reverts the phenotype133. In another study inhibition of Th17 differentiation leads to inhibition of IL-17 production and consequent exacerbation of infection showing the importance of IL-17 for immunity against C. albicans 134.

In addition to the Th lymphocytes, other important subsets of T cells for fungal immunity are CD8+ lymphocytes and regulatory T cells (Tregs). Antigen presentation via class I MHC mediates activation of CD8+ T cells and these activate cytotoxic T cells have an essential role in combating infectious diseases by killing target cells via release of cytotoxic molecules. CD8+ T cells recognize

peptides derived from fungi that are presented by class I MHC molecules and thereby target infected cells135.

Tregs represent a double-edged sword for antifungal defense. On the one hand, they are crucial for dampening immune responses and for conferring tolerance. Inflammation in mice exposed to Aspergillus conidia is controlled by the expansion and activation of Tregs which are capable of suppressing neutrophils136. Adoptive transfer of Tregs harvested from wildtype infected animals restors resistance to C. albicans reinfection and decreases inflammation in B7-2-deficient mice137. On the other hand, their function might be undesirable in certain settings, as depletion of Treg conferred improved resistance against disseminated candidiasis138.

B cell B-cells provide the humoral arm of the adaptive immune system. B cells develop in the bone marrow from a common lymphoid progenitor shared with other leukocytes such as T- and NK- cells, and some DC subsets. Throughout development, B cells migrate to different niches in the bone marrow. When mature B cells become part of blood lymphocytes, B cells constitute 5–25% of all lymphocytes. B lymphocytes protect against invading pathogens by directly reacting to fungal antigens. Fungal epitopes bind membrane-bound immunoglobulins on the surfaces of B cells. These specific interactions lead to a series of events that stimulate the expansion of that particular B cell population. As a result of B cell activation, secreted antibodies with the same binding specificities as those of the B cell receptors are produced139.

Immunoglobulins elicit protective immune responses in the host predominantly by targeting antigens in the fungal cell wall140. Monoclonal antibody C7, an antibody targeted against C. albicans after binding to the fungal adhesin Als3 prompted inhibition of fungal growth141. An additional effect described due to the antibody binding was the alteration in expression of genes important for iron uptake and iron homeostasis. Altered germination and adherence contributed to

a decreased fungal virulence. Only fungal mutants lacking the TPK1 gene, a gene important for iron uptake and defective in germination showed the capacity to escape from growth impairment imposed by the antibody binding142. Moreover, antibody binding to C. albicans exposes the fungal cells to increased binding of complement factors that in turn enhance the fungicidal potential of incoming phagocytes141.

Notably, immunoglobulins decrease the growth of the primary fungal pathogen H. capsulatum within macrophages by enhancing phagosomal maturation and increased lysosome-phagosome fusion143.

Throughout the production of IgM, B cells confer resistance to C. neoformans dissemination. C. neoformans infection in B and T cell-deficient Rag1-/- mice leads to facilitated fungal dissemination to the brain. In agreement to this, adoptive transfer of B cells to infected mice limits the spread of cryptococcosis144.

Final remarks to host immunity Innate and adaptive immune responses are intimately linked and controlled by sets of molecules and receptors that act in an orchestrated fashion to provide protection. Although our understanding of innate and cellular immunity against fungal pathogens continues to increase, many unresolved aspects remain. Importantly, we need to better understand how PRR signaling happens and how fungal pathogens evade host defense mechanisms. Thereby, we can devise better therapeutic approaches based on knowledge gained from host response studies and can tackle the hurdles from pharmacological treatment.

Treatment strategies

Regimen to treat invasive mycoses is limited to three structural classes of antifungal drugs: polyenes, azoles, and echinocandins. Both polyenes and azoles have been available in the clinic since 1980 while echinocandins are available for less than 20 years (Figure 5).

Figure 5. A timeline depicting milestones of antifungal drug development. [Image adapted from Roemer et al. 2014 145 ]

The oldest class of antifungal drugs composes the polyenes. Amphotericin B and nystatin are the most common drugs from this class. To take effect, polyenes bind to ergosterol, a membrane sterol that is unique to fungi. Upon binding, polyenes form pores in the fungal plasma membrane resulting in cell lysis146. An alternative mode of action proposed for amphotericin B shows aggregates of the drug and its target, ergosterol, at the lipid bilayer resulting in leakage and eventually killing of the fungal cells147. Until today, amphotericin B remains the antifungal drug with the broadest spectrum and hence is used to treat systemic infections despite severe cytotoxic side effects. Combinatorial therapy with 5-flucytosine is used in treatment with cryptococcosis and other less common mycoses148, while

for most other invasive mycoses, azoles and echinocandins are the standard choices of treatment.

The azoles are the most widely used class of antifungal drugs149. Azoles act by disturbing fungal cell membranes. They target the enzyme lanosterol 14α- demethylase preventing the biosynthesis of ergosterol. Lack of ergosterol consequently leads to loss of cell membrane integrity. Despite a good safety profile, application of azoles in combination with other drugs is restricted due to the ability of azoles to inhibit cytochrome P450145. Fluconazole, voriconazole, and posaconazole are most commonly used azoles. Fluconazole is fungistatic with broad activity against Cryptococcus and Candida spp., with exceptions of C. glabrata and C. krusei and molds in which it has limited activity. Voriconazole is fungicidal for A. fumigatus150. Compared to other azoles, posaconazole breaks ranks, as it displays in vitro activity against Mucor spp.151.

Echinocandins are the most recent addition to the antimycotic pharmacopeia. They hamper cell wall synthesis by blocking the β-glucan synthetase complex which results in cells lysis152. Echinocandins have become an important therapeutic option for candidiasis due to the broad fungicidal activity against Candida spp. and due to the low side effects against human cells, since a homologous target is missing. However, echinocandins show no activity against Cryptococcus or Mucor spp. Despite only a few years in use, increasing numbers of echinocandin-resistant fungal strains are a clinical concern153.

Over the past decades, the demand for antifungal drugs in modern healthcare has increased dramatically, but that demand is not reflected by the number of available therapeutics. Indeed, the number of therapeutic options for mycoses is remarkably limited when compared to those available for the treatment of bacterial infections. The rate of antifungal drug discovery though is insufficient to meet the urgent need154. The most common obstacle arises from the evolutionary proximity between fungi and humans, as compounds toxic to fungi tend to be toxic to humans as well. To address challenges in drug discovery, new

“out of the box” strategies are needed combining innovative screening methods with novel chemical libraries.

Aim of the thesis

Innate immune responses towards fungal pathogens remain an underrepresented area of research. Understanding the host processes governing the sensing of fungi and the subsequent immune response can show new strategies to emulate and develop new antifungal compounds.

The specific aims of this thesis are therefore to:

Investigate the mechanisms used by innate immune cells to tackle fungal infection in microenvironments.

To explore the mechanism of C. albicans adaptation to those niches and how do they contribute for survival fungi.

Explore new or off-target compounds for their antifungal activity.

Methodological considerations

This section briefly discusses the main experimental design of the thesis to allow a better understanding of the result and discussion sections. Further information is provided on why a particular method was chosen and on what potential advantages or drawbacks should be considered.

Fungal strains and growth

The C. albicans strain SC5314155 was used in the experiments for paper I-IV. Additional strains used: C. albicans mutants expressing GFP (CAI4 pENO1-GFP- CyC1t)156 (paper I and II) and Δefg1157 (paper II), C. tropicalis DSM 4238 (paper II), C. dubliniensis CD36/CBS7987 (paper III) and C. glabrata ATCC 90030 (paper III and paper IV). Clinical strains used in paper II and III were obtained from the strain collection of Umeå University Hospital, Umeå, Sweden which are all blood isolates.

Neutrophil isolation

Neutrophils were isolated from human blood of healthy volunteers obtained from the Blodcentralen part of the Laboratory Medicine, Clinical Immunology and Transfusion Medicine department at Umeå University Hospital according to the principles expressed in the Declaration of Helsinki. Neutrophils were isolated as described previously158 by a combination of two centrifugation on Histopaque and Percoll gradients. The viability of isolated cells was assessed using a cell counter by trypan-blue staining or by flow cytometry using propidium iodide.

Human mast cells lines

Several studies have established the human HMC-1159 cells as an appropriate model to investigate mast cell-related processes. By using this cell line, we avoid background variation combined with better accessibility and quick handling as compared to primary cell isolations. For paper I, mast cells (CBMCs) cells were differentiated from blood cells isolated from human cord blood. Pluripotent precursor cells isolated from cord blood were a kind gift from the Mast Cell Biology Group at Karolinska Institutet, Stockholm. Primary cells are more responsive and richer in granule content than cell lines.

Aerobic and low oxygen infection

Infections were mostly performed at 37 °C in 5 % CO2 but in paper II additional work was under hypoxic (1 % O2) and anoxic conditions (< 0.2 % O2). When working under these conditions, it is of great concern to not disturb the delicate environment while performing sampling or adding/removing material. To avoid breaches in experimental conditions liquid material used in anoxic experiments was depleted of oxygen and cells were introduced as a pellet before use in the anoxic chamber. Experimental plans were adjusted as such to keep handling of samples stored in the hypoxia incubator to a minimum in order to reduce the influx of O2. To better understand host-pathogen interactions in the human body which contains many oxygen-poor environments, we have to perform studies under hypoxic or anoxic conditions, even if these are very difficult to conduct.

Mouse strains

C57BL/6 and B6 albino female (B6(Cg)-Tyrc-2J/J) Jackson Laboratory-USA mice were purchased from Jackson Laboratory and were kept under a pathogen- free condition in the Umeå Center for Comparative Biology (UCCB) animal

facility. The albino strain was used for experiments involving the IVIS camera system since pigments in skin and fur hampered the read out.

Flow cytometry

We used flow cytometry as a tool to analyze host-microbe interactions. Flow cytometry allows for separation and distinction based on cellular and surface properties with high sensitivity and specificity. The cells or cellular components of interest were labeled with specific antibodies or dyes that do not alter their biochemical and physical properties.

In paper II, for instance, we used flow cytometry for assessing fungal and neutrophil cell viability, C. albicans cell wall composition, ROS production, and phagocytosis. We stained neutrophils from mice and humans using specific antibodies directed against neutrophil-specific surface molecules (Ly6G and CD66 respectively). Fungal cell wall components were stained with concanavalin A, a carbohydrate-binding lectin, to detect mannan; small compound calcofluor white to detect chitin; soluble dectin-1 antibody to detect β-glucan. ROS was detected using the cell-permeant H2DCFDA dye. The cells were stained with the reduced form of the dye but upon generation of intracellular ROS there is a cleavage of the acetate groups in the dye molecule and the nonfluorescent

H2DCFDA is converted to a highly fluorescent form. This kind of fluorescent probes is important for providing information on changes to the redox environment of the cell. However, their response might be affected by other chemicals in the milieu that might induce oxidation of the small molecule contributing to false positive measurements.

Protein quantifications

To determine the presence and concentration of different proteins we used two techniques: enzyme-linked immunosorbent assays (ELISA) and a multiplex immune assay system. In an ELISA, an antigen is immobilized on a plate surface and then complexed with an antibody that is linked to an enzyme. The incubation

of the substrate together of the conjugated enzyme can subsequently be detected and quantified. ELISAs are fast, simple and accurate assays that allow for the detection of proteins present in the supernatants of infection. The multiplex immune assay system uses antibodies coupled to magnetic beads to determine simultaneous proteins present in solution. The method has a higher sensitivity despite the higher cost than traditional ELISA. Both methods depend on specific antibody-antigen interactions.

Microscopic imagining - Immunofluorescence and electron microscopy

The interplay of either mast cells or neutrophils with C. albicans was visualized using microscopy techniques. In paper I, we performed infections of mast cells with C. albicans and focused particularly on the process of ETs formation. ETs are characterized by the co-localization of host DNA with hallmark cytoplasmic proteins entrapping the pathogen. We stained mast cell DNA using DAPI and used fluorescent antibodies against mast cell tryptase. The presence of these two markers allowed for the detection of MCETs entrapping the fungus. Complementary to this approach we visualized the interaction of mast cell and C. albicans using live cell imaging. We found and quantified two different mechanism that C. albicans uses to kill mast cells.

In paper II we performed both immunofluorescence microscopy and scanning electron microscopy (SEM) to detect NETs. For this purpose, neutrophil DNA was stained with DAPI and we used antibody staining for MPO and elastase as additional markers of NETosis. All three markers colocalized with the fungus and with bacteria confirming the presence of NETosis. Additional work in paper II using SEM demonstrated the NET response of neutrophils towards fungal biofilms. Finally, to determine differences in neutrophil antifungal response in abscesses from wildtype and S100a9-/- mice, we have used immunofluorescence microscopy. Abscesses were sectioned into 5 µm thick sections and neutrophils stained and quantified.

As the above examples demonstrate, microscopy is a valuable tool allowing to address a variety of scientific questions. We used microscopy to analyze specific immune responses, such as ETosis, to visualize how mast cells are killed by C. albicans and finally to determine the number of neutrophils in fungal abscesses. However, availability of antibodies for proteins of interest is limited and thus prevents usage of microscopy. The cost of equipment and materials, such as in particular antibodies, may be further determents.

ET quantification and DNA fluorescence assay

MCETs and NETs were quantified either microscopically or by using a fluorescence-based plate assay. In paper I, fluorescent microscopy pictures were transformed into binary pictures for analysis. Each image contained at least 100 cells and images were analyzed in a blinded fashion by independent researchers. Final scores were defined as MCETs per field of view and plotted by condition and infection end-point. Uninfected cells serve as a control. For NETosis in paper II, a semi-automated method was used. Ten images of the sample containing on average 100 neutrophils were captured randomly. After threshold adjustment, images were analyzed for the nucleus size using ImageJ software (version 2.1). Cells that exceeded the area of 100 µm2 were considered as cells undergoing NETosis. NET forming cells were presented as percentage of total cells.

Alternatively, ETosis was quantified using a DNA fluorescence assay. This assay uses a membrane-impermeable dye that is unable to penetrate live cells and hence only stains dead cells by intercalating with DNA to produce fluorescence. The relative fluorescence directly correlates to the amount of DNA released by dead cells, which can be expressed as a percentage of dead cells in comparison to triton-lysed controls.

Microscopic analyses remain the gold standard to define and quantify ETs, despite being a remarkably time-consuming and laborious procedure. Microplate assays are fast and reliable, however, they are based on fluorescent signals

quantified from dead cells. Thus, this assay does not discriminate, whether the cell in question died by releasing ETs or by other cell death pathways.

Cellular viability

Cell viability was determined by measuring ATP levels using the CellTiterGlow kit from Promega. This assay is present in all papers and was used to characterize the cellular viability of fungal and mammalian cells. The kit acts first by lysing the cell and followed by the staining in a luminescent base assay of the ATP molecules present. The luminescent signal is proportional to the amount of ATP present. The amount of ATP, in turn, is directly proportional to the number of living, metabolically active cells present in the culture. The assay is sensitive and fast to perform, and the luminescent signal is stable for several hours. Factors that affect the ATP content of cells may affect the relationship between cell number and luminescence, such as for example luminescence inhibition due to high cell densities.

Results and Discussion

Paper I - Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells

Candida albicans is part of the human commensal flora and colonizes the gastrointestinal, urogenital, oral-nasal cavity and skin. When host immunity is suppressed, C. albicans can disseminate to non-commensal niches, resulting in hazardous colonization and invasive disease. Due to their distribution in tissues facing external surfaces mast cells are among the first immune cells to get in contact with C. albicans. Mast cells are best known for their crucial role in inflammatory and allergic responses while little is known about their antifungal response. Here, we describe their contribution to anti -C. albicans defense. We have found that mast cells responded to C. albicans in a specific manner and these responses could be divided into time-specific occurrences. These responses comprised three discernable stages (Figure 6).

Figure 6. Mast cells elicited a temporal and distinct response upon encounter with C. albicans. [Image reproduced with permission from Lopes et al., 2015156].

1. Initial phase

The initial response occurred in the first 3 h and started with rapid degranulation of granule proteins as determined by the release of ß-hexosaminidase. Infected mast cells degranulated almost two-fold above the uninfected control. This rapid onset of response upon infection was able to temporally reduce fungal viability.

2. Intermediate phase response

Intermediate response (3-12 h) started with the secretion of pro-inflammatory cytokines- IL-8, migration inhibitory factor (MIF) and monocyte chemoattractant protein (MCP-1). These cytokines have been implicated in regulating leukocyte migration to the site of infection160,161. We decided to test the effect of these cytokines on the chemoattraction of neutrophils and monocytes. For this purpose, we used supernatants collected from C. albicans infected mast cells and assayed cellular migration towards these supernatants. Neutrophils, but not monocytes, were significantly chemoattracted by infected supernatants.

As part of their antifungal response, 6 h post-infection mast cells released MCETs (Figure 6). MCETs are composed of DNA and granular proteins such as tryptase and can ensnare C. albicans. The amount of MCETs in a given infection did not exceed 5 % of total cells. Due to low frequencies, we assume that MCETs act as danger signal rather than as antifungal mechanism since the release of proteases and other injurious cell constituents might contribute to exacerbating inflammatory processes even at very low concentrations.

Figure 6. Mast cells release MCETs. Shown is a representative micrograph C. albicans infected mast cells. MCETs (arrow) were identified by colocalization of extracellular laminar DNA (blue) with tryptase immunostaining (green) co-localizing with C. albicans (red). Scale bars, 10 µm. [Image reproduced from Lopes et al., 2015 156].

3. Late phase response

From 12 h onwards mast cells were no longer able to control fungal growth and significant mast cell death was triggered by C. albicans. Hyphae were either piercing the mast cell membrane from outside growing into mast cells or were growing out of mast cells upon the previous internalization of yeasts. Finally, this late phase was governed by the release of cytokines related to adaptive immunity (IL-16) anti-inflammatory cytokines (IL-1ra). The pro-inflammatory cytokine response at early time points post-infection seems to be counteracted by the release of the anti-inflammatory cytokines at later stages. Taken together, these data suggest that secretion of pro- and anti-inflammatory cytokines by mast cell

upon contact with C. albicans was a controlled process that was influenced by different stages of the infection.

Conclusions

The described processes point for a role of mast cells as tissues sentinels at initial infection phases. Mast cells launched a direct immune response and coordinate and the recruitment of other immune cells. In vivo studies with animal models which are deficient in mast cells could help to further assess the contribution of mast cells in orchestrating an antifungal defense.

Paper II - Evasion of immune surveillance under low oxygen enhances Candida albicans survival

A small proportion of all known microorganisms can infect humans and of those, only a minority is able to cause systemic disease. To do so, microbes need to adapt to different complex host niches which demand metabolic modifications and strategies to evade immune surveillance. Some of these adaptations involve adjusting to nutrient quality or scarcity and adapting to stresses, such as different oxygen levels. One of these omnipresent microbes is C. albicans which colonizes different niches of the human body. For successful colonization, the fungus must adopt and sustain different growth forms, such as biofilms, in order to persevere in distinct environmental settings. While it is known that some genes important for biofilm formation are also essential for hypoxia adaptation in Candida species162, it remains poorly understood, how different oxygen levels affect the virulence of microbes and how oxygen levels influence the immune response.

We describe how C. albicans modulated the host response by taking advantage of low oxygen levels which developed during infection. Notably, during C. albicans infection hypoxia was mainly created by the influx of neutrophils to the infected tissue, as depleting neutrophils abrogates hypoxia (Figure 8).

Figure 8. C. albicans infection and subsequent neutrophil infiltration induced a hypoxic state in an abscess model of infection Depletion of neutrophils (α-Ly6G) abrogated hypoxia induction when compared to non-depleted mice (C. albicans) on day 2. At day 3 neutrophil- depleted mice were hypoxic while non-depleted mice were not. Mice were depleted of neutrophils 1 day before infection by intraperitoneal injection of anti-Ly6G antibody and hypoxia was evaluated using an IVIS camera system. Depleted mice and non-depleted mice injected with PBS (sham) served as control.

Survival of C. albicans was higher in a low oxygen environment as compared to normoxic conditions, since the fungus was able to escape from neutrophil phagocytosis due to deficient dectin-1 recognition. The decrease in recognition of C. albicans by neutrophils was due to β-glucan masking in the cell wall of C. albicans under low oxygen conditions. The masking effect was caused by alteration of mannan and chitin amounts in the cell wall (Figure 9).

Figure 9. C. albicans escaped from PMN attack under anoxia a) Neutrophil phagocytosis of C. albicans was affected under anoxic conditions. Phagocytosis was measured after 1 h and 3 h infection with C. albicans and determined in flow cytometry as frequencies of cells in the double positive sub-gate and plotted here as a percentage of phagocytosed cells. Data from n ≥5 (3) b) C. albicans temporally resisted killing by PMNs in an anoxic environment. The fungal killing was

measured as CFU relative to fungal control at 1 h and 3 h post-infection. Data from n=4(3) c) C. albicans showed an increase in chitin levels in anoxia compared to normoxia after 2h infection. Thimerosal killed cells at time point 0 h served as a control (dashed line). Chitin was stained with CFW and measured by FACS. Data from n=3(3) d) C. albicans showed increased mannan levels in anoxia compared to normoxia after 2 h infection. Thimerosal killed cells at time point 0 h served as a control (dashed line). Chitin was stained with concanavalin-Alexa594 and measured by FACS. Data from n=3(3) e) C. albicans showed no difference in anoxic versus normoxic β-glucan exposure after 2h infection. Thimerosal killed cells at time point 0 h served as a control (dashed line). β-glucan was stained with Fc-hDectin-1a / Gt F(ab′)2 anti-human IgG conjugated to Alexa Fluor 488. Data from n=3(3) f) Dectin-1 recognition of anoxic C. albicans is decreased at 2 h post-infection. Flow cytometry of neutrophils for analysis of dectin-1 receptor exposure after anoxic and normoxic C. albicans infection. Plotted is the % of receptor exposure as determined as % from respective uninfected neutrophil control (anoxic and normoxic control).

Growth in low-oxygen environments results in slower fungal growth than under normoxic conditions. Despite this fitness cost, the transition of yeast to hyphal growth, an essential trait for C. albicans host invasion and virulence, was only temporally affected. We analysed the metabolic activity of anoxic and normoxic C. albicans by measuring ATP levels. Interestingly, adaptation to low oxygen environments was induced with a high metabolic rate within the first 8 h. We further observed this metabolic change in clinical isolates of different Candida spp. except for C. tropicalis. Interestingly, C. tropicalis also failed to induce hypoxia in the subdermal abscess model, indicating that only well- adapted Candida species induce hypoxic conditions. We decided to test, if low oxygen adaptation was an important trait for C. albicans virulence. Remarkably, during hypoxic infection with C. elegans C. albicans infection was rendered more lethal to the worms than during infections conducted under normoxic conditions (Figure 10).

Figure 10. Low oxygen environments promoted C. albicans virulence Hypoxia induced increased virulence in C. albicans infection of C. elegans. C. elegans were co-cultured with E. coli or C. albicans. Plates were incubated in normoxia or hypoxia conditions for 48 h and survival measured as the % of alive worms from the total worms plated. Data represents n=5 (10).

Conclusions

C. albicans responded to the alternation of oxygen availability during infection by adjusting the composition of the cell wall. These changes in cell wall components contributed to cell wall masking. This in turn altered neutrophil recognition of C. albicans resulting in decreased engulfment and increased C. albicans survival. The hampered functionality of neutrophils was supported by an infection model using worms indicating that C. albicans uses adaptive prediction under hypoxic conditions in the host to escape immune surveillance (Figure 11).

Figure 11. Summary of C. albicans interaction with neutrophils under low oxygen.

Paper III - Stable redox-cycling nitroxide tempol has antifungal and immune-modulatory properties

In the search for more treatment options for invasive mycoses, we applied biomimicry and exploited a process fundamental for neutrophil antifungal activity: production of oxygen species. Previous work has shown that pharmacological targeting of antioxidative pathways in fungal pathogens could prevent corneal infections163. We have tested tempol, a redox-cycling nitroxide as a new antifungal agent. Tempol is a highly soluble small molecule with remarkably low toxicity to human cells. It has proven anti-neoplastic and anti- inflammatory activity. Here, we present evidence for a considerable fungicidal effect against C. albicans. Systemic administration of tempol in mice resulted in high peak concentrations in blood (1.4 mg/ml) and in kidneys (1.25 mg/ml)164. In humans, topical treatment could be safely done with concentrations exceeding

68.9 mg/ml 165. In our assays, we determined ICs (0.15 – 0.68 mg/ml) and MFC (0.5 mg/ml) of tempol for C. albicans (Table 1).

Table 1. Tempol IC and MFC of C. albicans

Tempol ATP OD530nm Agar-spot assay Tested Concentration range (mg/ml) 0.05 - 17 0.05 - 17 -

ICFCa (AMB> 90 %) 0.68 0.5 -

ICFS b (AZL ≥ 50%) 0.15 0.3 - MFCc - - 0.5

a,b Inhibitory concentration (IC) was determined as the lowest concentration of tempol that

inhibited fungal growth at ≥90% aICFC = IC for fungicidal compounds) or ≥50% bICFS = IC for fungistatic compounds) b; Data from 3 independent experiments. c Minimum fungicidal concentration (MFC) was determined as the lowest concentration of tempol that killed at least 99.9% of the initial inoculum. Data from 3 independent experiments.

We pursued additional clues for tempol’s antifungal mode of action and analysed whole-genome transcriptomic responses of C. albicans to tempol using RNA-Seq. Several of the differentially expressed genes were involved in glycolysis or gluconeogenesis, stress responses and in responses to antifungal drugs, mainly azoles. Tempol activated several genes important for cell wall and cell membrane homeostasis. Additional metabolomics analyses of disturbed pathways showed interferences of tempol in pathways related to energy such as the glucose or Krebs cycle and upregulation of pathways related to alternative glucose pathways. Tempol caused an accentuated stress response in C. albicans similar to stress responses caused by other antimycotic drugs166.

In vivo, tempol could reduce the fungal burden of kidneys in an experimental model of systemic candidiasis at day 1 post-infection (Figure 12). However, that effect was not sustained in the following days. Different pharmacological formulation of tempol to reduce renal drug excretion, increased dosing or a

combination of both could probably increase tempol´s efficacy in vivo and match the promising in vitro results demonstrated by the compound.

Figure 12. Tempol reduced fungal burden at day 1 Mice infected intravenously with C. albicans (2.6 × 106 cells/ml) and treated with tempol (1.6 mg /gram of mouse /day) or PBS are shown. Tempol treatment reduced fungal burden in kidneys of infected mice to about 50% of control. Bars represent mean ± SD.

Besides reducing the fungal burden in mice, tempol treatment notably modulated immune responses in humans. We performed an infection and consequent treatment of human blood with tempol and measure the cytokine response triggered. Tempol contributed to an increased inflammatory response determined by increased levels of cytokines, such as IL-8, MCP-1, MIF, vascular endothelial growth factor (VEGF) and regulated on activation, normal T cell expressed and secreted (RANTES) (Figure 13). Interestingly, fluconazole has been shown to induce several of these cytokines during experimental candidiasis suggesting that tempol indeed shares properties of a common antifungal drug167.

Figure 13. Tempol induced a strong immune response upon infection of C. albicans in whole blood Human whole blood from 3 healthy volunteers were stimulated for 24 h with C. albicans (2 × 105 cells/ml) in presence or absence of 0.516 mg/ml tempol. The concentration of (A) IL-8 (B) MCP-1 (C) MIF (D) VEGF (E) IL-1Ra were measured in cell supernatants using Bio-plex

cytokine array. (A–E) Bars represent mean + SD. Significance was analyzed by one way-Anova with a Tukey post-test.

Conclusions

We demonstrated that tempol is a fungicidal agent. In vitro, tempol disrupted several fungal pathways, such as glycolysis, and induced a stress phenotype in the fungus. The immune modulatory activity of tempol is a very interesting property that might contribute to reduced resistance development under application of the substance. In vivo, tempol showed a promising initial effect in the treatment of candidiasis that might be potentiated with further drug development.

Paper IV - Antifungal application of non-antifungal drugs

Novel and efficient antifungal therapies are a necessity, since current antifungals are few in number, restrited to only 6 classes, and with a narrow target range. The side effects described for the standard antifungal drugs (SAD) range from mild to severe. In this study, we performed a screening of two compound libraries -the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library -for anti-Candida activity. The screening protocol was designed according to guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST)168. Briefly, C. albicans was grown in the presence of serial dilution of tested compounds for 6 h a 24 h. Growth inhibition induced by drugs was assessed by quantification of the optical density of cultures, by determining fungal ATP levels and by microscopy analysis of morphological changes.

A total of 844 drugs were screened resulting in 26 positive hits showing activity against C. albicans. Of those 26 agents, 12 were SADs and 7 were off-target drugs

with previously reported antifungal activity. The remaining 7 off-target drugs were unreported for antimycotic activity (Table 2).

Table 2. Identified drug hits with antimycotic activity [Image re-produced from Stylianou et al.169].

Identified Previously Previously Therapeutic Reference drug described described use no or source as antifungal as anti- Candida

Haloperidol HCl Yes No Antipsychotic 21, This study Trifluperidol 2HCl No No Antipsychotic This study Anemia, Stanozolol No No angioedema This study Melengestrol acetate No No Anticancer This study Megestrol acetate No No Anticancer This study Tosedostat No No Anticancer This study Amonofide No No Anticancer This study Methiothepin maleate No No Antipsychotic This study

Rapamycin Yes Yes Anticancer 26

Auranofin Yes Yes Antirheumatic 27 Bleomycin

Yes Yes Anticancer 40 sulfate

Disulfiram Yes Yes Anticancer 41

Artemisinin Yes Yes Antimalarial 42 Tamoxifen

Yes Yes Anticancer 43 citrate Tioconazole Yes Yes Antifungal NAa Oxiconazole Yes Yes Antifungal NA nitrate Ketoconazole Yes Yes Antifungal NA Climbazole Yes Yes Antifungal NA Miconazole Yes Yes Antifungal NA Myclobutanil Yes Yes Antifungal NA Fluconazole Yes Yes Antifungal NA

Amorolfine Yes Yes Antifungal NA Bifonazole Yes Yes Antifungal NA Sertaconazole Yes Yes Antifungal NA Itraconazole Yes Yes Antifungal NA Terbinafine HCl Yes Yes Antifungal NA NA, not applicable

Positive hits were further tested on type and clinical strains of C. glabrata and C. dubliniensis to test their efficacy against non-albicans strains. Both strains are frequent nosocomial pathogens with described resistance to SADs. All 7 identified off-target drugs showed potent antifungal activity against non-albicans Candida spp. As a reference, we used three off-target drugs with previously described antifungal activity (Table 3).

Table 3. Candida spp. susceptibility to the seven off-patent drugs with novel antifungal activity [Image re-produced from Stylianou et al.169].

) . n 0 0 i 9 3 6 a C 0 0 7 8 0 6 9 7 5 3 . . . r I 8 3 0 2 9 8 0 7 3 4 2 t ...... 6 s M 3 > > 3 3 3 2 > 0 3 0 2 l 7 a - c 3 i 0 0 5 9 7 3 6 3 n C 0 0 C 9 7 2 9 8 8 7 i 7 ...... I l 0 B 5 3 4 3 3 3 4 2 3 . . c U ( M > > > > > > > 3 > 0 3 . 0 0 9 3 ) 0 C 0 4 2 0 8 6 2 5 7 6 . . . I n 3 a 0 3 6 0 7 9 8 4 3 2 i ...... t 0 a M 3 > > 3 3 4 > 0 0 0 a 0 r r t 9 b s a C 0 0 e 5 9 7 3 6 l 0 0 C C 7 2 9 8 8 p 0 7 g ...... 0 I 5 y 5 T 1 . 3 4 3 3 3 4 2 . . t . C A ( M > > > > > > > 3 1 0 3 − 3 ) . 0 1 n 0 i C 0 0 4 × 8 6 a 9 5 0 0 I 2 3 4 3 0 r 0 2 8 8 4 9 ...... t . . 9 s M 0 0 3 1 3 4 1 0 0 < 8 l 3 a - c 0 3 i 6 0 5 9 7 3 7 0 7 n C 0 C . 1 2 9 8 8 5 . 2 i ...... I l 3 4 6 0 B 3 3 3 4 2 3 . . c U ( M > > > > > > > > 0 0 3 − 3 7 . 0 s 8 1 i 0 0 s 9 ) C 0 4 0 × 8 6 9 7 5 3 . n 7 I n 3 3 0 0 2 9 8 8 e 4 9 i ...... S i a M 0 > 3 3 3 4 2 0 0 < B n r i t C l s / b 6 0 0 e 9 5 3 9 7 7 u 3 C 0 0 0 2 9 8 8 5 p 8 6 d ...... I 6 0 y 7 D . 4 3 3 3 4 2 3 . . . t C C ( M 3 > > > > > > > 0 0 3 − 3 . 0 1 ) 0 n C 7 0 0 0 × 8 3 9 6 3 i I 3 4 3 4 3 0 3 0 8 a 9 ...... r 2 M 0 0 0 0 0 4 2 0 0 < t 2 s 9 l 7 a - c 3 i 0 0 2 9 3 n C C 0 0 0 2 8 1 5 7 0 i 6 . . . . I l 6 0 B 7 9 8 3 4 3 4 2 ...... c U ( M 3 > > 3 3 > > 3 0 0 3 − 3 . 0 1 0 C ) 8 6 0 0 × 3 7 9 1 0 I 4 4 3 3 3 0 3 0 n s 4 9 ...... i . n M 0 0 0 0 0 4 1 0 0 < a a r t c 4 i s 1 b 0 e 2 3 9 3 l C 0 0 0 2 8 p 8 5 7 5 0 6 a . . . I 6 0 0 y 7 9 8 3 . C 3 4 2 ...... t C S ( M 3 4 > 3 3 > > 3 0 0 3 9 6 7 2 8 7 0 0 9 8 5 . . . . 7 4 0 0 7 8 3 2 3 5 1 3 . . . e . . . 4 6 g 4 o 3 o o 3 9 o t t t t n o o o o

o o t t t t 3 a 3 3 3 t t

− − − − r ) 3 3 3 3 3 3 l 0 0 0 0 − − − − − − 1 n 1 1 1 0 0 0 0 0 0 m c 1 1 1 1 1 1 / × × × × n g × × 4 3 8 8 × × × × o . . . . µ C ( 6 7 3 6 6 4 2 7 4 9 e t e a t l e a l C t e l a t e t H c C n a m 2 t a e H e l g l n l c i o a o a o p n t d r l l e i i t l e d n a a o d c i i r s t l i o h g f r e y e s f t r o n o e g t a p o o z m s i u n p n n d u o f e a l a h e o i e o n f l g t l t r p s i a e e e a a n m u r o t

A H T S M M T A M A R

The clinical isolate and the type strain of C. albicans strains were affected to a similar extent by the 10 agents. Auranofin was efficient against both C. dubliniensis strains tested. Haloperidol, in contrast, inhibited the C. dubliniensis type strain (MIC, 3.76 µg/ml) but fail to inhibit the clinical isolate as efficiently. Together, the C. glabrata strains were the most resistant, however, they were nevertheless inhibited by 6 of the 10 agents tested. Remarkably, methiothepin maleate reached a MIC of 3.57µg/ml in both C. glabrata strains. The C. dubliniensis strains were in general slightly less susceptible than the C. albicans strains. Rapamycin was effective against all tested strains (MIC, <0.1 µg/ml).

The efficacy of the newly discovered compounds was compared to different SADs at a concentration of 1 µM. Notably, very similar growth inhibition rates were observed for the new off-target drugs and the tested SADs, indicating that our off- target drugs are similarly effective as currently available SADs.

Conclusions

Drug development demands high budgets and considerable efforts in terms of personnel and time. In our study, we aimed to provide alternative routes to reduce effective cost and time requirements by re-purposing already approved drugs. Hence, we proposed new starting points for the development of additional therapy options against invasive mycoses.

When medically adequate and advantageous for the patient, the use of these drugs could be beneficial either as a single option to treat cancer or as a prophylactic measure of a potential opportunistic infection. The high number of FDA-approved drugs to the low number of drugs tested for off-target effects additionally provides promising prospects of identifying future hits.

Outlook

This thesis highlights the importance of exploring the molecular interactions between immune cells and fungal pathogens at the niche level and how this interplay can influence the overall outcome of the infection.

We show how mast cells, a cell type known for its role in allergy and asthma, can serve an important role as sentinels and first responders upon entry of fungal pathogens. Mast cells further recruit neutrophils to orchestrate the second wave of antifungal responses. It will be interesting to establish a link between the amplitude of antifungal responses and the onset of asthma triggered by fungal cells or components.

Low-oxygen environments promote C. albicans virulence. Additional work in a gut model of infection could help elucidating the importance of this mechanism in one the C. albicans natural commensal niches.

Tempol is a molecule with previously unknown anti-Candida potential. New candidate molecules based on the structure of tempol with improved in vivo efficacy may be developed into new antifungal drugs with good safety profiles.

We have re-purposed 7 new agents for antifungal use. This work opens new doors to find new antifungal therapeutic potential in approved drugs. A retrospective cross-sectional study on patients treated with some of these drugs could help judge the drugs' potential in a clinical setting.

Acknowledgment

I would like to thank all who contributed with their support during this time. All the individual stories and unique moments we have shared were truly special.

Thank you, Gunnar Nilsson, for receiving me in your laboratory and Maria Ekoff for all the help during that month. In my short visit, I have received more than scientific knowledge. The joy and passion you have in your work and the camaraderie was inspiriting. I am grateful to Rolf for the collaborative effort. Without your constant availability, it would have been hard to accomplish so many successful experiments.

Thank you to members of Anders Sjöstedt group. Thank you for embracing me from my first days as a master student until now. Anders, Athar, Kjell, Igor Lena, Helena, Jeanette, Nasibeh you are a wonderful group of people and the familiar atmosphere you all have created wins people straight away. Must include in this group former members of the “fika corner” Anna, Olena, Kun. You have been always kind to me and your absence was always missed.

To former and present members of immunology. Thank you to Marie-Louise and Lucia for always having a smile in the corridor and all the wonderful suggestions during the seminars. To Anne for curating for our well-being always with a smile. To Lena for always expediting all matters at light speed and with extreme kindness. Thank you Viqar and Radha I could not have thought about better people to share all these years of teaching. Thank you, Kristina my co- supervisor for all the great pep talks during all these years. Your positive energy aura is contagious. Thank you, Mattias, for the all the advices and humors’ attitude. Priscilla, Andy, Shrikant and Wolly you are cheerful bunch always willing to help. Thank you for making this journey more interesting.

Special thanks CU laboratory, former and present members. Work ethics, dedication, and commitment just to name a few things I have learned while in

your company. Thank you, Maria and Marie, Nathalie, Lucas, Marc, Anna, Saeed, Sujan, and Ava. I am especially thankful to Cecilia and Emily for all the dedication and never giving up on me and the projects. I have learned a lot from both and cherish the friendship that was born during those times. Madhu the best thing about science is the smart and engaging people we meet along the way, thank you for being a friend from day 1. Thank you, Hanna. Your work ethics and non-compromising scientific integrity have had quite an influence on me. Finally, thank you, Constantin, for taking the chance on me. Your scientific deep knowledge was always impressive, but your biggest asset is giving space for students to grow. Always cheered the non-imposing and honest dialogue we had. Thank you for the open-door policy to discuss science and teaching me how to be a conscious researcher and never compromise the work quality. My future development will always reflect the effort you have put in me.

To Per and Sandra for all the support, love and indoctrination into the Swedish way of life. Thank you for always making me feel at home when you were around. I care deeply about both of you.

My last paragraph and final words are for my family. To my brother from Cyprus, Marios efharisto µαλάκας for the companionship, the support and all the love. There will always be a special place in my heart for you. Hvala ti Marija što si uvijek bila tu za nas i za djecu. Ja puno poštujem sav trud koji si učinili i našla vremena da nas posjetiš i pomogneš. Hvala Nenade, ako bih imao mlađeg brata, voljeo bih da je on kao ti. Hvala Mari i Jovanu. Uvijek ste pokušavali da budete prisutni tijekom ovih godina da biste podržali svoju kćerku i razmazili svoje unuke. U portugalskom kažemo da ako poljubiš one koje voliš onda osjećaš to na sebi. I zahvalan sam za svu ljubav koju ste nam svi dali. Ao meu traquina Peter um beijo do tio que gosta muito dele. Para a Daniela obrigado por toda a ajuda durante estes anos. Ao meu querido irmão Nuno por toda a ajuda e todo o amor que nunca vou conseguir retribuir. Aos meus pais pelos sacrifícios que fizeram para me ver chegar aqui, a minha admiração, respeito e amor. Amo-vos. A Klara e Matias tanto de mim e tanta da minha felicidade passa por vocês os dois. Para vocês o mundo!!! Kristina, o meu pilar, o meu porto de abrigo. Amo-te!

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