Department of Clinical Microbiology

Molecular Infection Medicine Sweden (MIMS)

Umeå University

Umeå 2015

Pharmaceutical And Immunological Challenge Of Fungal Pathogens

Marios Stylianou

Pharmaceutical And Immunological

Challenge Of Fungal Pathogens

Marios Stylianou

Doctoral thesis Department of Clinical Microbiology Molecular Infection Medicine Sweden (MIMS) Umeå University Umeå 2015

Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-308-3 ISSN: 0346-6612 Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Print & Media Umeå, Sweden 2015 Cover illustration: Re-produced and modified from Lopes et al 2015.

As for me , all I know is that I know nothing

Socrates

To my Wife and my Parents, the heroes of my life.

i

Table of Contents Table of Contents i Publications included in the thesis iii

Publications not included in the thesis iv

Abstract v

Abbreviations vii Introduction and Background 1

1.0 Human fungal pathogens and pathogenicity 1 1.1 Candida spp. 1 1.2 C. albicans polymorphism 2 1.2.1 Two-way Yeast to Hyphal transition 3 1.3 C. albicans evasion of human defense 5

2.0 Human Innate Immunity 9 2.1 Neutrophils 9 2.2 Mast cells 10 2.2.1 Mast cells in allergies 11 2.2.2 Mast cells and infections 13

3.0 Antimycotics and their mode of action 15 3.1 Polyenes 15 3.2 Azoles 16 3.3 Allylamines 17 3.4 Echinocandins 18 3.5 Resistance to antimycotics 19 3.6 Alternative strategies for novel antimycotics 19

Material and Methods 22 Fungal strains 22 Candida albicans GFP construct 22 Human mast cells 22 Isolation of human neutrophils 23 Human neutrophil and monocyte migration 23 Sytox green-based cell death assay 23 Mast cell degranulation assay 24 Cytokine quantification assay 24 Cellular viability 24 High-content analysis of yeast and hyphal morphotypes 22

Aims 26 Results and Discussion 28

ii

Paper I 28 Paper II 31 Paper III 35

Concluding Remarks 39 Acknowledgements 40 References 42

iii

Publications included in the thesis

Paper I

Lopes JP**, Stylianou M**, Nilsson G, Urban CF: Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells. Scientific reports 2015; 5:12287.

**equal contribution

Paper II

Stylianou M, Uvell H, Lopes JP, Enquist PA, Elofsson M, Urban CF: Novel high-throughput screening method for identification of fungal dimorphism blockers. Journal of biomolecular screening 2015; 20:285-291.

Paper III

Stylianou M, Kulesskiy E, Lopes JP, Granlund M, Wennerberg K, Urban CF: Antifungal application of nonantifungal drugs. Antimicrobial agents and chemotherapy 2014; 58:1055-1062.

iv

Publications not included in the thesis

Paper IV

Björnsdottir H, Welin A, Stylianou M, Christenson K, Urban F, Forsman H,

Dahlgren C, Karlsson A, Bylund J. Cytotoxic peptides from Staphylococcus

aureus induce ROS-independent neutrophil cell death with NET-like feature.

Manuscript

v

Abstract

Incidences of fungal infections are on the rise in immunosuppressed people.

Predominant causative agents for these mycoses are species of the genus

Candida, including Candida albicans, Candida glabrata and Candida

dublieniensis. Despite a wide range of emerging pathogens, C. albicans

remains the leading cause. According to recent epidemiological studies,

blood stream infections with C. albicans cause annually ~55% mortality in

approximately 300,000 patients from intensive care units worldwide.

Furthermore, the percentage of morbidity linked to oral, esophageal and

vulvovaginal mycoses cause by C. albicans reach up to 90%. Reasons for

these medical concerns are the lack of efficient diagnostics and antifungal

therapy.

Here, we therefore sought to find novel antifungal strategies inspired by

innate immune cells, such as neutrophils. These phagocytes are able to block

the fungal pathogenicity. Neutrophils are bloodstream leukocytes serving as

the first line of defense against pathogenic microbes. It has been shown that

neutrophils have a strong antifungal activity by impairing the conversion of

the dimorphic C. albicans from yeast to hyphal form (Y-H). Consequently,

we raised the question whether other immune cells, such as mast cells, with

less phagocytic cabapilities may have similar activity to neutrophils.

Mast cells are tissue-dwelling cells. Mucosal tissue is rich in mast cells and

usually constitutes the entry ports for fungal pathogens into the human

body. A contribution of mast cells in antifungal defense is, thus, very likely.

We human explored mast cell functions upon encounter with fungal

pathogens. Interestingly, human mast cells show a transient potential to

impair fungal viability. To understand the mechanism behind this

impairment we analyzed the human mast cell functions in more detail. We

found that human mast cells challenged with C. albicans, immediately

degranulate and secrete distinct cytokines and chemokines in an

orchestrated manner. The chemokines secreted attract neutrophils. Mast

vi

cells moreover are able to internalize fungal cells and to ‘commit suicide’ by

releasing extracellular DNA traps that ensnare the pathogen.

The effectiveness of future antifungals is depended on targeting the pathogen

virulence with more efficiency.

The dimorphism of C. albicans is proven to be essential its virulence.

Blockage of this switching ability could render the pathogen avirulent.

Consequently, we screened for compounds that mimic the neutrophils anti-

dimorphic activity by screening small chemical molecule libraries that block

Y-H transition. The screening of big chemical libraries requires a reliable,

reproducible and rapid high-throughput screening assay (HTS). We

developed an HTS assay based on automated microscopy and image

analysis, thereby allowing to distinguish between yeast and filamentous

forms. In order to find the ideal Y-H blocker, we also evaluated the cell

viability via the count of ATP levels when challenged with the respective

small chemical molecules.

Drug development is an elaborate and expensive process. We therefore

applied our screening setup to identify antidimorphic/antifungal activity in

compounds from two different chemical libraries including FDA-approved

drugs. The study disclosed 7 off-patent antifungal drugs that have potent

antimycotic activity, including 4 neoplastic agents, 2 antipsychotic drugs and

1 antianemic medication.

In a nutshell, we aimed to mimic the anti-dimorphic/antifungal activity of

neutrophils with small chemical molecules. Furthermore, we elucidated how

immune cells contribute to antifungal defense to exploit these mechanisms

for the development of novel antifungal therapies. Thus, this thesis provides

novel tools for the discovery of more efficient compounds, identifies

previously unknown antifungal aspect of off-patent FDA-approved drugs and

highlights the interplay of mast cells with pathogenic fungi with the aim to

define new screening strategies.

vii

Abbreviations ABC ATP-binding cassette

ALS Agglutinin-like sequence

AmpB Amphotericin B

CAT Catalase

CFW Calcofluor white

CPH1 Candida pseudohyphal regulator

EFG1 Enhanced filamentous growth

ERG Ergosterol

FHL Factor–H-like protein

GAS Group A Streptococci

GFP Green fluorescent protein

HCS High content screening

HMCs Human mast cells

HSL Homoserine lactone

HTS High-throughput screening

HWP1 Hyphal wall protein

IFN Interferon

KO Knockout

LWR Length to width ration

MAPK Mitogen activated protein kinase

MC Mast cell

MCT Mucosal mast cell

MCETs Mast cell extracellular traps

MCP-1 Monocyte chemoattract protein 1

MDRs Multi-drug resistance

MIF Migration inhibitory factor

MOS Mean object shape

MTC Connective tissue mast cell

NETs Neutrophil extracellular traps

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NRG1 Negative regulator of glucose repressed genes

PAMPs Pathogen -associated molecular pattern

PRRs Pattern recognition receptors

RFG1 Repressor of filamentous growth

ROS Reactive oxygen species

SCF Stem cell factor

SOD Superoxide dismutase

TLR Toll-like receptor

TUP1 Thymidine uptake

YWP1 Yeast wall protein

Y-H Yeast to hypha

1

Introduction and Background

1.0 Human fungal pathogens and pathogenicity

Fungal infections in humans are not perceived adequately as major disease

and increasing health problem despite the annual high morbidity and

mortality due to mycoses. Notably, more than 1.5 billion people globally

encounter superficial mycoses afflicting skin and nails. In addition, 50-75%

of women in their childbearing years experience at least one episode

vulvovaginal mycoses, while approximately 75 million women annually face

several relapsing incidents1. More importantly, mortality counts resulting

from invasive mycoses are comparable to tuberculosis or malaria whereby

more than 90% of lethal mycoses are caused by Cryptococcus, Candida,

Aspergillus or Pneumocystis1. Amongst these, the Candida spp. are the most

frequent etiologies of invasive opportunistic infections in

immunosuppressed individuals2.

1.1 Candida spp.

Candida albicans, Candida glabrata and Candida krusei are the most

prominent causes of human mycoses while C. albicans is always in "pole

position”2. Incidences of severe, opportunistic infections by Candida

albicans are continiously increasing worldwide. C. albicans is now ranked as

the second most-frequent cause of nosocomial infections along with

Staphylococcus aureus and Pseudomonas aeruginosa3. Notably, C. albicans

causes more than 50% of all nosocomial blood stream infection

(candididemia) cases4 with a mortality rate of ~36% in intensive care units 5.

An epidemiological study from USA describes that candiduria and systemic

candidiases were escalated 2-3 fold within 5 years6. Half of the non-albicans

candidiases are caused by C. glabrata which is, nonetheless, the most

frequently isolated fungal pathogen from HIV-patients diagnosed with oral

thrush 7; 8. Importantly, C. glabrata and C. krusei are treatment-refractory

against fluconazole, the most commonly used antifungal drug. C. glabrata

2

has evolved resistance to fluconazole by drug target mutation, whereas C.

krusei is naturally-resistant. In that line, hospitalized individuals are

frequently administered with fluconazole due to suspicion for a mycose or as

routine prophylactic procedure allowing an increase with severe mycoses

caused by C. glabrata or C. krusei 7; 9; 10.

1.2 C. albicans polymorphism

Virulence to cause invasive or superficial infections is closely related to the

capability of C. albicans to reversibly switch between budding yeast and

filamentous forms (Y-H transition)11. Filaments exist as pseudohyphae or

true hyphae (figure 1). Pseudohyphae are characterized by constrictions of

the septum as a chain of unseparated yeast cells with dissimilar cell walls11.

On the other hand, a true hypha grows apically from the mother cell which

periodically can form branches. This polarized growth has perfectly parallel

cell walls without constrictions to the septae11.

Figure 1: Candida albicans morphotypes12. C. albicans filamentation is defined as

switching from yeast (lower left) to hyphae (lower right) or to pseudohyphae (upper).

[Re-printed with permission of the Nature Publishing Group, Licence 3687551119070]

3

1.2.1 Two-way yeast to hyphal transition and virulence

Upon host susceptibility yeast can escape from the commensal niche, such as

the gut and the oral cavity, to other tissues by switching to hyphae. These

filaments have been demonstrated to be essential for dissemination, whereas

the yeast is rather considered the proliferative form13. Both are, however,

required for colonization, virulence and biofilm formation. In vivo and in

vitro experiments with several morphology-regulating transcription factor

mutants have shown to be avirulent and incapable to form biofilms14-16.

Candida pseudohyphal regulator (CPH1) is a transcriptional factor and part

of the conserved mitogen activated protein kinase (MAPK) transduction

pathway (figure 2). CPH1 knockout (KO) strain shows a significant reduction

of hyphal filamentation15; 16. Moreover, enhanced filamentous growth (EFG1)

is a transcriptional activator of hyphal transition. Lack of this gene results in

impairment of hyphal induction (figure 2) 15-17. However, under certain

conditions such as in response to serum EFG1 KO strains have shown to

form pseudohyphae. Interestingly, CPH1 and EFG1 double KO irreversibly

failed to filament resulting in a complete loss of virulence15-17.

The Y-H transition is not only dependent on transcriptional activators, but

also on transcriptional repressors such as thymidine uptake (TUP1)18; 19

(figure 2), and negative regulator of glucose-repressed genes (NRG1) and

repressor of filamentous growth (RFG1) 15; 20; 21. Nrg1p, Rfg1p and Tup1p are

proteins with DNA-binding domain which upon activation results in

suppression of hyphal-associated gene expression and consequently in

blockage of hyphal growth15;20;21.

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Figure 2: Regulatory network of Y-H transition in Candida albicans 15. [Granted Re-

printing permission from American Society for Microbiology]

Farnesol, a quorum-sensing molecule, is secreted by C. albicans to disturb

the Y-H transition via TUP1 activation22-24. It is also assumed to participate

in yeast relocation from biofilms in order to disseminate the infection. In

addition to farnesol, homoserine lactone (HSL) has antidimorphic activity by

blocking hyphal growth. HSL has been observed to be produced and secreted

from Pseudomonas aeruginosa in co-infections with C. albicans22-24. HSL

influences the transcriptional upregulation of TUP1, NRG1 and of yeast wall

protein (YWP1), leading to inhibition of Y-H transition and biofilm

impairment23.

Taken together, the Y-H transition in several fungi and particularly in

C. albicans is an essential virulence trait of the pathogen to establish

successful colonization and infection. Consistent with this notion,

C. albicans cells are incapable to invade human cells, to escape phagocytosis,

and to form biofilms when Y-H transition is disturbed resulting in an

5

avirulent state. Therefore, one could say that the reversible Y-H switching is

the “Achilles´ heel’’ of C. albicans.

1.3 C. albicans evasion of human defense

Even though human immune responses to microbial invasion are mostly

victorious, some pathogens are able to overcome attacks, such as

phagocytosis, oxidative burst or released antimicrobial peptides25. Fungal

pathogens in turn are armed or likewise have evolved various mechanisms to

oppose the human immune system26. The latter reacts to fungal invasion

primarily by the recruitment of the first line of defense namely neutrophils

and macrophages27;28.

Fungal cell wall components, such as mannan, β-(1.6) and (1.3)-glucan, are

major pathogen molecular associated patterns (PAMPs) for innate immunity

(figure 3). PAMPs are recognized by different pattern recognition receptors

(PRRs), such as Dectin-1, Dectin-2, toll-like receptors (TLR) TLR2 and

TLR429-32 (Figure 4). C. albicans can evade detection by either masking the

recognition site or by escaping from macrophages’ ensnaring and

phagolysosomal toxicity. For example, Dectin-1 from human cells senses β-

glucan from the fungal cell wall which C. albicans camouflages by the use of

mannoproteins33. In addition, C. albicans yeast cells might also escape from

phagolysosomes in human macrophages by induction of hyphal growth

resulting in cell penetration and pyroptosis. Interestingly, in human

neutrophils this escape route for C. albicans is blocked34; 35.

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Figure 3: Candida albicans cell wall architecture 36. [Re-printed with permission of the

Nature Publishing Group, Licence 3690810405720]

A different escaping mechanism of C. albicans cells is the production of

various superoxide dismutase proteins (Sod) to oppose neutrophil and

macrophage ROS37. C. albicans possesses cytosolic and mitochondrial Sods

to detoxify ROS. Upon superoxide (O2-) production, the primary ROS during

the oxidative burst from the host cell, C. albicans expresses Sods which

convert O2- to hydrogen peroxide (H2O2), while in turn catalase protein

(Cat1p) decomposes H2O2 to H2O and O238. To date, Sod1p (cytoplasmic),

Sod2p (mitochondrial), Sod3p (cytoplasmic), Sod4p, Sod5p and Sod6p (cell

surface) have shown to prevent host’s hazardous oxygen radicals37-39.

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Figure 4: Candida albicans cellular components recognized by human immune system36. [Re-printed with permission of the Nature Publishing Group, Licence 3690810405720]

The fact that Sods 4-6 are located on the cell surface indicates that they play

a role in protection against host-originated ROS stress. Indeed, Sod4p and

Sod5p have shown to counteract the oxidative burst as demonstrated by

reduced cellular viability of SOD4 and SOD5 double-KO mutants when

interacting with macrophages37. In good agreement, Sod5p is induced under

hyphal growth, which is essential for escape from macrophage

phagolysosomes38; 40.

Another example of a C. albicans defense strategy is the characteristic

evasion of the complement system. The human complement system is

mediated by three pathways, the classical, the alternative, and the lectin

pathway. The classical pathway initiates via an antibody-antigen complex,

while, the alternative pathway is antibody-independent and induced by

PAMPs on microbial surfaces41. The lectin pathway is activated via binding of

host-derived lectin to fungal mannose- and mannan-related PAMP

molecules. The lectin pathway, as the other two pathways, drives the

generation of C3 convertase to hydrolyse C3 to C3a and the C3b opsonin.

8

Factor H and factor H like protein-1 (FHL) mediate further activation of C3b.

C. albicans encodes the pH-regulated protein-1 (Pra1p) which is released

from both yeast and hyphal cells to bind Factor H and FHL42. This prevents

the respective activation of C3 upon recognition of fungal cells.

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2.0 Human innate immunity

The human immune system is categorized as innate and adaptive branch.

Adaptive immunity is characterized by slow reaction (>4 days) but with a

specific immunological memory. When innate immune responses fail to

entirely clear microbial invaders the adaptive branch takes over and by

building up specific memory, it can activate fast responses upon re-infection

for efficient and rapid removal of “memorized” microbes. However, innate

immunity being the first line of defence does not require prior exposure, but

is rather rapid and efficient. The innate branch consists of three main

barriers the primary-physical (skin), the chemical (stomach acidity) and the

cellular barrier (immune cells) 41. Neutrophils are part of the innate

immunity and defined as professional phagocytes supported by the mucosal

immune cells, such as mast cells, whose function against pathogens is poorly

explored.

2.1 Neutrophils

The neutrophils are the most abundant leukocytes circulate in the blood

stream. They carry a plethora of cytoplasmic granules and have a

characteristic multilobed nucleus (figure 5). Neutrophils are terminally-

differentiated, non-dividing cells which after their maturation process, called

granulopoiesis, in the bone marrow are released to the blood stream. To

date, the exact life-span of neutrophils is not unambiguously determined;

moreover it depends on several variables, such as the variety of stimuli.

Nevertheless, according to current literature it ranges between 6 hours to 5

days approximately43-45. During this period they are fully-equipped to deal

with pathogens. Parts of their antimicrobial arsenal include the secretion of

ROS, peptides and proteases46. The ROS production also enhances

phagocytosis by boosting the release of various enzymes and antimicrobial

peptides to the phagolysosome47; 48.

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Figure 5: Neutrophil with the characteristic lobulated nucleus (red) and granules

(yellow)49. [Granted re-printing permission from Wikiversity Journal of Medicine]

In addition to above defense tools, neutrophils are also able to prevent

pathogenicity by “sacrificing” themselves, via a novel cell death process.

Upon contact to pathogens, neutrophils undergo a programmable molecular

signalling and intracellular rearrangement to eventually release neutrophil

extracellular traps (NETs)50. NETs are web-like structures composed of a

nuclear DNA scaffold decorated with cytoplasmic and granular material51. By

producing NETs Neutrophils entrap microbes and in the case of pathogenic

fungi, such as C. albicans, NETs have a fungistatic mechanism. Mainly the

NET–bound protein-calprotectin, a zinc-chelator, drives C. albicans to

growth arrest, when it is entangled in NETs52. Eventhough NETosis (NET-

forming process in analogy to apoptosis) is a very defined and distinct form

of cell death and continuously new triggering stimuli are revealed the exact

molecular details and regulatory networks remain poorly defined.

2.2 Mast cells

Mast cells (MCs) are tissue-resident leukocytes originated from

hematopoietic progenitors (figure 6). The respective progenitors are primed

with stem cell factor (SCF) which binds on SCF receptors/CD117/tyrosine-

protein kinase also termed c-kit53-55. After differentiation and maturation

MCs leave the peripheral blood to be further differentiated into two dinstict

subtypes, the mucosal MC (MCT) and the connective tissue MC (MCTC)56; 57

(table 1). The basic differences of MCT and MCTC are their residency to

11

different tissues and granular composition as well as the chymase expression

which is absent in MCT56; 57.

Figure 6: Hematopoietic tree and mast cell maturation41. [© 2007 Janeway's

Immunobiology, Seventh Edition by Murphy et al. Reproduced by permission of Garland

Science/Taylor & Francis Group LLC.]

2.2.1 Mast cells in allergies

MCs have strong IgE-binding capacities via the expression of the high-

affinity IgE receptor, FcεRI, whereas other IgE-receptor-associated cells,

such as eosinophils, express IgE-binding receptor FcεRII with lower affinity

than FcεRII 57; 58. IgE as well as other secreted allergic triggers, such as

cytokines, anaphylatoxins or neuropeptides59, have beyond doubt connected

12

MCs with allergic responses. Indeed, human MCs undergo IgE binding upon

activation with various stimuli, such as antigen/allergen (peanut, pollen and

latex), allowing mediator production57; 60. The mediator secretion is classified

into two distinct mechanisms, the preformed mechanism and the newly-

synthesized molecule mechanism56;57;61. Preformed mediators are “ready-to-

go” molecules such as histamine, heparin, serotonin, cathepsin G and TNF-

α. Examples of newly-synthetized mediators are nitrogen and oxygen

radicals, different cytokine and chemokines (TNF-α, MCP-1 and MIP-1α) as

well as various lipid mediators (Prostagladin D2 and leukotrienes)56; 57; 61.

Table 1: Differences of mast cell subtypes 57. [Re-printed with permission from Springer

Publishers, Licence 3687600957650].

Feature MCTC cell MCT cell

Structural features Grating/lattice granule ++ – Scroll granules Poor Rich

Tissue distribution Skin ++ – Intestinal submucosa ++ + Intestinal mucosa + ++ Alveolar wall – ++ Bronchi + ++ Nasal mucosa ++ ++ Conjunctiva ++ +

Mediator synthesized Histamine +++ +++ Chymase ++ – Tryptase ++ ++ Carboxypeptidase ++ – Cathepsin G ++ – LTC4 ++ ++ PGD2 ++ ++ TNF-α ++ ++ IL-4, IL-5, IL-6, IL-13 ++ ++

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2.2.2 Mast cells and infections

MCs have versatile roles beyond allergies, as they actively participate in

different autoimmune diseases such as rheumatoid arthritis, asthma and

systemic sclerosis56;57. Furthermore, MCs are associated to inflammatory

diseases, such as, graft-versus-host disease, fibrotic disease and ischemic

heart disease as well as in various infectious diseases 56; 57; 62. Human MCs

immediate response against GAS (Group A Streptococci) comprises ROS

production and the release of in vitro and in vivo antimicrobial molecules,

similar to Neutrophils 63.

Figure 7: Role of mast cell responses to different microbial pathogens62.[Re-print

permission from Plos publisher]

PRRs expressed by MCs include TLRs, such as TLR2 and TLR4, well-defined

receptors for recognition of bacteria and fungi. MCs also express non-TLRs,

C-type lectin receptors, such as Dectin-1 and Mincle, for the identification of

fungal components (figure 7)64-66. The antibacterial contribution of MCs

includes the secretion of proteases and tumour necrosis factor alpha (TNF-

α). In vivo experiments highlighted a key role of MCs in innate immunity

against bacterial pathogens, such as for instance Escerichia coli, Klebsiella

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pneumonia and Listeria monocytogenes, as indicated by a considerable

decrease of mouse survival in infected mast cell-deficient mice compared to

wild-type littermates67. Similar to NETs, mast cell extracellular traps

(MCETs), have been described, at first as a response to S. pyogenes68.

MCETs ensared and killed the bacteria (figure 8)69. Again, in analogy to

NETs, MCETs are composed by a DNA scaffold decorated with granular

proteins and antibacterial components, such as tryptase and LL-3768.

Figure 8: Mast cell extracellular traps. Staphylococcus aureus cells (black arrows)

ensnared by MCETs (white arrows) (modified from Jens Abel et al 2011)69. [Re-printed with

permission from Journal of Innate Immunity, Licence 3693791275161]

MCs mediated responses to viruses are poorly investigated, MCs recognize

viruses or viral double-stranded RNA via TLR3 which mediates the secretion

of antiviral type I interferons (IFNs) 70. Interestingly, in vitro and in vivo

MCs are reported to trigger antiviral properties of CD8+ T cells.

In summary, data exists which highlights the importance of MCs in defence

and clearance of bacteria and viruses, whereas MC-fungus interactions

virtually remain a “clean sheet”71; 72.

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3.0 Antimycotics and their mode of action

An increase in the number of immunosuppressed patients due to cancer,

organ or bone-marrow transplantation and cystic fibrosis set the ground for

the emergence of opportunistic fungal pathogens. The lack of rapid

diagnostics and efficient antimycotics, results in emerging fungal

infections73. The development of a new class of antimycotics requires a better

understanding of Candida colonization, infectivity and virulence. Current

fungal antibiotics are few with a narrow target range31;32. The most-

frequently used classes of antifungals are polyenes, azole, allylamines and

echinonocandins (figure 9).

Figure 9: Fungal cell anatomy and antifungal targets74; 75. Polyenes and azoles affect

directly or indirectly the ergosterol integrity as well as the β-glucan synthesis of the cell wall.

Other drugs target DNA and protein synthesis.[Adapted from Shankar et al 2013]

3.1 Polyenes

Polyenes aim for ergosterol, the functional analog of cholesterol in

mammalian cell membranes and biosynthesis of this molecule is essential for

fungal growth. Therefore, it is an ideal target for antifungal agents 74. The

mode of action is characterized by channel formation in the plasma

16

membrane upon binding of polyene molecules to ergosterol leading to ion

leakage76. Amphotericin B (AmpB) and nystatin are the most common

examples from this class of antifungals (figure 10).

A B

Figure 10: Nystatin (A) and AmpB (B) chemical structures.

AmpB is predominantly used to treat systemic mycoses, whereas nystatin is

applied for the therapy of oral thrush 75. Albeit its antifungal potency, AmpB

causes notable toxic side effects. As AmpB is able to interact with sterols, it

not only interacts with ergosterol, but also with cholesterol, although with

lower affinity. As a result, AmpB can be cytotoxic and consequently cause

renal and hepatic insufficiency2; 77.

3.2 Azoles

Azoles are synthetic compounds with a broad specificity against mycoses and

thus frequently used. Fluconazole is the most common member of the azole

family (figure 11)2. Azoles act by disturbing fungal cell membrane elasticity.

The membrane fluidity is balanced due to the presence of saturated fatty

acids and ergosterol. Fluconazole interacts with 14α-demethylase which

prevents the transition of lanosterol to ergosterol during biosynthesis of the

molecule. This causes loss of ergosterol and consequently loss of integrity of

the fungal cell membrane. Although fluconazole can inhibit 14α-demethylase

in the human cytochrome P450, it can not cause severe adverse effects.

17

Figure 11: Fluconazole chemical structure.

Fluconazole is very potent against C. albicans. However, there is a serious

concern regarding the emergence of resistant strains78. Morover, fluconazole

is inefficient against some fungal species.79; 80. C. krusei is naturally-resistant

to fluconazole, C. glabrata has a reduced sensitivity to fluconazole and

fluconazole-resistant Aspergillus strains are emerging. Nonetheless, new

fluconazole and itraconazole derivatives, such as voriconazole and

posaconazole, have proven to have high efficacy against different forms of

candidiasis and aspergillosis81; 82.

3.3 Allylamines

Allylamines target fungal squalene epoxidation of the ergosterol biosynthetic

pathway. Allylamines, such as terbninafine, cause non-competitive

disturbance of squalene epoxidase enzymatic activity83. Therefore,

allylamines have no function as enzyme-competitive-inhibitors towards

squalene epoxidase, but rather as squalene accumulators leading to

ergosterol deficiency thereby to cell membrane impairement83. Allylamines,

like terbinafine (figure 12) are effective against onychomycosis due to

dermatophytes or Candida spp84. It is mostly administered locally and

occasionally orally. Adverse effects ( acute uritaria, anorexia and epigastric

pain) caused by this drug have been mainly reported from long-term usage

of oral administration85.

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Figure 12: Terbinafine chemical structure

3.4 Echinocandins

Echonochadins comprise the newest family of antimycotics and are

compounds partially chemically synthesized and partially derived from

natural products. Echinochadins are cyclic hexapeptide compounds

connected to acyl fatty acid chains86. These side chains function as linkers to

the fungal cell wall. Echinocandins such as caspofungin (figure 13), block the

enzyme complex β-1.3-D-glucan synthase and thereby β-glucan synthesis,

which leads to cells lysis due to exposure to osmotic pressure87; 88.

Figure 13: Caspofungin chemical structure.

Caspofungin has a wide antifungal sprectrum, particularly towards Candida

spp. On the other hand, it has a limited to negligible effect against other

fungi, such as the basidiomycete Cryptococcus neoformans. Generally, it is

the drug of choice for incidences of life-threatening mycoses in case of azole

inefficacy or as part of combinatorial therapies. Notably, there are low side

19

effects due to a lacking target homologue in human cells, but the increasing

numbers of echinocandin-resistant fungal strains are considerably reducing

the possibilities of usage 89.

3.5 Resistance to antimycotics

The increasing cases of resistance to antimycotics have been highlighted and

gave rise to the investigation and understanding of resistance mechanisms.

Fungal pathogens either evolve mechanisms to resist antifungal exposure or

are selected due to natural resistance through prophylactic drug

administration. Examples of resistant fungal isolates are identified in all

commonly-used antifungal classes. Some clinical isolates from C. albicans

were reported to carry mutations in Δ5,6 sterol desaturase (ERG3) resulting

in decreased susceptibility to AmpB and azoles due to the replacement of

ergosterol by other sterols, such as 3β-ergosta-7, 22-dienol and 3β-ergosta-

8-enol90.

In contrast, C. glabrata elevates the concentration of ergosterol resulting in

descreased susceptibility to azoles and AmpB. Elevation of ergosterol levels

was achieved by increasing expression of ERG11 the gene coding for

microsomal cytochrome P-450 14α-demethylase90. Interestingly, the natural

resistance of C. krusei to azoles originates from expression of membrane

transporters- multidrug resistance (MDRs) efflux pumps - ABC1 and ABC2 -

which use azoles and other toxic compounds as substrates91; 92. MDR efflux

pumps are carried by all fungi, whereas different fungi express specific MDR

mechanisms93;94. Upon drug exposure, MDRs efflux the drug to decrease

intracellular drug concentrations and to consequently eliminate the

antifungal activity.

3.6 Alternative strategies for novel antimycotics

Due to the numerous drawbacks of the current antifungal agents the

identification of novel compounds is urgently needed. Since, the fungicidal

and fungistatic approach of the current drugs can promote the development

20

of resistant strains; the importance of new antifungal approaches is evident.

The reversal Y-H transition of C. albicans and other species of the Candida

clade is essential for virulence in order to establish successful colonization,

biofilm formation or immune evasion14;16;23. Thus, interference of this

transition constitutes an optimal target for new therapies. A chemical

compound that can block the yeast to hyphal switch will drive C. albicans to

remain in a commensal morphotype, the yeast form95. Examples of such

antidimorphic compounds are not numerous and to date none of the

identified hits has reached the clinical level. Indeed, it is a very challenging

strategy since an efficient Y-H transition blocker should not be fungicidal or

fungistatic therefore leaving the cellular viability undisturbed.

A successful detection of new antifungal agents is mainly based on the

quality of high-throughput screening assay (HTS). An ideal HTS has to be

rapid, reproducible and reliable. HTS methods targeting the identification of

antidimorphic compounds have been previously presented96-98. The basis of

these studies was the construction of hyphal specific reporter strains. Green

fluorescence protein (GFP), for instance, was placed downstream of the

hyphal specific protein (HWP1) promoter and upon promoter activation, due

to hyphal switching, GFP is expressed97. Subsequently, antidimorphic

compound cause decrease of fluorescent levels.

Approaches related to reporter strains can lead to false-negative or false-

positive hits. For instance, there is a high possibility that the identified

compound has blocked the fluorescence by affecting GFP, rather than the

morphological transition. Alternatively, a chemical molecule disturbs the

HWP1 promoter activation, but not the Y-H transition, given that HWP1 is

not an essential Y-H transition gene. More, assays have been described,

using for instance beta-galactosidase as reporter, with similar uncertainties

as mention above96.

21

In summary, the identification of antidimorphic compounds that disturb the

Y-H transition, but not the fungal cellular viability per se can form the basis

of a new class of antifungals. The identification of antidimorphic compounds

is dependent on the quality of the HTS, a major goal of this thesis. However,

until an applicable medication is developed, a long and expensive road filled

with challenges lies ahead.

22

Materials and Methods

Fungal strains

The fungal strains used for the thesis are all from the Candida clade.

Candida albicans type strain SC5314, Candida albicans clinical strain

UCB3-7922, Candida glabrata type strain ATCC 90030, Candida glabrata

clinical strain UCB3-7268, Candida dubliniensis type strain CD36/CBS7987

and Candida dubliniensis clinical strain UCB-3892. The clinical isolates

were from the strain collection of the Norrland University Hospital Umeå.

Fungal strains were cultured (o/n) in synthetic complete medium + 2%

glucose (SC medium).

Candida albicans GFP construct

For the needs of paper I C. albicans GFP constitutively expressing strain

(CAI4 pENO1-GFP-CyC1t) was generated. The sequence was ordered from

Genscript and integrated to pCaEXP by PstI and XbaI restriction enzymes.

pCaEXP was linearized by Stu1 restriction enzyme and introduced to C.

albicans CAI4 strain by homologous recombination to the RP10 locus99-102.

The transformation to the CAI4 genome was confirmed by sequencing.

Human mast cells

Human MC line (HMC-1) and cord blood-derived MCs (CBMCs) have been

used for paper I. HMC-1 was cultured at 37°C with 5% CO2 with the Roswell

Park Memorial Institute medium RPMI (RPMI, Life technologies) enriched

with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin

(Lonza)103. CBMCs are differentiated from CD34+ cells which were isolated

by positive selection according to the manufacturer’s protocol (Miltenyi

Biotec). CD34+ cells cultured for 4 weeks in StemPro-34 SFM medium

(Invitrogen) enriched with 10 ng/ml of IL-6 and IL-3 at 37°C with 5%

CO2104.

23

Isolation of human neutrophils

Neutrophils were isolated from venous blood of healthy donors with two

gradient steps for experiments presented in paper I. The first step uses

Histopaque 1119 (Sigma-Aldrich) to differentiate white blood cells from

erythrocytes and plasma. The second one uses different Percoll (Amersham)

concentrations that separates neutrophils from remaining cell types105.

Neutrophil and monocyte migration assay

Primary neutrophils/monocytes cell line (U937) were stained with

fluorescent BCECF-AM (3.3 μM Sigma-Aldrich) cytoplasmic dye.

Neutrophils/monocytes migration was tested in a transwell system and

presented in paper I. The neutrophils/monocytes were seeded in the upper

chamber of transwell inlets attached to 24-well/plate (BD Falcon, HTS

FluoroBlok Insert, 3.0 μM pore size)106. Collected supernatants from mast

cells infected with different C. albicans concentrations were tested for

chemotactic activity towards PMNs/monocytes. The supernatants were

placed into the bottom wells and migration from the transwell inlet into the

bottom well was monitored every minute for 3o min or 90 min, respectively,

using fluorescent signals from BCECF-AM dyed cells at 37°C, 5% CO2.

Migration was calculated as percentage of 100 % control for which the total

amount of fluorescent neutrophils/monocytes was place into the bottom

well.

Sytox green-based cell death assay

For paper I mast cell death was monitored using the cell-impermeable DNA

dye sytox green. MCs (5 × 104 cells/well) were infected with C. albicans or

left uninfected in a black 96-well plate106. Sytox green was added to all wells

and cell death was quantified according the intensity of fluorescent signal. As

a 100% lysis control MCs were lysed with triton X-100. As a negative control

the cells were left untreated. The assay was monitored for 16 hours at 37°C,

24

5% CO2 and the MC cell death was calculated as percentage of the fluorescent

signals from the 100% lysis control.

Mast cell degranulation assay

MC degranulation is part of paper I and it is based on β–hexosaminidase

secretion107. MCs (1 × 105 cells/well) were infected with different C. albicans

concentrations and the collected supernatants from these infections were

assayed for degranulation. For this purpose, the supernatants were

incubated for 2 hours with 2.738 mg/ml 4-P-nitrophenyl-N-acetyl-β-D-

glucosaminide. Subsequently, the β–hexosaminidase release from infected

MCs in comparison to the UC was measured by means of absorbance (A405).

Cytokine quantification assay

In paper I, the release of cytokines was tested in 24-well plates. Supernatants

were collected from C. albicans-infected mast cells (1 × 106 cells/well) and

from uninfected controls. The supernatants (50 μl) were screened for 27-plex

and 21-plex panels (Bio-Rad Inc., USA) from a microplate reader Bio-Plex

200 (Bio-Rad Laboratories). The quantification was based on respective

standard curves according to manufacturer’s recommendations.

Cellular viability

Cell viability was measured by means of ATP levels. This assay is part of

paper I-III and was used to characterize the cellular viability of fungal cells

in response to either mast cells or chemical compounds. The assay is based

on the lumiscent CellTiter-Glo Promega kit. C. albicans (5 × 104 cells/well)

were challenged with MCs (5 × 104 cells/well), at a multiplicity of infection of

1 (MOI1) or with different concentrati0ns of chemical compounds.

Subsequent to infection, MCs were lysed with Triton X-100. After the

mammalian or chemical challenge equal volumes of fungal cell suspensions

were added and incubated for 15 min. The luminescent signals were recorded

using a Tecan Infinite F200 microplate reader.

25

High content analysis of yeast and hyphal morphotypes

The development of a high-throughput screening assay discriminating yeast

to hyphal C. albicans cells is presented in paper II. C. albicans cells (SC5314)

and C. albicans KO strains Δefg1 and Δedt1 were grown overnight at 30oC in

SC medium. The next day a subculture (1 × 107 cells/ml) from each strain

was inoculated at 30oC for 3h. Cells were washed and resuspended

(2 × 105 cells/ml) in 1X PBS. Cells (50 μl) were seeded in 96 well plate (black

with transparent bottoms) and RPMI 1640 was used to make-up the volume

(200μl). The cells were incubated for 3, 6 and 24 h and fixed with 2%

paraformaldehyde (PFA).

Subsequently, the cells were stained with 0.1% calcofluor white

(CFW/Sigma-Aldrich), chitin-specific, and the plate was scanned with a

fluomicroplate microscope (HCA-Cellomics ArrayScan VTI, Thermo

Scientific). The automatically captured images from the microscope were

analysed with the high content analysis software. According to the individual

cells the cellular size and shape were determined. The sizes and shapes were

defined as length to width ratio (LWR) and mean object shape (MOS=

c2/4π)*area). The method validity as a HTS was also defined by the Ζ′ factor

calculation for LWR and MOS 108.

26

Aims

A professional phagocyte from humans, the PMN, is able to block

morphological transition of C. albicans from yeast to hyphal growth.

Neutrophils achieve this either by internalization of C. albicans yeast-form

cells or by the release of NETs. Inspired by this notion, we raised two

questions: First, do other immune cells contribute to anti-candida activity in

a similar fashion? MCs are tissue-dwelling immune cells which have PRRs

for microbial PAMPs, release ROS and proteases for destruction of microbes.

Although an emerging body of literature describes antibacterial responses of

MCs, their interaction with and response to fungal pathogens is virtually

unexplored. We therefore investigated how MCs recognize and respond to C.

albicans. Secondly, is it possible to identify chemical molecules which

interfere with Y-H transition and thus mimic the antifungal activity of

Neutrophils? We addressed this question by i) developing a new, reliable

HTS screening assay for antidimoprhic compounds and by ii) screening

libraries of off-patent and off-target FDA-approved drugs for antidimorphic

as well as fungistatic activity. Repurposing screens, such as ours, are

promising, since it is time-consuming and excessively expensive to develop a

drug from scratch.

27

Paper I

Human MCs recognize C. albicans and release neutrophil-recruiting, pro-

inflammatory and anti-inflammatory cytokines in a timely orchestrated

manner. MCs transiently kill C. albicans and form MCETs to trap the fungal

pathogen. In turn, C. albicans is able to escape from MCs at later time

points.

Paper II

A new HTS assay is presented to identify antidimorphic compounds, from

large chemical compound libraries, that are not fungistatic.

Paper III

Repurposing screen: Identification of antifungal activity in off-patent drugs

licensed to serve other purposes.

28

Results and Discussion

Paper I

Opportunistic pathogen Candida albicans elicits a temporal

response in primary human mast cells

This study investigated how human MCs (hMCs) respond towards C.

albicans. The repsonse could be divided into three time phases; the

immediate (0-3 hours), intermediate (3-12 hours) and late response (>12

hours) (figure 14). hMC degranulation was detected as early as 1 hour post

infection (immediate phase) reaching 80 % above uninfected control (UC).

In addition, within 6 hours hMCs secreted considerable amounts of IL-8,

significantly above UC. IL-8 is a chemokine, strongly associated to

neutrophil migration. Importantly, infected hMCs released macrophage

migration inhibitory factor (MIF) but not monocyte chemoattractant protein

(MCP-1). Those two cytokines have opposing roles: MIF leads to inhibition

of monocyte chemotaxis, whereas MCP-1 induces monocyte

chemoattraction. MIF release was significantly above UC whereas MCP-1

release upon C. albicans stmulation was rather below UC suggesting that

hMCs do not recruit monocytes upon C. albicans infection. We functionally

analysed recruitment of neutrophils and monocytes using a migration assay

in which the respective infection supernatants served as a chemotractant for

PMNs or monocytes. Interestingly, hMCs possess antifungal activity given

that 6 hours post infection C. albicans cell viability has been reduced

approximately to 50 % compare it to the fungal growth control(figure 15 B).

29

Figure 14: Orchestrated responses of human mast cell upon encounter with

Candida albicans. [Re-produced with permission from Nature publishing group109]

During the intermediate phase MIF concentration increased, whereas IL-8,

though decreased, remained significantly above UC. Importantly, hMCs

release MCETs which begins from the intermediate phase and lasts until the

late phase. Even though hMCs could not terminate fungal growth, the fungal

cells were entrapped within the MCETs according to microscopic

investigation. Finally, the late phase demonstrates the potency of hyphal

forms to cause MC lysis originating either from extracellular and

intracellular space. The cell death in hMCs is C. albicans-mediated either

due to hyphal penetration or due to induction of MCETs (figure 15 A + C). In

addition to MIF, MCP-1 and IL-8 secretion, at late phase, the cytokines IL-16

(adaptive immunity-related) and IL-1rα (anti-inflammatory protein) were

released.

30

Figure 15: Mast cell anticandida and C. albicans antimast cell activity. MCET

secretion upon C. albicans stimulation (A). Transient antifungal activity of MCs which is not

dependent on MCETs as determined with DNase digest of DNA traps (B). . MC cell death

induced by C. albicans is time and concentration dependent (C).[Re-produced with permission

from Nature publishing group109]

This study illustrates the responses of hMCs upon interaction with C.

albicans. MCs secrete cytokines related to innate and adaptive immune cells.

In addition, hMCs show a transient antifungal activity which is independent

from MCET production. From the pathogen point of view, C. albicans

displays antimast cell activity leading to mast cell lysis by hyphal invasion

either from outside-to-inside or from inside-to-ouside. Conclusively, hMCs

indeed serve as tissue-sentinels for commensal fungal pathogen. It will be

beneficial for the development of new antifungal agents to gain more insight

into MC responses to other fungal pathogens.

31

Paper II

Novel high-throughput screening method for identification of

fungal dimorphism blockers

Inspired by the weakness of non-filamentous C. albicans to cause disease, we

aimed to develop a high-throughput screening (HTS) method to identify

non-fungicidal molecules that break C. albicans Y-H transition. The high

content screening (HCS) is a cell-based assay using image analysis via an

automated fluorescent microscope in order to be able to visually

discriminate yeast from hyphal growth. The analysis was subsequently

incorporated into an algorithm to allow automated analysis of acquired

images.

Mutant C. albicans strains that are unable to generate hyphae served as key

references, namely Δefg1 and Δedt1. These mutants continue growing as

yeast-form cells under otherwise hypha-inducing conditions and thus should

be distinguishable from growing wild-type hyphae applying the image

analysis and undelying algorithms. We used the preservative thimerosal to

kill C. albicans as additional control. Farnesol is a quorum sensing molecule

released by C. albicans to inactivate hyphal induction and to promote

apoptosis110. Farnesol can therefore contribute to regulation of colony

density. The molecule is additionally immune-modulatory activating the

innate, however attenuating the adaptive immune response to C. albicans,

respectιvely111. In our setup, farnesol blocked the hyphal transition and did

not affect yeast growth at 3 and 6 h. Therefore, we included treatment of C.

albicans with farnesol as a positive test compound in our assay112.

Amongst several different parameters from the HCS we chose length width

ratio (LWR) and mean object shape (MOS) which sufficiently described

yeast-shaped and hyphal cells for proper discrimination. LWR is the quotient

of length and width of an object. Circular objects thus have an LWR of 1,

whereas rod-shaped objects have values above 1 depending on the maximum

length. MOS is defined as the ratio of circumference squared to 4π*area

32

(MOS = [(c2/4π)*area]). Circular objects therefore have a MOS of 1 and

filamentous of >1.5. Wild-type C. albicans grown under hypha-inducing

conditions at 3 hours and 6 hours resulted in LWR and MOS values above

1,5, while grown under yeast-inducing conditions resulted in LWR and MOS

values below 1.5 (figure 16). The control conditions including dead, mutant

and farnesol-treated C. albicans additionally showed LWR and MOS values

below 1.5. Hence, we used a threshold of LWR and MOS of 1.5 to clearly

define yeast or hyphal cells, respectively.

Figure 16: Distinction of yeast and hyphal cells. Different LWR and MOS values for the

indicated strains and conditions are shown. LWR after 3 hours (A) and 6 hours (B) as well as

MOS after 3 hours (C) and 6 hours (D). [Re-produced with permission from Sage publisher113

To distinguish fungistatic or fungicidal compounds from agents that purely

inhibited morphotype transition without affecting yeast growth we added an

additional layer to the assay. We tested for C. albicans viability using ATP

quantification. Only metabolically active and thus living cells produce and

33

retain measurable ATP levels (figure 17). This assay is reliable, rapid and is

not affected by the hyphal morphotype. Serial dilution and plating for colony

counting cannot be applied for quantification of hyphae, since growing cells

do not separate and hyphal filaments additionally tend to clump. Both

features lead to false results in quantification methods based on colony

counting. The ∆edt1 and ∆efg1 strains as well as farnesol-treated C. albicans

resulted in ATP levels close to untreated control. Dead C. albicans and

farnesol-treated overnight resulted in negligible ATP levels (figure 17).

Until today, other assays have been suggested for detection of antidimorphic

molecules96-98. However, the described assays are dependent on reporter

strains which are based on the promoter of hyphal wall protein 1 (HWP1).

HWP1 is expressed under hypha-inducing conditions and repressed during

yeast growth. As reporters placed downstream of the promoter the open

reading frames (ORFs) for either green fluorescent protein (GFP)97; 98 or

beta-galactosidase (lacZ)96 were used.

Figure 17: The cellular viability of Candida albicans when challenged with

farnesol and thimerosal and the cellular viability of Candida albicans mutant

yeast-locked strains. Candida albicans cellular viability was recorded after 3 (A), 6 (B) and

24 (C) hours. Farnesol blocks the Y-H switching, but retains the cellular viability: in contrast

thimerosal kills the cells. For screening purposes farnesol represents an ideal antidimorphic

compound, thimerosal mimics a fungistatic / fungicidal agent. [Re-produced with permission

from Sage publisher 113]

An advantage of our method is the fact that it is applicable for type fungal

strains and not dependent on genetically-modified strains, such as GFP and

lacZ reporter strains. During development we accounted for the antifungal

34

susceptibility testing (AFST) guidelines of the European committee on

antimicrobial susceptibility testing (EUCAST) which is considered standard

for this type of analyses. In the two step screening approach the positive hits

are detected by means of C. albicans LWR and MOS values for cells derived

from image analysis. Subsequently, the positive hits were tested for

fungistatic / fungicidal activity by ATP quantification. Thus, this method can

reproducibly distinguish fungistatic / fungicidal agents from solely

antidimorphic compounds. Importantly, our method was reported as

suitable and valid for HTS given that Ζ′ factor calculation of LWR and MOS

for 3 hours was scored between 0.5 – 1 describing the method as an excellent

assay108.

Conclusively, the long-term goal of this screening is to identify a new

generation of virulence-blocking agents with the potential to be developed

into novel, efficient antifungal drugs. Remarkably, the method is applicable

for compound screenings against any pathogenic fungus that changes

morphology from roundish to filamentous forms or vice versa, such as

certain non-albicans species, Aspergillus spp.or Histoplasma spp..

35

Paper III

Antifungal application of nonantifungal drugs

To date, the standard antifungal drugs (SAD) are only few in numbers with

partially severe side effects. In this study, C. albicans was challenged with

844 patented drugs and the positive hits further tested on C. dubliniensis

and C. glabrata on both type and clinical strains at 6 and 24 hours

incubation time (table 2).

Despite, C. dubliniensis phenotypical and genetical similarities to C. albicans

it is less pathogenic due to the lack of important virulence factors, such as

Agglutinin-Like Sequence (ALS)114. Though lacking the Y-H transition C.

glabrata though it lacks the yeast to hyphal transition it remains an

important pathogen mainly due to the high resistance against the most

frequent antifungal, fluconazole115. Seven non-antifungal drugs from four

different categories were shown to have potent antifungal activity against the

tested Candida spp. using three different, independent assays.

Drug serial dilutions, cell concentrations, incubation times and growth

susceptibilities were performed according to EUCAST guidelines with minor

modifications116. The minimum inhibitory concentration (MIC) was

determined according to absorbance (A450) and cellular viability by means of

ATP levels117. However, since both readout assays regarding C. albicans

growth showed comparable results with small variations, the susceptibility

testings of the clinical strains were carried out with ATP measurements

exclusively. Furthermore, five non-antifungal drugs with a proved antifungal

activity served as controls.

After challenge with the novel candida off-patent drugs, C. albicans type and

clinical strain UCB3-7922 showed up to 50% susceptibility whereas C.

dubliniensis growth inhibition mostly ranged 30%. C. glabrata growth was

affected in three of the drugs up to 30% but it is resistant to Auranofin

control which showed to have up to 100% anticandida activity towards C.

albicans and C. dubliniensis. The novel antifungal compounds activity was

36

also tested in comparison to different SADs (table 3). All the drugs were

tested in 1µM, below the maximal reachable concentration in humans,

incubated at equal time and which led to comparable growth inhibition.

The de novo designing of drugs presupposes very long time and high

budgets. Here, we report a drug re-purposing study which provides new

antimycotic options to protect immunocompromised patients from

opportunistic fungal pathogens. So far only a small proportion of the entity

of FDA-approved drugs has been tested. There is a high likelihood that more

off-target antifungal drugs can be identified with our methods.

Table 2 : Candida spp. susceptibility to seven novel antifungal off-patent drugs

[Re-produced with permission from ASM publisher117]

MIC MIC0.3 MIC MIC0.3 MIC MIC0.3 MIC MIC0.3 MIC MIC0.3 MIC MIC0.3

Haloperidol HCl 6.4 × 10−3 to 3.76 3.76 0.46 3.76 0.38 3.76 0.38 > 3.76 0.38 >3.76 3.76 >3.76 >3.76

Trifluperidol 2HCl 7 × 10−3 to 4.00 4.00 0.40 >4.00 0.40 >4.00 >4.00 > 4.00 0.40 >4.00 >4.00 >4.00 >4.00

Stanozolol 3.3 × 10−3 to 3.29 >3.29 0.33 >3.29 0.33 >3.29 3.29 >3.29 3.29 >3.29 >3.29 >3.29 3.29

Melengestrol acetate 6.8 × 10−3 to 3.97 3.97 0.37 3.97 0.40 >3.97 3.97 >3.97 1.80 >3.97 3.97 >3.97 3.97

Megestrol acetate 6 × 10−3 to 3.85 3.85 0.39 3.85 0.39 >3.85 3.85 >3.85 3.85 >3.85 3.85 >3.85 3.85

Tosedostat 4 × 10−3 to 4.00 >4.00 4.00 >4.00 4.00 >4.00 4.00 >4.00 4.00 >4.00 4.00 >4.00 2.00

Amonafide 2.8 × 10−3 to 2.83 >2.83 1.40 >2.83 2.83 >2.83 2.83 >2.83 1.40 >2.83 >2.83 >2.83 >2.83

Methiothepin maleate 7 × 10−3 to 3.57 3.30 0.31 3.30 0.36 >3.57 0.36 >3.57 0.36 3.57 0.36 3.57 0.36

Auranofin 4 × 10−3 to 6.78 0.68 0.08 0.61 0.07 0.68 0.04 0.62 0.04 1.10 0.62 >3.73 3.73

Rapamycin 9 × 10−3 to 9.14 0.002 <9 × 10−3 0.002 <9 × 10−3 0.009 <9 × 10−3 0.01 <9 × 10−3 0.50 0.04 0.09 0.009

UBC3-7268(clinical strain)

Antifungal agent Concn range(μg/ml)

C. albicans C. dubliniensis C. glabrata

SC5314(type strain)

UBC3-7922(clinical strain)

CD36/CBS7987(type strain)

UBC3-3892(clinical strain)

ATCC 90030(type strain)

37

38

Table 3: Standard antifungal drugs versus off-patent drugs. [Re-produced with

permission from ASM publisher117]

Drugs MIC MIC0.3

Standard antifungal

Tioconazole 0.39 μg/ml

Oxiconazole nitrate 0.40 μg/ml

Ketoconazole 0.50 μg/ml

Climbazole 0.29 μg/ml

Miconazole 0.40 μg/ml

Fluconazole 0.30 μg/ml

Amorolfine 0.32 μg/ml

Myclobutanil 0.29 μg/ml

Bifonazole 0.30 μg/ml

Sertaconazole 0.40 μg/ml

Itraconazole 0.70 μg/ml

Terbinafine HCl >1 μM

Nystatin >1 μM

Off-target antifungal

Haloperidol HCl 0.38 μg/ml

Methiothepin maleate 0.36 μg/ml

Auranofin 0.68 μg/ml

Trifluperidol 2HCl 0.40 μg/ml

Stanozolol 0.30 μg/ml

Melengestrol acetate 0.40 μg/ml

Megestrol acetate 0.39 μg/ml

Tosedostat >1 μM

Amonafide >1 μM

39

Concluding Remarks

• In response to C. albicans hMCs display a temporal antifungal

activity andsecrete chemokines to attract neutrophils but not

monocytes. C. albicans dimorphism causes mast cell lysis via hyphal

formation.

• A novel highthroughput screening method rapidly, reproducibly and

reliably dinsctints yeasts from hyphae and therefore can serve as the

basis for the development of a new class of antifungals.

• The seven novel off-patent antifungals with potent antifungal

activity provide new options for clinicians to treat patients with a

primary, immunosuppressive disease (cancer) and a potential,

secondary opportunistic mycose.

40

Acknowledgements

A successful PhD thesis, at least in natural sciences, requires tremendous

sacrifices, positive and collaborative working environment and the support

from all the beloved ones, family and bros.

Especially I would like to thank my wife. Kardoula mou without your

sacrifices and support the PhD studies would still be a dream.

I would like to thank my Parents for their countless help, I am really grateful for that.

I would like to thank my brothers for all the support.

I would like to thank my friends in Cyprus for all the support.

Koumpare (Cotsios) You are a member of my family and a real buddy!!!

Though Brothers don’t thank each other i want anyway to Thank You!!!

Koumparos (Costas) whatever I say will be just not enough so I keep it

laconic and say a big Thank You!!!!

File Stephane Pavlide, you came in my life just at the right time!!! Thanks for

the help, support and beautiful discussions!!! Thanks bro!!!

Daskale (Apostolos Giorgakis) I feel blessed for you being in my life!!! Thank You!!!

Pedro really thanks for always being there for me. I really appreciate it!!!

Thanks bro!!!

Kristina thanks for all the provided help to me and my wife!!! Thank you!!!!

Per thanks for being in my life and helping with everything. Thanks bro!!!

41

Madhu though I know you for only half a year, for the first minute the

chemistry worked. Thanks for the nice discussions and support. Thanks

bro!!!

Sujan though I met you just a month ago already the friendship started from time 0´.Thanks bro!!!

Melis / Venki and Joseph we first met almost 10 years ago and still we

support each other like family members do. Thanks buddies!!!

I would like to thank the Urban group members (current and former) for

constructive and productive meetings and discussions. Ava thanks for the

very nice collaboration, Hanna thanks for the very nice discussions, Anna

thanks for the very funny time we had, Marc thanks for all the advices,

Sandra, Emily, Cecilia and Emil thanks for the nice time we had.

I would like to thank the groups of Mikael Elofsson, Krister Wennerberg and Johan Bylund for the very good collaboration.

I would like to thank the Journal Club members. The groups of AS, SB,

UvPR, AJ and ÅG for the constructive discussion time.

I would like to thank my studies examiner Anders Sjöstedt for all the help!!!! A big Thank You!!!

Last but of course not least I would like to thank my BOSS, Constantin

Urban. Thank you for being the Father of my PhD studies. A huge Thank

You!!!

42

References

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2. Pfaller MA, Diekema DJ: Epidemiology of invasive candidiasis: a persistent public health problem. Clinical microbiology reviews 2007; 20:133-163.

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