1. Department of Clinical Microbiology
Molecular Infection Medicine Sweden (MIMS)
Umeå University
Umeå 2015
Pharmaceutical And Immunological
Challenge Of Fungal Pathogens
Marios Stylianou
2. Pharmaceutical And Immunological
Challenge Of Fungal Pathogens
Marios Stylianou
Doctoral thesis
Department of Clinical Microbiology
Molecular Infection Medicine Sweden (MIMS)
Umeå University
Umeå 2015
3. 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.
4. As for me , all I know is that I know nothing
Socrates
To my Wife and my Parents, the heroes of my life.
5.
6. 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
7. ii
Paper I 28
Paper II 31
Paper III 35
Concluding Remarks 39
Acknowledgements 40
References 42
8. 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.
9. 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
10. 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
11. 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.
12. 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
13. viii
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
14. 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
15. 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]
16. 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.
17. 4
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
18. 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.
19. 6
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 O2
38. To date, Sod1p (cytoplasmic),
Sod2p (mitochondrial), Sod3p (cytoplasmic), Sod4p, Sod5p and Sod6p (cell
surface) have shown to prevent host’s hazardous oxygen radicals37-39.
20. 7
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.
21. 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.
22. 9
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.
23. 10
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
25. 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 ++ ++
26. 13
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
27. 14
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.
28. 15
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
29. 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.
30. 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.
31. 18
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
32. 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
33. 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.
34. 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.
35. 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%
CO2
104.
36. 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,
37. 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.
38. 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.
39. 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.
40. 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.
41. 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).
42. 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.
43. 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.
44. 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
45. 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
46. 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
47. 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..
48. 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
49. 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.
52. 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.
53. 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!!!
54. 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!!!
55. 42
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66. 1Scientific Reports | 5:12287 | DOI: 10.1038/srep12287
www.nature.com/scientificreports
Opportunistic pathogen Candida
albicans elicits a temporal
response in primary human mast
cells
José Pedro Lopes1,2,3,*
, Marios Stylianou1,2,3,*
, Gunnar Nilsson4
& Constantin F. Urban1,2,3
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 expectedly among the first immune cells to get into contact
with C. albicans. However, mast cell-fungus interaction remains a neglected area of study. Here
we show that human mast cells mounted specific responses towards C. albicans. Collectively, mast
cell responses included the launch of initial, intermediate and late phase components determined
by the secretion of granular proteins and cytokines. Initially mast cells reduced fungal viability and
occasionally internalized yeasts. C. albicans could evade ingestion by intracellular growth leading
to cellular death. Furthermore, secreted factors in the supernatants of infected cells recruited
neutrophils, but not monocytes. Late stages were marked by the release of cytokines that are known
to be anti-inflammatory suggesting a modulation of initial responses. C. albicans-infected mast cells
formed extracellular DNA traps, which ensnared but did not kill the fungus. Our results suggest
that mast cells serve as tissue sentinels modulating antifungal immune responses during C. albicans
infection. Consequently, these findings open new doors for understanding fungal pathogenicity.
Severe mycoses are rising in modern health care, mainly due to the use of catheters and immunosup-
pressive treatments1
. The most prevalent fungal pathogen2
, Candida albicans is also part of the human
commensal flora. C. albicans commensally 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. C.albicans-associated mycoses have an annual
prevalence of 300 000 with an associated mortality up to 55% in European intensive care units3,4
. 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 tissue-dwelling cells derived from hematopoietic progenitors. These cells migrate from
the blood to the skin, airways or the gastrointestinal tract where final differentiation is induced by sur-
rounding structural cells. Mast cells are known for triggering hypersensitivity reactions at the body inter-
faces with external environments. They respond to stimuli by rapidly degranulating their cytoplasmic
vesicles leading to selective and differential mobilization of granule contents into the milieu.
The role of mast cells in asthma and inflammatory disorders is an intensively active area of research,
however, comparably little is known about the role of these cells in host defence5
. Mast cells have
been shown to participate in the killing of bacteria6–8
, whereas their antifungal defence response is
1
Department of Clinical Microbiology, Umeå University, Umeå, Sweden. 2
Umeå Centre for Microbial Research
(UCMR). 3
The Laboratory for Molecular Infection Medicine Sweden (MIMS). 4
Department of Medicine, Karolinska
Institutet and Karolinska University Hospital, Stockholm, Sweden. *
These authors contributed equally to this work.
Correspondence and requests for materials should be addressed to C.F.U. (email: constantin.urban@umu.se)
received: 02 March 2015
accepted: 19 June 2015
Published: 20 July 2015
OPEN
67. www.nature.com/scientificreports/
2Scientific Reports | 5:12287 | DOI: 10.1038/srep12287
virtually unexplored9–12
. Interestingly, mast cells express molecules, for instance inflammatory cytokines,
myeloid-attracting chemokines, and pattern recognition receptors that were demonstrated to be involved
in antifungal responses in other cells. Toll like receptors (TLRs), such as TLR2 and TLR4 are in addi-
tion to mediate responses against bacteria13–15
well-established contributors for detection and clearance
of fungi16,17
. Activation of C-type lectin receptors, such as dectin 1, by fungal components also results
in mast cell activation18,19
. This suggests a possible involvement of mast cells in antifungal immunity.
Specific ligands for such receptors can activate mast cells causing release of reactive oxygen species20
and different vasoactive mediators, for instance histamine, prostaglandins, leukotrienes, and tryptase,
which are injurious to the microbes21
. While most of these mediators promote inflammation, they are in
addition responsible to recruit other immune cells22,23
.
We chose C. albicans to study fungal-mast cell interactions, since C. albicans is a commensal and a
frequent human pathogen. This dual role enables a more detailed understanding of fungal pathogenic-
ity, innate immune response and immune tolerance. We found that human mast cells have a versatile
and timed response upon fungal encounter. Mast cells first degranulated β -hexosaminidase and were
able to transiently reduce 30% of C. albicans viability up to 3 h post infection. In intermediate responses
mast cells released pro-inflammatory cytokines, such as interleukin-8 (IL-8) and supernatants of
C. albicans-infected mast cells were chemoattractive to neutrophils. In late responses mast cells secreted
IL-16 and anti-inflammatory IL-1ra and released mast cell extracellular traps (MCETs) that ensnared,
but probably did not kill C. albicans. In addition, the fungus could cause mast cell death by different
mechanisms.
Ultimately, our work contributes to the understanding of the role of mast cells in modulating the
innate immune response against opportunistic pathogenic fungi.
Results
C. albicans induced rapid degranulation in mast cells. Mast cells contain large amounts of
enzymes in their granules21
, particularly proteases or lysosomal enzymes like β –hexosaminidase24
. These
enzymes are involved in inflammation onset25,26
and in defence against microbes27–29
. Degranulation is
therefore a putative mechanism mast cells may employ to respond to C. albicans infection. Therefore,
we measured β -hexosaminidase, a routinely used marker for mast cell degranulation, during infection of
mast cells with C. albicans. Indeed, mast cells degranulated and released β -hexosaminidase in response
to C. albicans after 1 h of infection in a dose-dependent manner (Fig. 1A). This indicates that mast cells
recognized the fungus and mounted an early and direct response.
Mast cells mounted a unique cytokine response upon C. albicans infection. To test mast cell
immune modulatory responses we infected human mast cell line-1 (HMC-1) cells with C. albicans and
subsequently analysed culture supernatants for presence of cytokines. We found 5 cytokines that were
differentially released from mast cells in a time-dependent manner following infection with C. albicans.
An early cytokine response (6 h post infection) involved release of IL-8, a strong neutrophil chemoat-
tractant (Fig. 1B). Comparably, cord blood-derived mast cells released similar amounts of IL-8 upon C.
albicans infection (Fig. S1).
Upon C. albicans stimulation, mast cells additionally secreted macrophage migration inhibitory fac-
tor (MIF), a pro-inflammatory, stress-response cytokine crucial for sustaining an inflammatory milieu
(Fig. 1C)30
. Interestingly, secretion of monocyte chemoattractant protein 1 (MCP-1), one of the key
chemokines inducing migration and infiltration of monocytes/macrophages was not released (Fig. 1D).
Mast cells therefore are likely to contribute to neutrophil, but not to macrophage recruitment upon
C. albicans infection. At later time points (12 and 24 h), the cytokine profile revealed the release of
IL-16, a chemokine linked to chemoattraction of CD4+
T lymphocytes31
(Fig. 1E). The pro-inflammatory
cytokine response at early time points post infection seems to be counteracted by release of the
anti-inflammatory cytokine IL-1ra at 24 h (Fig. 1F). Taken together, these data suggests that secretion of
pro- and anti-inflammatory cytokines was a controlled process that was influenced by different stages
of the infection.
Human neutrophils but not monocytes were chemoattracted towards C. albicans-infected
mast cells. As some of the chemokines from our multiplex screening are relevant in host immune cell
recruitment, we next tested the chemoattractive potential of supernatants from C. albicans-infected mast
cells towards neutrophils and monocytes. Mast cells were infected for three time points and supernatants
harvested. The chemotactic potential of the supernatants was tested using fluorescently labelled neutro-
phils in a transwell system (Fig. 2A). Chemoattractant fMLF was used as positive control32
.
Notably we found, that C. albicans-infected supernatants induced migration of neutrophils similar to
fMLF, whereas C. albicans alone or uninfected mast cells induced significantly lower neutrophil migra-
tion. Chemotaxis was significantly above controls with supernatants collected after 12 h or longer. The
migration assay shows slightly delayed neutrophil chemotaxis compared to the cytokine release assay
revealing increased IL-8 already after 6 h of infection (Fig. 1B). However, neutrophil migration might
be influenced by other chemokines that were not analysed with the multiplex assay used. On the other
hand analysis of monocyte chemotaxis corroborated the cytokine multiplex results. Monocyte-attractant
chemokine MCP-1 was secreted by uninfected mast cells, however not induced upon C. albicans infection
68. www.nature.com/scientificreports/
3Scientific Reports | 5:12287 | DOI: 10.1038/srep12287
Figure 1. C. albicans induced mast cell degranulation and cytokine release in a MOI-dependent manner.
(A) HMC-1 cells were infected with opsonized C. albicans yeasts (MOI 0.1, 1 and 10) for 1 hour, after
which ß-hexosaminidase release was measured from supernatants of infection. (B–F) Shown are 5 cytokines
at 6, 12 and 24 h post infection that were released differentially from different supernatants of mast cells
infected with C. albicans (MOI 0.1 and 1) or of mast cells left uninfected. ß–hexosaminidase percentage
release was defined by the amount of ß-hexosaminidase release from infected cell divided by spontaneous
ß-hexosaminidase release from uninfected cells (% of ß –hexosaminidase release/% of ß –hexosaminidase
control). Significance for (A–F) was analysed by Tukey one-way ANOVA *P ≤ 0.05. Data are presented as
means of n = 4 (4) ± SD (ß-hexosaminidase release assay) and n = 3 (3) ± SD (Cytokine Multiplex).
69. www.nature.com/scientificreports/
4Scientific Reports | 5:12287 | DOI: 10.1038/srep12287
of mast cells (Fig. 1D). In contrast, monocyte-inhibitory MIF was increasingly released by infected mast
cells over time (Fig. 1C).
Taken together, we confirmed our findings regarding mast cells cytokine-release following fungal
infection by a functional migration assay revealing that mast cells secrete neutrophil chemoattractants.
Despite releasing extracellular traps mast cells only transiently control C. albicans via-
bility. The release of extracellular DNA traps is part of the innate immune response to infection.
Extracellular traps have been observed in mast cells in response to auto-inflammatory skin diseases
and upon bacterial infection6,14,33
. Here, we investigated the potential release of MCETs in response to
fungi. After 6 h, C. albicans-infected mast cells released MCETs composed of DNA and granular proteins
ensnaring the fungus (Fig. 3A arrow, Movie S1), whereas these structures were absent in uninfected con-
trol (Fig. 3B). Primary mast cells similarly released extracellular traps upon C. albicans stimulation (Fig.
S2A, arrow). Quantification of MCETs in a blinded fashion revealed that extracellular trap formation
increased over time being significantly different from uninfected controls, but rarely exceeded 5% of the
total amount of cells (Fig. 4A). Infection with higher MOIs led to MCET formation (Fig. S2B, arrow)
without further increase in number (data not shown).
To account for mast cell antifungal activity, we quantified ATP levels correlating with the presence
of metabolically active fungal cells. Within 3 h of incubation C. albicans viability was reduced by 30%.
However, this antifungal effect was transient and declined after 6 h, possibly due to fungal overgrowth.
In a similar assay, we tested the antifungal activity of MCETs by degrading their DNA backbone with
DNase. The nuclease was added before infection of mast cells with C. albicans and not removed during
the whole assay to ensure degradation of any emerging MCET. We did not observe a significant differ-
ence in fungal viability in the presence of DNase as compared to samples without DNase (Fig. 4B). We
conclude that mast cells display moderate antifungal activity, which appears to be MCET-independent.
The traps, nevertheless, ensnared C. albicans (Fig. 3A).
Figure 2. Neutrophils but not monocytes differentially migrated in response to supernatants from mast
cells infected with C. albicans. Supernatants collected from C. albicans-infected mast cells (MOI 0.1) at 6 h,
12 h and overnight infection were used as chemoattractants to neutrophils and monocytes in a transwell
system. End-point cell migration was plotted per condition, per time as ratio of migrated cells using as 100%
control cells added to the lower compartment without inserted transwell system. (A) Neutrophil migration
is increased over time towards supernatants of infection but not to C. albicans and HMC-1 alone (controls).
(B) Monocytes show no significant chemoattraction towards supernatants of infected mast cells. Variations
between neutrophil or monocyte migration over time towards supernatants of infection and C. albicans
control were analysed for statistical significance using a one-way ANOVA with Tukey post-test. As positive
control for migration we used fMLP causing chemotaxis significantly above background of approximately
45% after 30 min for neutrophils and 13% after 90 min for monocytes. These values are indicated as a
horizontal, dashed line in the graphs of the figure. Data are presented as means of n = 5 (3) ± SD.
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5Scientific Reports | 5:12287 | DOI: 10.1038/srep12287
Figure 3. C. albicans induced MCETs in a time-dependent manner. Mast cells were infected for 6 h with
C. albicans with an MOI 0.1 (A) or left uninfected (B). Shown are representative micrographs of indirect
immunofluorescence from fixed and permeabilized samples with DNA (blue), mast cell tryptase (green) as
well as C. albicans (red) stained samples. MCETs were identified by co-localization of extracellular laminar
DNA with tryptase immunostaining (arrows). Scale bars, 10 μ m.
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6Scientific Reports | 5:12287 | DOI: 10.1038/srep12287
Figure 4. Quantification of cellular death and antifungal activity of mast cells. (A) The graph depicts
the relative amount of MCETs per micrograph of C. albicans-infected mast cells (MOI 0.1) as compared to
the uninfected mast cell control at two different time points. At both time points analysed (6 h and 10 h)
the variation between MCETs compared to uninfected samples was analysed for statistical significance. (B)
The graph represents the viability of fungal cells after normalization to the biological replicates 100% growth
control. C. albicans viability is reduced in mast cell infection (MOI 1) up to 3 h. (C) C. albicans-induced mast
cell death in a time and dose-dependent manner as determined with Sytox green. The Y-axis represents the
relative amount of dead cells after normalization to the mast cell lysis control. (A) Significance was analysed by
t-test and by Tukey one-way ANOVA (B) *P ≤ 0.05. (B) Data are presented as means of at least six replicates
(A) and n = 3 (6) ± SD. (C) For cell death significance was analysed by Bonferroni two-way ANOVA *P ≤ 0.05
comparing to the mast cell uninfected control at each time point. Data represents n= 4 (5) ± SD.
