Project
Zebrafish as a novel model host for morphological and undiscovered immunl characteristics of in vivo Candida albicans biofilms
In nature, most microorganisms exist predominantly as a community of cells, called a
biofilm, where they adhere to and live on various surfaces. Cells present in such a
biofilm acquire different characteristics compared to planktonic cells, such as altered
survival, growth and virulence. The human fungal pathogen Candida albicans is also
well-known for its ability to form biofilms, both on mucosal surfaces as well as on a
variety of implants. C. albians biofilm cells are known for their significant resistance
to different classes of antifungals and removal and/or replacement of the device is
often the sole solution for the treatment. To study biofilm formation, different in vitro
Candida biofilm model systems have been developed, but they can hardly mimic the
situation in vivo. Therefore, several in vivo central venous catheter C. albicans biofilm
models have been developed in rats (1), in rabbits (2) and mice (3). In our laboratory
we have developed an alternative simple subcutaneous model system in rats Ricicová
et al. (2010) (4). In this CREA project we want to use the zebrafish larvae as model
system to study C. albicans biofilm development. The zebrafish larvae have been
used to study C. albicans infections caused by planktonic cells and proved to be
a useful host model organism to study pathogenesis (5,6). Noteworthy, the zebrafish
larval model provides advantages compared to mammalian systems from its small
size and transparency, permitting high throughput screens, chemical genetic screens,
and non-invasive whole animal visualization of host pathogen interactions. In this
project we will investigate the undiscovered role of the innate immune system during
C. albicans biofilm development on microspheres in the novel model host organism -
zebrafish.
Scientific goals
The first main scientific goal is to develop and characterize C. albicans biofilms
formed on polystyrene microspheres in zebrafish embryo as a host model organism.
Notably, in already existing in vivo biofilm models, the visualization of biofilms is
followed post-mortem. Therefore, the innovative aspect of this objective is the usage
of optically transparent zebrafish embryos, which will permit the first real-time
fluorescence visualization of in vivo C. albicans biofilms formed on microspheres,
which is not as easily accessible as in rodent models. It is noteworthy to mention that
the adult zebrafish candidemia model does not permit the visualization of infection in
real time or morpholino (MO)-directed gene knockdown, both of which techniques
are available with the larval host (6). This model will provide an easier tool to study
in vivo different stages of biofilm development over time.
The second main scientific goal is to elucidate, for the first time, the role of innate
immune system during in vivo C. albicans biofilm development in zebrafish embryos.
It is noteworthy to mention, that zebrafish have similar signaling through Toll-like
receptors to that in humans, express similar cytokines and have macrophages,
neutrophils, dendritic cells, mast cells, eosinophils, T cells and B cells (7). The
innovative aspect of this objective is to characterize the as yet not known role of the
innate immune system during in vivo C. albicans biofilm development. Additionally,
we will provide a unique opportunity to address the molecular nature of in vivo
interactions between C. albicans cells and immune cells in the context of a live host.
Research methods
Zebrafish as a novel host organism to study C. albicans biofilms in vivo.
Zebrafish at the prim25 stage (approximately 36 h postfertilization) will be injected
with polystyrene microspheres through the otic vesicle into hindbrain ventricle.
Afterwards, zebrafish will be infected with Candida cells (1x107 cells/ml) of wild
type C. albicans GFP-expressing cells (WT-GFP). Biofilm development will be
monitored immediately after the injection (30 min), period of adhesion (90 min), early
(4 h), intermediate (12 h) and maturation (24 h) stage by confocal microscopy and by
quantification of colony forming units (CFUs). In order to validate the proposed
model, we will use C. albicans bcr1/bcr1 and C. albicans efg1/efg1 cph1/cph1 GFP
expressing mutant strains as controls. Bcr1 is a transcription factor responsible for
mature in vitro and in vivo biofilm development and a key regulator of biofilm
specific genes, such as ALS3 and ECE1 (8). C. albicans Efg1 and Cph1 are important
transcription factors required for expression of genes involved in morphogenesis,
a key step in biofilm formation. All experiments will be compared to a control
zebrafish, which will contain only microspheres and will be injected only with
phosphate buffered saline (PBS).
Examination of C. albicans colonization and invasion in zebrafish.
The ability to colonize and invade tissues within the host is critical for C. albicans
infection, also because of the ability to switch from yeast to the more virulent hyphae
form. As it is already mentioned above, morphogenesis is an important step during
biofilm development. Upon microspheres implant and subsequent injection of C.
albicans we expect the cells to form biofilms on microsphres but at the same time to
disseminate into different compartments of the host. Therefore, we will perform
histological analyses and colony forming units count (immediately after injection, 30
min, 90 min, 4 h, 12 h and 24 h of infection) to examine C. albicans dissemination
within zebrafish acording to Chao et al. (2010) (6).
The role of innate immune system during in vivo C. albicans biofilm formation in
zebrafish.
To asses the role of innate immune response during C. albicans biofilm formation we
will use transgenic fli1:EGFP fish with EGFP-expressing macrophage-like cells and
endothelium (9,10) and mpx:GFP fish with EGFP expressing neutrophils (11).
Zebrafish will be injected with microspheres and subsequently infected with C.
albicans wild type cells expressing yCherry (a version of mCherry that is codon
optimized for Candida spp.) (5). C. albicans bcr1/bcr1 and C. albicans efg1/efg1
cph1/cph1 containing the yeast optimized mCherry gene will be used as controls.
Different time points will be investigated - immediately after C. albicans injection, 30
min, 90 min, 4 h, 12 h and 24 h of biofilm formation. Subsequently, we will apply
confocal scanning laser microscopy to coimage fungi together with innate immune
cells and we will determine the innate immunity-fungus interactions.