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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.

Date:1 Oct 2013 →  30 Sep 2015
Keywords:Zebravis
Disciplines:Microbiology, Systems biology, Laboratory medicine