Biomedical Engineering Reference
In-Depth Information
of promoter elements responsible for the regulation of
MDR1
(Rognon et
al. 2006). Other studies are focussed in the study of the effect of tetracycline
when the TET-inducible promoter system (Park and Morschhauser 2005) is
used as a tool to analyze the effect of certain antifungal agents. For that, RLuc
is also fused to the promoter regions of
MDR1
,
ERG11
and
UPC2
(regulator
of sterol biosynthesis) (Oliver et al. 2007) and assayed for light emission
upon treatment with different antifungal agents. The fi nal conclusion from
these studies is that tetracycline alters the drug susceptibility in
C. albicans
and other fungal pathogens such as
A. fumigatus
and
C. neoformans
and the
need to be careful when using this system, especially in studies that involve
antifungal drugs (Oliver et al. 2008).
Recently, our group has established a connection between a pre-
treatment of
C. albicans
with sublethal doses of azoles (fl uconazole) and the
functional consequences of this treatment in a subsequent interaction with
phagocytic cells. In this study the
RLUC
gene is fused to several
C. albicans
genes involved in oxidative stress response and luminescence signals are
quantifi ed in response to sublethal doses of fl uconazole, concluding that
these responses protect the yeast against killing mediated by phagocytes
(Arana et al. 2010).
Applications in the Interaction with the Host Immune Cells
Ex vivo
studies of host-fungal pathogens interactions have been used as
infection models. In general, host cells (cell lines or primary cells) are
grown in the presence of isolated microorganisms in order to analyse
both, host cell and fungal pathogen responses (Fradin et al. 2003, Lorenz
et al. 2004, Thewes et al. 2007).
From the fungal pathogen point of view, adaptation is essential at all
stages of pathogenesis, as a failure to respond to the changing environment
conditions of the host would lead to elimination of the microorganism.
Bioluminescent reporters have been used during last years to monitor the
expression of genes involved in detoxifying reactive oxygen and nitrogen
species (ROS and RNS) inside the host cells, one of the microenvironments
that
C. albicans a
nd other fungal species
,
must face as a consequence of
recognition by host cells. Recent studies have quantifi ed these responses
in
C. albicans
using
different reporters by using, generally, single-reporter
assays as well. Concretely, the expression of
RLUC
transcriptional fusions
involved in the response to oxidative (
TRR1
,
GRE2
) and nitrosative stress
(
YHB1
) have been analysed in the presence of different oxidant agents (
in
vitro
) and when the fungal cells are recognized by phagocytic cells such as
neutrophils or macrophages (
ex vivo
) (Arana et al. 2007, Arana et al. 2010).
In
vitro
and
in vivo
expression has been also addressed by using GFP as reporter
