Research activities of the Galar Fungail 2 network
Research activities conducted within the Galar Fungail 2 network were
directed towards dissecting critical interactions between C. albicans,
the leading cause of human systemic fungal infections, and host immune defenses
during disease development. In this context we have developed several research
programmes in order to understand:
(1) How specific C. albicans signal transduction pathways co-ordinate
broad cellular responses to specific signals;
(2) How C. albicans responds to host defences during disease establishment
and progression
(3) How the mammalian host responds locally and systemically to C. albicans
infections
This research was conducted using post-genomics, molecular and cellular
approaches in different contexts that are relevant to the interaction of
C. albicans with the host: biofilms, reconstituted human epithelia,
macrophages and animal models when necessary.
Through these research programs, major progresses have been made in the understanding of the molecular events that are involved in the interplay between Candida albicans and the host during the progression of disease. These advances have been made possible thanks to the availability of the genome sequence of C. albicans and of post-genomics tools that provided the necessary environment to investigate in depth the biology of the pathogen in the context of its interaction with the host.
Signaling networks and the regulation
of virulence attributes
Research conducted by GF2 has contributed significantly to the understanding
of major signaling networks regulating the expression of genes encoding
C. albicans virulence attributes, especially the ability to switch
from the yeast to hyphal growth forms, and their interplay with metabolic
functions. Partner 4’s investigation of cell surface sensors has uncovered
a novel regulator of morphogenesis, invasion and cell wall biogenesis, namely
Msb2, a cell surface signaling mucin that participates in one of the two
branches of the Hog1-mediated stress-adaptation MAP kinase pathway in C.
albicans. Elucidation of the Hog1 network in C. albicans suggests
divergence and specialization in yeast and filamentous fungi. Such rewiring
is reinforced by the global analysis of the role of the key regulator, Hog1,
in stress adaptation in C. albicans that has been conducted by
Partner 3. This work involved the global comparison of stress- and Hog1-regulated
functions in C. albicans with benign model yeasts (S. cerevisiae
and Sz. pombe). This has been followed up with global analysis
of stress- and Hog1-regulated proteins using proteomics. This work has shown
that Hog1-mediated signalling plays a key role in stress regulation at both
transcriptional and post-transcriptional levels.
Refinement of our understanding of the gene networks that are placed under
the control of key regulators of morphogenesis has been obtained, in particular
through the characterization of transcriptional targets of the chromatin
remodeling enzyme Hos2 by Partner 6 and the pH responsive regulator Rim101
by Partner 5. While Partner 6’s results land support to an interplay
between the regulation of morphogenesis and metabolic functions, Partner
5’s observations indicate that cell surface proteins of the ALS family
of adhesins are differentially regulated in response to environmental cues
relevant to host-pathogen interaction. In this regard, the characterization
of the promoter region of the ALS3 gene by Partner 3 has showed
that expression of this gene in response to morphogenetic signals is mediated
by an intricate interplay between positive and negative regulation. Importantly,
a novel regulator of morphogenesis that may link positive and negative regulation
has been uncovered by Partner 1. Indeed, the Yak1 protein kinase has been
involved in the initiation and maintenance of hyphal growth and necessary
for the up-regulation of only a subset of hypha-induced genes, possibly
through inhibition of the Tup1 pathway that is known to negatively regulate
hyphal emergence and maintenance in C. albicans.
Dynamics of the cell wall proteome
GF2 has made unprecedented advances in the description of the regulation
of dynamic cell wall (CW) architecture in C. albicans using a combination
of biochemical and genetic approaches. Novel CW extraction procedures and
mass spectrometry methods have been developed by Partner 9 and 7 that have
enabled the identification of a significant number of CW proteins previously
postulated through in silico analysis of the C. albicans genome.
These technological improvements provide an unprecedented handle on the
dynamics of the C. albicans cell wall when exposed to environmental variations
relevant to host-pathogen interactions. For instance, Partner 9 has shown
that the CW proteome of C. albicans is exquisitely sensitive to changes
in ambient oxygen concentration and iron availability and that the responses
to hypoxic conditions and iron restriction are related.
These studies of the CW proteome have been complemented through analysis
of C. albicans mutants with defects in different CW proteins. Partner 5
has established a collection of C. albicans strains with null mutations
in genes encoding 46 GPI-anchored proteins. Some of these mutants show alterations
in their sensitivity to CW-damaging agents or antifungals and in the composition
and/or structure of the CW. In addition, detailed studies of the Pir1, Pga13,
Pga31, Pga59, Pga62, Sap9, Sap10, Sun 41 and Sun42 CW proteins conducted
by Partners 1, 2, 7 and 9 have shown the involvement of these proteins in
CW maintenance, cell separation, cell viability and virulence in C.
albicans. Sap9 and Sap10 are of particular interest since they represent
the first example of GPI-anchored regulatory proteases associated with virulence
of pathogenic fungi.
C. albicans
interplay with the host: a genome-wide approach
GF2 has made pioneering studies to obtain the first detailed in vivo transcriptional
profiles of C. albicans cells during infection of mice (organ samples)
and patients (clinical samples) and from ex vivo and in vitro infection
models (blood, liver and Reconstituted Human Epithelium models). This novel
approach developed by Partner 2 of comparing in vivo transcript profiling
data with that from detailed alternative infection models for two distinct
(liver and oral) forms of candidosis has been a major achievement and resulted
in a number of important scientific outcomes. Firstly, it has shed new light
on both pathogen and host biological processes and the delicate interplay
between the two. Secondly, it has identified novel infection-associated
genes with unknown function. For example, the EED1 gene was identified
as associated to epithelial infection and appeared essential for maintenance
of hyphal elongation as eed1 mutants showed transient cell elongation on
epithelial tissue, which enabled only superficial invasion. Once inside
an epithelial cell, eed1 mutant cells could proliferate as yeasts
or pseudohyphae but remained trapped intracellular. These results suggest
that the adaptive response and morphology of C. albicans play specific
roles for host-fungal interactions during mucosal infections. Thirdly, this
comparative approach has confirmed that our ex vivo models accurately reflect
the situation in vivo and has had an ethical benefit by reducing the scientific
communities reliance on animal experimentation. Other models relevant to
infection have been developed, especially those dedicated to the study of
biofilm formation, and have enabled the identification by Partner 1 and
4 of novel genes necessary for the formation of C. albicans biofilms
that are important source of therapeutic failures due to elevated antifungal
resistance.
C. albicans interplay with the host: evaluating
local responses
In parallel to the investigation of genome-wide responses to interactions
with the host, GF2 has performed local analysis of stress responses in C.
albicans. Partner 3 has established single cell profiling (SCP), a
novel approach that uses yEGFP fusions to C. albicans promoters
diagnostic of specific stress responses in order to investigate the local
environment encountered by C. albicans during its interaction with
the host. Through SCP, it has been shown directly that C. albicans
cells activate a robust oxidative stress response following phagocytosis
by neutrophils. However, phagocytosis by macrophages induces only mild oxidative
stress responses by C. albicans cells. Once C. albicans
cells have established infections in the kidney, they are not exposed to
significant oxidative stresses despite the close proximity of immune cells
to the infecting fungal cells. Partner 3 has also examined the metabolic
responses of C. albicans to its host, focusing on central carbon
metabolism because this is critical for the growth of the pathogen in vivo.
This work has shown that C. albicans regulates its central metabolic
pathways in a niche-specific fashion during disease establishment and progression.
These data significantly advance our understanding of how the pathogen responds
to its host.
With a view to monitoring the dynamics of C. albicans infections
without having to sacrifice the infected mice, GF2 has developed an innovative
and sensitive bioluminescent reporter system. Partners 1 and 3 have demonstrated
the utility of this specialized luciferase reporter in vitro. This new cell-surface
exposed luciferase reporter was shown to be several orders of magnitude
more sensitive than other C. albicans reporters, to be measurable
without having to lyse the fungal cells, and to be responsive to the regulation
of the C. albicans promoters that are fused to it. Therefore, this
reporter is both sensitive and reliable and has multiple uses in vitro including
high throughput analyses of gene regulation, and in large scale genetic
screens. Partners 1 and 8 are currently testing the utility of the reporter
in in vivo infection models and have shown that it can be used to monitor
cutaneous and subcutaneous C. albicans infections over time.
Host interplay with C. albicans:
a genome-wide approach
GF2 has initiated genome-wide approaches to investigate host responses to
C. albicans infections. For instance, Affymetrix microarrays have
been used by Partner 6 to compare the transcript profiles of mouse liver
in the presence and absence of C. albicans infections, showing that genes
encoding pro-inflammatory molecules, receptors (TLR- 2, MyD88 , IL1R and
CD14), acute-phase proteins, chemokines and their receptors were highly
expressed at early stages of the infection. In contrast genes associated
with metabolism and transport were down-regulated in response to infection.
These in vivo observations are reminiscent of those made by Partner 2 using
qRT-PCR and immunoassays of specific responses (cytokines, immune receptors,
granule proteins) of human leukocytes, PMN, RHE in the presence and absence
of PMN exposed to C. albicans.
Host interplay with C. albicans:
involvement of cellular components
Another important contribution of GF2 lies in the analysis of the contribution
of different host components to infection. For instance, Partner 8 has investigated
the role the glucocorticoid-induced tumor necrosis factor (TNF) receptor-related
gene (GITR) that is known to modulate immune response through activation
of co-accessory signals in T cells. Partner 8 has shown that the interaction
of GITR with its ligand (GITRL) negatively regulates susceptibility to systemic
candidiasis. This is likely due to GITR-dependent regulation of TLR4 and
MyD88 functions that play important roles in regulating innate immunity
to opportunistic infections.
The host machinery involved in the endocytosis of C. albicans hypha
has also been studied. Using live cell-imaging, Partner 1 has monitored
the recruitment of clathrin to the hyphae entry sites in the very early
steps of C. albicans internalization. Colocalization of the recruited
clathrin and E-cadherin, a receptor for the Als3 fungal adhesin, with C.
albicans hyphae was consistently observed during entry. Furthermore,
using siRNA it was shown that clathrin, dynamin or cortactin depletion decreased
C. albicans endocytosis. These data suggest that, during the early
stages of C. albicans internalization, the clathrin-dependent endocytosis
machinery of the host cell is recruited, similar to what is observed during
bacterial infections.
Towards immune intervention against C. albicans
infections
Vaccinal approaches for the prevention of C. albicans infections
have been investigated by GF2. An analysis of the interaction of the C.
albicans mannoprotein MP65 with dendritic cells (DC) has demonstrated
that MP65 induced DC maturation by increasing co-stimulatory molecules and
decreasing CD14 and FcgR molecule expression. The latter effect is partly
mediated by TLR2 and TLR4 with involvement of the MyD88-dependent pathway.
MP65 enables DC to activate T cell response: its protein core is essential
for induction of T cell activation, while its glycosylated portion primarily
promotes cytokine production. The mechanisms involved in induction of protective
response against C. albicans could be mediated by the MP65 antigen,
suggesting that MP65 may be a suitable vaccine candidate. Other studies
of Partner 8 have shown that b-glucan in the fungal cell wall is an antibody-sensitive
target and that antibodies to this component have a promising therapeutic
potential against fungal pathogens.