Galar Fungail 2

Interaction of fungal pathogens with host cells: a post-genomic approach

What is Galar Fungail 2 ?

Introduction

Galar Fungail 2 is a Marie Curie Research and Training Network dedicated to the study of fungal pathogens and their interactions with host cells using post-genomic approaches. Galar Fungail 2 was launched in January 2004 in the framework of the Marie Curie actions of the 6th Framework Programme of the European Commission. Galar Fungail 2 has ended its activities in December 2007.

Fungal infections are an increasingly significant medical problem in the European Community. Most women suffer a Candida infection in their lifetime, and some of these infections are deep-seated and recurrent. Candida albicans also is a common cause of life-threatening systemic fungal disease. These infections are associated with a high mortality because of poor diagnosis and the lack of effective systemic antifungal drugs. Hence there is a clear need for more effective antifungal therapies. However, the development of new therapies is limited by a poor understanding of C. albicans pathogenesis and of the host responses to this important pathogen.

The Galar Fungail 2 network comprised 9 European academic laboratories, 8 of which participated in the Galar Fungail network funded within the 5th Framework Programme of the European Commission. The expertise that these groups had gained in the genomics and post-genomics of C. albicans has been used to develop research programmes focusing on the study of the interaction of C. albicans with host cells and to train young scientists (6 PhD students and 3 post-doctoral fellows) in the most up-to-date aspects of molecular medical mycology.

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.

Training activities of the Galar Fungail 2 network

One of the main objectives of the Galar Fungail 2 network was to provide an advanced training in molecular medical mycology to young European scientists. Five post-doctoral fellows and six PhD students were employed within the Galar Fungail 2 network. Apart from the training they got in their repective laboratories, they all received two other main types of training: research training courses, and transferable skills training courses.
The GF2 Network provided an outstanding balance of interdisciplinary 3-4 days research training courses that covered topics in fungal molecular and cell biology (state-of-the-art molecular tools, etc.), immunology (antibody technologies and cytokine assays) and genomics. The genomics courses included microarray techniques, proteomics technologies and data analysis tools. The courses combined hands on practical work with lectures and tutorials from leading scientists in the field.
In addition, the GF2 Network provided a comprehensive set of training courses on transferable skills. These included Commercialisation and Intellectual Property, Time and Project Management, Statistics, Ethical Issues in Science, Scientific Writing, Preparing a PhD Thesis, Oral Presentation Skills, and Career Development. These strong courses combined lectures from experts in each field, with case studies and discussion groups. The courses were regularly attended by all of our researchers, and they provided very positive feedback for most of these courses.

International networking of the concerned scientific community

Galar Fungail II has been very proactive in driving International networking of the concerned scientific community – at the European level and a more global level. We have:
1) Initiated the FUNNET network, which links European network and consortia working on medically important fungal pathogens (Partners 1 and 3).
2) Organised the first FUNNET meeting in Crete in September 2006 (Partner 3).
3) Played an active role in the second FUNNET meeting in Gosau in September 2007 (all GF2 partners)
4) Organised two FEBS Advanced Courses on Human Fungal Pathogens in 2005 and 2007 (Partner 1).

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