CanTrain - Host-pathogen interaction systems as tools to identify antifungal targets in C. albicans and C. dubliniensis

EC contribution: : € 2.689.991
Duration: : 48 months
Starting date: : 01/03/2005
Funding scheme: : Marie-Curie Research Training Network
Keywords: : fungal infections, Candida sp., target screening, nutrient sensing, host-pathogen, morphogenesis, proteome analysis
Contract/Grant agreement number: :MRTN-CT-2004-512481
Project web-site: : http://www.cantrain.be/

Background:

One of the major tasks the Life Science industry faces in the coming years is to validate a plethora of targets for treatment of various diseases and the identification of novel lead compounds to generate novel pharmaceutics specifically acting on the targets identified. The "CanTrain" network will train both early and experienced researchers in the methodologies necessary for drug development, including target identification, target validation, development of cell-based screening assays all of which is required for drug screening and lead compound identification. For this purpose "CanTrain" addresses infectious diseases caused by fungal pathogens, especially strategies for the identification of novel antifungal drugs. Fungal infections have increased largely in the recent years, mostly due to immunocompromised patients in intensive care units. Despite intensive therapy, a high proportion of these patients contract systemic mycoses. CanTrain attempts to establish approaches leading to novel anti-fungal compounds by combining the expertise of 11 partners in cellular sensor systems and signalling pathways controlling morphogenesis and virulence, model systems for host-pathogen interaction, assay development and drug screening technologies. This network will not only give insight into mechanisms of host-pathogen interactions in general, but also bridge the gap between the identification of potential target proteins from the pathogen required for colonisation and infection of the host. CanTrain will enable the identification of antifungal compounds by establishing intelligent cell-based screening assays considering the host environment. These cell based screening assays will employ reporter genes activated by signalling pathways required for pathogenesis. The experimental approaches of CanTrain comprise state-of-the-art technologies, including global transcriptome and proteome analysis of pathogens (Candida albicans and Candida dubliniensis) during the infection process using in vitro host-pathogen generated tissue systems. The knowledge originating from this network will provide highly qualified individuals for the challenging tasks in life science aimed at antifungal drug discovery.

Problem:

The current project addresses an area with a great significance for human health. It is intended to identify and characterize potential novel targets for development of potent antifungals with little or no side effects. The need and timeliness for such research comes from the following facts:

The incidence of fungal infections has considerably increased worldwide. These are serious diseases which often have a very deleterious effect on the survival of immuno-compromised patients, including those receiving chemotherapy, undergoing organ transplants, or infected with HIV. The increasing incidence of fungal infections is largely due to opportunistic pathogens such as Candida spp., which is the fourth most common cause of bloodstream infections in the United States. They are similarly widespread in Europe, and are associated with mortality up to 38%. Up to 75 % of women will experience at least one episode of acute, or in many cases, chronic, candidal vaginitis during their lifetime, which clearly has significant medical, social and economic ramifications.
Large medical costs (the estimated excess medical costs attributed solely to nosocomial candidemia exceed 800 million dollars per year in the United States alone).
Current, commonly used drugs have problems with emerging resistance and in some cases (e.g. amphotericin B) severe side effects. The development of new drugs directed against novel molecular targets is critical in addressing these problems.
Novel antifungal targets should ideally not have a host orthologue, should allow development of efficient screening systems to identify specific inhibitors of possible targets and be easily accessible by the inhibitor. Based on these criteria prime targets for antifungals should be identified amongst cell wall components as well as the sensors required for expression of membrane receptors and transporters.
The genome of C. albicans is fully sequenced, therefore genome-wide methods of analysis are applicable to it.

Aim:

The main objective of this network is to train both early and experienced researchers in the methodologies of drug development starting from target identification, target validation, development of screening assays and drug screening up to the identification of lead compounds. This will be achieved in the field of infectious diseases caused by fungal pathogens. The aim is to develop new screens and cell-based assays for identifying novel antifungal substances. The training gained is transferable to all fields involving drug screening.

The major research objectives can be outlined as follows:

Objective 1 - To identify and characterize C. albicans and C. dubliniensis membrane transporters and sensors as well as downstream components which are important for the expression of virulent traits. To perform comparative genomics of C. albicans and C. dubliniensis to identify genes absent in the less virulent C. dubliniensis.
Objective 2 - To study the virulence of wild type and respective isogenic mutant C. albicans and C. dubliniensis strains using human reconstituted tissue systems and mouse macrophages as model systems.
Objective 3 - To study the molecular basis of host-pathogen interaction and virulence using transcriptional profiling, proteomics and biochemical approaches.
Objective 4 - To develop new cell-based assays for identifying potential novel antifungal substances in the context of host-pathogen interaction, including assay validation with clinical C. albicans and C. dubliniensis isolates and known antifungal drugs and screening using combinatorial compound libraries.

Expected and obtained results:

A: scientific part

1. We have bridged the gap between environmental stimuli inducing infection mechanism and the signal transduction pathways triggered by these stimuli. We have characterized a G protein-coupled receptor that is important for the yeast-to-hyphae transition on solid medium. Currently we are characterizing the pathway by which this receptor triggers this morphogenesis. In this aspect we have recently identified a genetic interaction between the phosphodiesterases and this receptor, a phenotype that does not occur in Saccharomyces cerevisiae. We have also characterized the CaSPS-sensing pathway, a pathway by which amino acids induce their own uptake or induce the expression of a number of proteases.

2. Several potential new targets for antifungal drug discovery in both C. albicans and C. dubliniensis have been identified. These include the fungal specific phosphodiesterase Pde2, the trehalose-6-phosphate phosphatase Tps2, various components of the SPS sensing system, the AUF genes, which are genes belonging to one family that are upregulated during adhesion on human tissue.

3. In order to find these novel targets and also in order to characterize the pathways involved, we have developed proteomics and transcriptome analysis under various conditions. These include in vitro, ex vivo and in vivo host-pathogen interaction systems. One example is a newly developed in vivo biofilm system using a subcutaneous rat model system. This analysis also includes a comparative genomics approach comparing C. albicans and C. dublinienis. Two important differences that we have observed is the role of methionine for morphogenesis in these two species and the different substrate specificities of the potassium transporters. Finally novel reporter constructs and a C. albicans specific two-hybrid system have been developed.

4. Apart from the pathogen, we have also looked at the host site in order to understand the reaction of the host upon an infection. Both macrophage and dendritic cell systems have been investigated and the results are currently being analysed.

5. We have also eveloped different screening assays in order to screen for novel antifungal drugs. An example of a screening system is based on the G protein-coupled receptor Gpr1, that we mentioned above. A deletion of this receptor results in sensitivity to a low concentration of histatin 5, a naturally occurring antifungal. We are using a wild type strain in the presence of histatin 5 to find compounds that inhibit the growth, which are possible antagonists of this Gpr1 protein. Other types of screening will start soon.

B: Training part

Apart from the biannual workshops were all the ESR and ER students presented their results, we have also organized several training courses in which most of them participated. These include:

Real time PCR course
In vitro biofilm course
Host-pathogen interaction of C. albicans using human epithelial models
Bioinformatics and microarray analysis course
C. albicans - macrophage interaction course

Two more training courses are scheduled. In addition, we will also organize a FEBS sponsored advanced practical and lecture course in Madrid in February 2009 (see www.febs.org).

Potential applications:

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Coordinator:

Prof. Patrick Van Dijck
Department of Molecular Microbiology, VIB
Laboratory of Molecular Cell Biology, K.U. Leuven
Insitute for Botany and Microbiology
Kasteelpark Arenberg 31
3001 Leuven, Belgium
Tel. +32 16321512
Fax +32 16321979
Patrick.vandijck@bio.kuleuven.be

European Commission