Metabolic adaptation and nutrient acquisition of Paracoccidioides spp. during host interactions: Search for targets and new therapeutic approaches

The number of life-threatening fungal infections is steadily increasing. Due to limited diagnostic and therapeutic opportunities invasive fungal infections may cause lethality rates of more than 50%. Paracoccidioides brasiliensis is an ubiquitous fungus in Latin America. This fungus has a dimorphic life-style with a filamentous growth morphology at lower and a yeast morphology at elevated temperatures. In soil the filamentous form is the prevalent morphology and asexual conidia are formed from hyphae. These conidia are easily inhaled by farm workers. After inhalation condia may germinate and form persisting yeast cells in the lung.
It has been assumed that in Latin America -and especially in Brazil- approximately 10 million people carry a latent infection with Paracoccidioides spp. and in 1 - 2% of cases the fungus is no longer controlled by the immune system, which leads to acute infections that, when untreated, are life-threatening.

In order to persist and proliferate within host tissues the acquisition and utilisation of host-provided nutrients is indispensable for all pathogenic organisms. In terms of P. brasiliensis, previous studies have shown that regulation of metabolism and metabolic physiology among hyphae and yeasts substantially differs. Even more, at least four virulent P. brasiliensis subspecies (S1, PS2, PS3 and 01-like) have been described and first preliminary analyses indicate that these subspecies differ in their metabolic preferences when entering the yeast form. However, the impact on virulence, fungal proliferation and therapeutic options has not been studied yet.

Therefore, this projects deals on the identification and characterisation of the metabolic physiology of the different P. brasiliensis subspecies to gain a deeper understanding on the impact and interplay of metabolic pathways during the infection process. Identification of essential metabolic processes provides new targets for antifungal therapies that may not only be suitable for combating P. brasiliensis, but also other invasive life threatening fungal infections.

By an generating bioluminescence imaging system for Paracoccidioides species, it will be possible to establish an in vivo imaging approach that allows the real-time visualisation of disease progression in temporal and spatial resolution using murine model systems of Paracoccidiomycosis. This system will be of high value to investigate the niche-specific metabolic capacity of Paracoccidioides spp. that is required proliferate within the host.

To assess the impact of distinct metabolic pathways key enzymes will be silenced by gene knock down approaches, which will be performed in combination with bioluminescence reporter expression. The resulting mutants will be tested in alternative and murine infection models to investigate the fungal proliferation rate and dissemination.

To identify new compounds targeting key metabolic pathways, a library of natural products available at the Brazilian partners will be screened. This includes the direct inhibition of purified enzymes as well as inhibition analyses of fungal growth under in vitro conditions. Interesting candidates will subsequently become analysed for potential cytotoxic effects on cell lines and eventually in murine models.

By performing these complex analyses the project not only aims in broadening the understanding of metabolic adaptations during the infection process, but also tries to refine the understanding of Paracoccidioides spp infection process and will identify new compounds that target essential metabolic pathways and could be suitable to prevent and cure invasive fungal infections.

Previous proteomic analyses on strains from four different P. brasiliensis subspecies (S1, PS2, PS3 and 01-like) implied significant differences in metabolic physiology. As an example, Pb01 tends to display increased fermentative capacity. Resistance to oxidative stress appears more developed in Pb01, Pb2 and Pb339, whereas the pentose phosphate pathway is strongly represented in Pb2. Furthermore, antigenic proteins, such as GP43 and a 27-kDa antigenic protein were less abundant in Pb01 and Pb2. These data and their impact on virulence will be analysed in more detail.
Transcription profiles will be recorded by qRT-PCR from selected strains during the dimorphic switch and data will be correlated with determination of enzymatic activities. Key enzymes will be heterologously expressed either in prokaryotic or eukaryotic expression system. These enzymes will be used for comparative biochemical characterisations to identify species-specific differences and will subsequently be used to screen for specific inhibitors from natural product libraries.
To test for the importance of distinct metabolic pathways antisense constructs will be generated to silence these pathways in P. brasiliensis. Transformation will involve a DNA transfer via Agrobacterium tumefaciens. Furthermore, a constitutive luciferase expression system will be simultaneously integrated. By flanking the silencing construct at one border with the resistance marker and at the second border with the luciferase construct a rapid assessment of clones with integration of the complete transformation cassette will be possible. Proteome profiles will be recorded to determine the changes induced by gene silencing. The transformation procedure will generate bioluminescent strains that will be suitable for in vivo imaging of disease progression in murine infection models. This allows assessment of attenuated virulence and enables drug efficacy studies in murine infection models.

Who will benefit from this research?

The principal economic and societal beneficiaries will be all those who gain from improved understanding of metabolic physiology and adaptation of Paracoccidioides species during establishment of Paracoccidiodomycosis in the infected host; principally, the following: (1) Regulatory bodies concerned with economic costs due to severe illness of rural workers. (2) Organizations providing health-monitoring services. (3) The pharmaceutical industry. (4) Hospitals and medical doctors involved in diagnosis and treatment of disease. (5) Diseased patients and people at risk. (6) Researchers and collaborators who are directly involved in the project.

How will they benefit from this research?

(1) Assuming that 10 million people in Latin America, and especially in Brazil carry a latent infection with Paracoccidioides spp. an outbreak of disease would overwhelm the costs available in the health system. Therapy with currently available antifungals is expensive and needs to be continued for about 2 - 3 years. New antifungals with increased efficacy against Paracoccidioides spp. could significantly reduce length of therapy, reduce costs and bring workers earlier back to work.

(2) It is anticipated that results from this project will provide more awareness of this dreadful disease, leading to a change in obligation to report. This, in turn, could accelerate early diagnosis, which is important for a successful outcome of therapeutic approaches.

(3) The screening approach will base on new classes of compounds that will be tested for their antifungal potential. Despite specific analyses on Paracoccidioides species, compounds will also be tested against a broad spectrum of other medically important fungi. The project will also involve cytotoxicity studies and new compounds with low cytotoxicity may be of interest for the development of new antifungal drugs, which could be of interest to the pharmaceutical industry.

(4) Currently, mortality rates of patients with severe Paracoccidiodomycosis are high. The organism seems to disseminate via bloodstream and the lymphatic system and may persist in cryptic niches. Bioluminescence imaging approaches will help to identify these niches, supporting therapeutic approaches and will shorten the duration of therapy.

(5) Obviously, patients suffering from Paracoccidiodomycosis and people at risk will directly benefit from a deeper understanding of disease establishment, progression and therapy. While we assume that new drugs may become available to combat this disease, we will also provide information on the speed of disease progression and putative cryptic reservoirs of infection. Moreover, new insights from this project may increase the awareness of the public for this disease leading to increased willingness to seek medical advice.

(6) Staff working on this project will gain valuable new research skills as well as transferable skills like oral and written communication, collaboration, networking, and working as part of a team. Due to direct interaction of researchers from both countries, new research perspectives will be exchanged, increasing the skill especially of the junior staff in this project. These skills will improve their employment prospects in all sectors and their contribution to the economic competitiveness of the developing country.