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

危及生命的真菌感染的数量正在稳步增加。由于诊断和治疗机会有限，侵袭性真菌感染可能导致超过50%的致死率。巴西副球孢子菌（Paracoccidioides brasiliensis）是拉丁美洲普遍存在的一种真菌。这种真菌具有二型生活方式，在较低温度下具有丝状生长形态，在升高的温度下具有酵母形态。在土壤中，丝状形式是普遍的形态，无性分生孢子由菌丝形成。这些孢子很容易被农场工人吸入。在吸入后，condia可能会在肺部发芽并形成持久的酵母细胞。据推测，在拉丁美洲，特别是在巴西，大约有1000万人携带副球孢子菌属的潜伏感染。并且在1 - 2%的情况下，真菌不再受免疫系统控制，这导致急性感染，如果不治疗，则会危及生命。为了在宿主组织内持续存在和增殖，获得和利用宿主提供的营养物对于所有病原生物体是必不可少的。就巴西假单胞菌而言，先前的研究表明菌丝和酵母之间的代谢和代谢生理调节有很大不同。甚至，已经描述了至少四种毒性巴西假单胞菌亚种（S1、PS2、PS3和01样），并且第一初步分析表明，这些亚种在进入酵母形式时的代谢偏好不同。然而，对毒力、真菌增殖和治疗方案的影响尚未研究，因此，本项目涉及鉴定和表征不同P. brasiliensis亚种的代谢生理，以更深入地了解感染过程中代谢途径的影响和相互作用。必需代谢过程的鉴定为抗真菌治疗提供了新的靶点，所述抗真菌治疗可能不仅适用于对抗巴西青霉，而且适用于对抗其他侵入性威胁生命的真菌感染。将有可能建立一种体内成像方法，使用副球孢子菌病的鼠模型系统在时间和空间分辨率上的疾病进展的时间可视化。该系统对研究拟球孢子菌的生态位特异性代谢能力具有重要价值。需要在宿主体内增殖。为了评估不同代谢途径的影响，将通过基因敲除方法沉默关键酶，该方法将与生物发光报告基因表达组合进行。由此产生的突变体将在替代和小鼠感染模型中进行测试，以研究真菌增殖率和传播。为了确定针对关键代谢途径的新化合物，将筛选巴西合作伙伴提供的天然产物库。这包括纯化酶的直接抑制以及体外条件下真菌生长的抑制分析。感兴趣的候选物随后将分析其对细胞系的潜在细胞毒性作用，并最终在小鼠模型中进行分析。通过进行这些复杂的分析，该项目不仅旨在扩大对感染过程中代谢适应的理解，而且还试图完善对副球孢子菌感染过程的理解，并将鉴定出靶向基本代谢途径的新化合物，治愈侵袭性真菌感染。

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.