Bacterial oxalotrophy as an alternative biocontrol approach for the fight against pulmonary aspergillosis

Abstract

Although fungi are estimated to kill more than 1.5 million people every year worldwide, the issue of fungal pathogenesis is largely neglected. Moreover, the rise of emergence of multi-resistant fungal pathogens worldwide is a major threat for human health. This is notably the case of the opportunistic fungal pathogens of the genus Aspergillus. The prevalence of Aspergillus-related infections, also known as aspergillosis, has dramatically increased in the last few years. Aspergillus species, such as A. fumigatus, A. flavus, A. nidulans, A. terreus or A. niger, are known to cause a vast spectrum of respiratory diseases, ranging from mild allergies to life-threatening invasive infections. Interestingly, the formation of calcium oxalate crystals has been previously reported in the latter cases. Oxalic acid is known to play a key role in the pathogenesis of plant fungal pathogens, such as for instance Sclerotinia sclerotiorum. However, a link between the production of oxalic acid and pathogenicity has not been made yet in the case of Aspergillus. Oxalic acid is commonly produced by soil fungi, along with other low molecular weight organic acids. In soils, oxalic acid generally occurs in the form of calcium oxalate crystals. Despite its chemical stability and low solubility, calcium oxalate is rarely found in the geological records, something that has been suggested to be the results of its metabolization by oxalotrophic bacteria. Oxalogenic fungi are known to interact with oxalotrophic bacteria in soils within the oxalate-carbonate pathway, where fungi, along with plants, are the source of oxalate, and oxalotrophic bacteria are its sink. Oxalotrophy is concomitant with a pH increase, which eventually leads to the precipitation of calcium carbonate, if the pH increases above a value of 8.4. The aim of the present thesis was to translate the metabolic interaction between oxalogenic fungi and oxalotrophic bacteria occurring in soils to human health. Specifically, we developed and assessed a novel biocontrol strategy for the treatment of pulmonary aspergillosis based on the manipulation of the environment through bacterial oxalotrophy, a process we named environmental interference. For this, the influence of the composition of the culture medium on the production of low molecular weight organic acids was tested for selected fungal strains, as well as their interaction with non-oxalotrophic and oxalotrophic bacterial strains. The fungal strain Aspergillus niger was selected because of its systematic production of oxalic acid in all culture media tested, and because of its medical relevance. The first demonstration of the principle of environmental interference in-vitro was made by showing the biocontrol exerted by the oxalotrophic bacterial species Cupriavidus oxalaticus on the growth of A. niger. The use of soil bacteria was shown to be problematic, as they induce important cellular damage. Therefore, the genome of C. oxalaticus was analyzed in order to better understand the oxalotrophy metabolism in this model bacterial species. This highlighted the presence of an operon containing all the genes that are required for the degradation of oxalate. These data will be used for the design of an entirely enzymatic degradation pathway of oxalate that will be more suitable as a potential therapeutic option. Finally, the fungal:fungal:bacterial interaction between A. niger, Candida albicans and C. oxalaticus was investigated. This interaction is relevant in the case of pulmonary co-infection in immunocompromised patients and patients suffering from cystic fibrosis. The interaction of both opportunistic fungal pathogens and the oxalotrophic bacterium depended on the inoculation mode (simultaneous versus sequential). To conclude, while the presented results on the biocontrol concept of environmental interference are promising, a preclinical in-vivo demonstration of this concept in a murine infection model is crucial for the development of a potential clinical application. Indeed, a more comprehensive approach by integrating the immune system is necessary in order to better comprehend the interplay between the host, the pathogen and the lung microbiota in disease development.