Volatile sulphur compounds produced by Pseudomonas aeruginosa synergize with Aspergillus fumigatus in vivo enhancing the pathobiology of co-infection

Objectives
Co-infection with Pseudomonas aeruginosa and Aspergillus fumigatus is associated with a more rapid decline in Cystic Fibrosis pulmonary function, more frequent hospitalisations and more respiratory infections, all leading to worse prognosis (1). Recently it was reported that P. aeruginosa produced dimethyl sulphide (DMS), a volatile sulphur compound (VSC), promotes A. fumigatus growth in vitro (2). The aim of this study was to investigate the role of VSCs for A. fumigatus growth and the relevance of P. aeruginosa derived VSCs in the pathogen-pathogen interaction during co-infection.

Methods
Gas chromatography mass spectrometry was used to identify the major VSCs derived from methionine catabolism that can be exploited as S-source. Production and assimilation of these VSCs was then characterised using an in vitro volatile setup. A leukopenic murine model of pulmonary aspergillosis was used to investigate the sulphur sources utilised by A. fumigatus during infection of the mammalian lung. The alternative mini-host model of Galleria mellonella allowed investigating the influence of P. aeruginosa volatiles on A. fumigatus during co infection in vivo. 

Results
Here we show that A. fumigatus produces a number of VSCs during methionine catabolism which can be exploited as sole sulphur source. These VSCs are assimilated into metabolism as H2S, as proved by the inability of a cysBΔcysDΔ strain to utilise them. Major VSCs released are DMS and DMDS, which were previously reported to be produced by P. aeruginosa and to promote A. fumigatus growth in vitro. We demonstrate that pure and Pseudomonas derived DMS and DMDS are exploited as S source by A. fumigatus and promote growth on low concentration of organic S-source.

The cysBΔcysDΔ strain cannot utilise any inorganic S-source, including DMS and DMDS, but is completely virulent in both a leukopenic model of pulmonary aspergillosis and the Galleria model. This strongly suggests that A. fumigatus exploits organic S sources during growth in the tissues. In the Galleria model, co-infection with P. aeruginosa and A. fumigatus results in increased mortality. Interestingly, co infection with wild-type A. fumigatus enhances mortality to a much higher degree than co-infection with the cysBΔcysDΔ strain. This suggests that P. aeruginosa is producing DMS in vivo and this can be exploited as an additional sulphur source by A. fumigatus wild-type, but not the mutant strain, promoting its growth and in turn increasing mortality. Further investigations are ongoing to investigate this hypothesis.

Conclusion
Aspergillus fumigatus exploits organic sulphur sources during in vivo growth. Increased mortality caused by A. fumigatus – P. aeruginosa co-infection can partially be attributed to the positive effect of P. aeruginosa derived VSCs on A. fumigatus growth due to its utilization as an additional S-source.

7 / Volatile sulphur compounds produced by Pseudomonas aeruginosa synergize with Aspergillus fumigatus in vivo enhancing the pathobiology of co-infection