Secondary metabolites produced during Aspergillus fumigatus and Pseudomonas aeruginosa biofilm formation

In Cystic Fibrosis (CF), mucus plaques are formed in the patient's lung, which creates a hypoxic condition and a propitious environment for colonization and persistence of many microorganisms. There is clinical evidence showing that Aspergillus fumigatus can co-colonize/infect CF patients with Pseudomonas aeruginosa, which has been associated with lung function decline. Several reports have showed that P. aeruginosa produces several compounds with inhibitory and anti-biofilm effects against A. fumigatus in vitro; however, little is known about the fungal compounds produced in counterattack. In this work we annotated fungal and bacterial secondary metabolites (SM) produced in mixed biofilms in normoxia and hypoxia conditions. We detected nine SMs produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion were increased by the fungal presence. The roles of the two operons responsible for phenazines production (phzA1 and phzA2) were also investigated and the results showed that phzA1 and ΔphzA2 mutants are able to produce partial sets of phenazines when in hypoxia and in the presence of the fungus. We detected a total of 20 SMs secreted by A. fumigatus either in monoculture or in co-culture with P. aeruginosa. All these compounds are secreted during biofilm formation either in normoxia or hypoxia. However, only eight compounds (demethoxyfumitremorgin C, fumitremorgin, ferrichrome, ferricrocin, tricetylfusigen, gliotoxin, gliotoxin E, and pyripyropene A) were detected detected during the biofilm formation by the co-culture of A. fumigatus and P. aeruginosa upon both normoxia and hypoxia conditions. Overall, we showed how diverse is SM secretion during A. fumigatus and P. aeruginosa mixed culture and how this can affect biofilm formation both in normoxia and hypoxia.在囊性纤维化（Cystic Fibrosis, CF）患者的肺部会形成黏液斑块，这会营造出低氧环境，并为多种微生物的定植与持续存活提供适宜的微环境。已有临床研究证据表明，烟曲霉（Aspergillus fumigatus）可与铜绿假单胞菌（Pseudomonas aeruginosa）共定植或感染CF患者，且该现象与患者肺功能下降密切相关。多项研究显示，铜绿假单胞菌在体外可分泌多种对烟曲霉具有抑制及抗生物被膜活性的化合物，但目前对于烟曲霉的反击性分泌代谢产物尚缺乏深入了解。本研究对常氧与低氧条件下混合生物被膜中产生的真菌及细菌次级代谢产物（secondary metabolites, SM）开展了注释分析。我们共检测到铜绿假单胞菌分泌的9种次级代谢产物，其中吩嗪类化合物与多种绿脓菌素（pyoverdin）类似物为其主要分泌产物，且真菌的存在可提升这些物质的分泌水平。本研究同时探究了负责吩嗪类物质合成的两个操纵子phzA1与phzA2的功能，结果显示，在低氧且存在真菌的条件下，phzA1突变株与ΔphzA2突变株仍可合成部分吩嗪类化合物。我们共检测到烟曲霉在单培养或与铜绿假单胞菌共培养体系中分泌的20种次级代谢产物，上述化合物均会在常氧或低氧条件下的生物被膜形成阶段被分泌。然而，仅8种化合物——去甲氧基烟曲霉素C（demethoxyfumitremorgin C）、烟曲霉素（fumitremorgin）、铁色素（ferrichrome）、铁载体素（ferricrocin）、tricetylfusigen、胶霉毒素（gliotoxin）、胶霉毒素E（gliotoxin E）及吡咯菌素A（pyripyropene A）——在烟曲霉与铜绿假单胞菌共培养形成的生物被膜中，于常氧及低氧条件下均被检测到。总体而言，本研究阐明了烟曲霉与铜绿假单胞菌共培养过程中次级代谢产物分泌的多样性，以及该过程如何在常氧与低氧条件下影响生物被膜的形成与发展。