The influence of Aspergillus fumigatus fatty acid oxygenases PpoA and PpoC on caspofungin susceptibility

Aspergillus fumigatus can cause Invasive Pulmonary Aspergillosis (IPA). Fungicidal azoles and fungistatic caspofungin (CAS) are the first- and second-line therapies, respectively, used to treat IPA. Treatment of A. fumigatus with CAS or micafungin induces production of the oxylipin 5,8-diHODE by the fungal oxygenase PpoA. Here we investigated the influence of ppo genes, that encode fatty acid oxygenases responsible for oxylipin biosynthesis, on CAS tolerance. PpoA and PpoC influence on CAS tolerance is mediated by MpkA phosphorylation and protein kinase A (PKA) activity. RNAseq transcriptional profiling and label-free quantitative proteomics of the ppoA and ppoC mutants showed that differentially expressed genes and proteins are related to secondary metabolites and carbohydrate metabolism. We also characterized two clinical isolates, CM7555 and IFM61407, that have decreased and increased susceptibility to CAS, respectively. CM7555 does not have increased oxylipin production in the presence of CAS while the oxylipin induction upon CAS exposure is increased in the IFM61407, suggesting oxylipins are not the single mechanism involved in CAS tolerance in these isolates. Upon CAS exposure, CM7555 has higher MpkA phosphorylation and PKA activity than IFM61407. Our results reveal different aspects and genetic determinants involved in A. fumigatus CAS tolerance.

烟曲霉（Aspergillus fumigatus）可引发侵袭性肺曲霉病（Invasive Pulmonary Aspergillosis, IPA）。杀真菌性唑类药物与抑真菌性卡泊芬净（caspofungin, CAS）分别为治疗IPA的一线与二线疗法。采用CAS或米卡芬净（micafungin）处理烟曲霉时，真菌加氧酶PpoA会诱导产生氧脂类物质5,8-二羟基十八碳二烯酸（5,8-diHODE）。本研究探究了编码负责氧脂类生物合成的脂肪酸加氧酶的ppo基因对CAS耐受性的影响。PpoA与PpoC对CAS耐受性的调控作用，经由MpkA磷酸化及蛋白激酶A（PKA）活性介导。对ppoA及ppoC突变体开展RNA测序（RNAseq）转录组分析与无标记定量蛋白质组学分析后发现，差异表达基因与差异表达蛋白均与次生代谢产物及碳水化合物代谢相关。本研究还表征了两株临床分离株CM7555与IFM61407：前者对CAS的敏感性降低，后者则升高。当暴露于CAS环境时，CM7555的氧脂类生成量未出现上调，而IFM61407的CAS诱导氧脂类生成量则有所提升，这表明氧脂类并非这两株分离株CAS耐受性的唯一相关机制。在CAS暴露条件下，CM7555的MpkA磷酸化水平及PKA活性均高于IFM61407。本研究结果揭示了烟曲霉CAS耐受性涉及的多个层面及相关遗传决定因素。