Lapp Katrin, Vödisch Martin, Kroll Kristin, Strassburger Maria, Kniemeyer Olaf, Heinekamp Thorsten, Brakhage Axel A
Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany.
Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany ; Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena Germany ; Integrated Research Treatment-Center - Center for Sepsis Control and Care, University Hospital Jena, Jena Germany.
Front Microbiol. 2014 Sep 11;5:469. doi: 10.3389/fmicb.2014.00469. eCollection 2014.
Aspergillus fumigatus is a saprophytic mold that can cause life-threatening infections in immunocompromised patients. In the lung, inhaled conidia are confronted with immune effector cells that attack the fungus by various mechanisms such as phagocytosis, production of antimicrobial proteins or generation of reactive oxygen intermediates. Macrophages and neutrophils can also form nitric oxide (NO) and other reactive nitrogen intermediates (RNI) that potentially also contribute to killing of the fungus. However, fungi can produce several enzymes involved in RNI detoxification. Based on genome analysis of A. fumigatus, we identified two genes encoding flavohemoglobins, FhpA, and FhpB, which have been shown to convert NO to nitrate in other fungi, and a gene encoding S-nitrosoglutathione reductase GnoA reducing S-nitrosoglutathione to ammonium and glutathione disulphide. To elucidate the role of these enzymes in detoxification of RNI, single and double deletion mutants of FhpA, FhpB, and GnoA encoding genes were generated. The analysis of mutant strains using the NO donor DETA-NO indicated that FhpA and GnoA play the major role in defense against RNI. By generating fusions with the green fluorescence protein, we showed that both FhpA-eGFP and GnoA-eGFP were located in the cytoplasm of all A. fumigatus morphotypes, from conidia to hyphae, whereas FhpB-eGFP was localized in mitochondria. Because fhpA and gnoA mRNA was also detected in the lungs of infected mice, we investigated the role of these genes in fungal pathogenicity by using a murine infection model for invasive pulmonary aspergillosis. Remarkably, all mutant strains tested displayed wild-type pathogenicity, indicating that the ability to detoxify host-derived RNI is not essential for virulence of A. fumigatus in the applied mouse infection model. Consistently, no significant differences in killing of ΔfhpA, ΔfhpB, or ΔgnoA conidia by cells of the macrophage cell line MH-S were observed when compared to the wild type.
烟曲霉是一种腐生霉菌，可在免疫功能低下的患者中引起危及生命的感染。在肺部，吸入的分生孢子会遇到免疫效应细胞，这些细胞通过吞噬作用、抗菌蛋白的产生或活性氧中间体的生成等多种机制攻击真菌。巨噬细胞和中性粒细胞还可以形成一氧化氮（NO）和其他活性氮中间体（RNI），这也可能有助于杀死真菌。然而，真菌可以产生几种参与RNI解毒的酶。基于烟曲霉的基因组分析，我们鉴定出两个编码黄素血红蛋白的基因FhpA和FhpB，它们已被证明在其他真菌中将NO转化为硝酸盐，以及一个编码S-亚硝基谷胱甘肽还原酶GnoA的基因，该酶将S-亚硝基谷胱甘肽还原为铵和谷胱甘肽二硫化物。为了阐明这些酶在RNI解毒中的作用，我们构建了FhpA、FhpB和GnoA编码基因的单缺失和双缺失突变体。使用NO供体DETA-NO对突变菌株进行分析表明，FhpA和GnoA在抵御RNI中起主要作用。通过与绿色荧光蛋白产生融合蛋白，我们发现FhpA-eGFP和GnoA-eGFP都位于烟曲霉所有形态型的细胞质中，从分生孢子到菌丝，而FhpB-eGFP定位于线粒体中。因为在感染小鼠的肺部也检测到了fhpA和gnoA mRNA，我们通过使用侵袭性肺曲霉病的小鼠感染模型来研究这些基因在真菌致病性中的作用。值得注意的是，所有测试的突变菌株都表现出野生型致病性，这表明在应用的小鼠感染模型中，解毒宿主来源的RNI的能力对于烟曲霉的毒力不是必需的。同样，与野生型相比，巨噬细胞系MH-S的细胞对ΔfhpA、ΔfhpB或ΔgnoA分生孢子的杀伤没有显著差异。
