PDF(Pubmed)
Abstract：
Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the Mucorales order. Among the Mucor species complex, Mucor circinelloides is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance. With this new model, phenotypic analyses of gene deletion mutants were conducted to define Erg3 and Erg6a as key biosynthetic enzymes in the M. circinelloides ergosterol pathway. Erg3 is a C-5 sterol desaturase involved in growth, sporulation, virulence, and azole susceptibility. In other fungal pathogens, erg3 mutations confer azole resistance because Erg3 catalyzes the production of a toxic diol upon azole exposure. Surprisingly, M. circinelloides produces only trace amounts of this toxic diol and yet, it is still susceptible to posaconazole and isavuconazole due to alterations in membrane sterol composition. These alterations are severely aggravated by erg3Δ mutations, resulting in ergosterol depletion and, consequently, hypersusceptibility to azoles. We also identified Erg6a as the main C-24 sterol methyltransferase, whose activity may be partially rescued by the paralogs Erg6b and Erg6c. Loss of Erg6a function diverts ergosterol synthesis to the production of cholesta-type sterols, resulting in resistance to amphotericin B. Our findings suggest that mutations or epimutations causing loss of Erg6 function may arise during human infections, resulting in antifungal drug resistance to first-line treatments against mucormycosis.
OBJECTIVE: The Mucor species complex comprises a variety of opportunistic pathogens known to cause mucormycosis, a potentially lethal fungal infection with limited therapeutic options. The only effective first-line treatments against mucormycosis consist of liposomal formulations of amphotericin B and the triazoles posaconazole and isavuconazole, all of which target components within the ergosterol biosynthetic pathway. This study uncovered M. circinelloides Erg3 and Erg6a as key enzymes to produce ergosterol, a vital constituent of fungal membranes. Absence of any of those enzymes leads to decreased ergosterol and consequently, resistance to ergosterol-binding polyenes such as amphotericin B. Particularly, losing Erg6a function poses a higher threat as the ergosterol pathway is channeled into alternative sterols similar to cholesterol, which maintain membrane permeability. As a result, erg6a mutants survive within the host and disseminate the infection, indicating that Erg6a deficiency may arise during human infections and confer resistance to the most effective treatment against mucormycoses.
OBJECTIVE: The Mucor species complex comprises a variety of opportunistic pathogens known to cause mucormycosis, a potentially lethal fungal infection with limited therapeutic options. The only effective first-line treatments against mucormycosis consist of liposomal formulations of amphotericin B and the triazoles posaconazole and isavuconazole, all of which target components within the ergosterol biosynthetic pathway. This study uncovered M. circinelloides Erg3 and Erg6a as key enzymes to produce ergosterol, a vital constituent of fungal membranes. Absence of any of those enzymes leads to decreased ergosterol and consequently, resistance to ergosterol-binding polyenes such as amphotericin B. Particularly, losing Erg6a function poses a higher threat as the ergosterol pathway is channeled into alternative sterols similar to cholesterol, which maintain membrane permeability. As a result, erg6a mutants survive within the host and disseminate the infection, indicating that Erg6a deficiency may arise during human infections and confer resistance to the most effective treatment against mucormycoses.
摘要：
毛菌病是由毛菌病顺序中的多种异质物种引起的新兴真菌感染。在Mucor物种复合体中，毛霉是毛霉菌病患者中最常见的病原体，尽管其临床意义，目前还缺乏已建立的基因组操作技术来进行分子发病机制研究。在这项研究中,我们产生了一个自发的尿嘧啶营养缺陷型菌株，并开发了一个遗传转化程序来分析赋予抗真菌药物抗性的分子机制。有了这个新模型，进行了基因缺失突变体的表型分析，以将Erg3和Erg6a定义为circinelloides麦角甾醇途径中的关键生物合成酶。Erg3是参与生长的C-5固醇去饱和酶，孢子形成,毒力,和唑类易感性。在其他真菌病原体中，erg3突变赋予唑抗性，因为Erg3在唑类暴露后催化产生有毒的二醇。令人惊讶的是,circinelloides仅产生痕量的这种有毒的二醇，由于膜甾醇成分的改变，它仍然对泊沙康唑和伊沙武康唑敏感。这些改变严重加重了er3Δ突变，导致麦角甾醇耗尽，因此,对唑类药物的高度敏感。我们还确定了Erg6a是主要的C-24固醇甲基转移酶，其活性可以部分由旁系同源物Erg6b和Erg6c挽救。Erg6a功能的丧失将麦角甾醇的合成转移到胆甾型甾醇的生产上，导致对两性霉素B的耐药性。我们的研究结果表明，在人类感染期间可能会出现导致Erg6功能丧失的突变或表现突变，导致抗真菌药物对针对毛霉菌病的一线治疗产生耐药性。
目的：毛霉物种复合体包含多种已知可导致毛霉菌病的机会病原体，潜在致命的真菌感染，治疗选择有限。针对毛霉菌病的唯一有效的一线治疗包括两性霉素B和三唑泊沙康唑和伊沙康唑的脂质体制剂，所有这些都是麦角甾醇生物合成途径中的目标成分。本研究发现M.circinelloidesErg3和Erg6a是产生麦角甾醇的关键酶，真菌膜的重要组成部分。缺乏任何这些酶导致麦角固醇减少，因此，对麦角甾醇结合多烯如两性霉素B的抗性，特别是，失去Erg6a功能构成了更高的威胁，因为麦角固醇途径被引导到类似于胆固醇的替代甾醇中，保持膜的通透性。因此,erg6a突变体在宿主内存活并传播感染，表明Erg6a缺乏症可能在人类感染期间出现，并赋予对粘液菌病最有效治疗的抗性。
目的：毛霉物种复合体包含多种已知可导致毛霉菌病的机会病原体，潜在致命的真菌感染，治疗选择有限。针对毛霉菌病的唯一有效的一线治疗包括两性霉素B和三唑泊沙康唑和伊沙康唑的脂质体制剂，所有这些都是麦角甾醇生物合成途径中的目标成分。本研究发现M.circinelloidesErg3和Erg6a是产生麦角甾醇的关键酶，真菌膜的重要组成部分。缺乏任何这些酶导致麦角固醇减少，因此，对麦角甾醇结合多烯如两性霉素B的抗性，特别是，失去Erg6a功能构成了更高的威胁，因为麦角固醇途径被引导到类似于胆固醇的替代甾醇中，保持膜的通透性。因此,erg6a突变体在宿主内存活并传播感染，表明Erg6a缺乏症可能在人类感染期间出现，并赋予对粘液菌病最有效治疗的抗性。
