- 作者列表："Valero C","Colabardini AC","Chiaratto J","Pardeshi L","de Castro PA","Ferreira Filho JA","Silva LP","Rocha MC","Malavazi I","Costa JH","Fill T","Barros MH","Wong SSW","Aimanianda V","Wong KH","Goldman GH
:Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance.IMPORTANCEAspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.
烟曲霉菌是引起肺部真菌病的主要原因。唑类药物作为治疗和预防侵袭性曲霉菌病的主要抗真菌药物已使用多年。然而，在过去的 10 年里，有几篇关于烟曲霉唑类药物耐药的报道，需要新的策略来对抗侵袭性曲霉菌病。卡泊芬净对其他人类致病真菌有效，但仅对 A 有效。烟曲霉对卡泊芬净的抗性 A。烟曲霉与编码药物 β-1,3-葡聚糖合成酶靶酶的 fksA 基因突变有关。然而，高卡泊芬净浓度的耐受性，这种现象被称为卡泊芬净矛盾效应 (CPE)，对随后的适应和耐药性进化也很重要。在此，我们通过筛选 CPE 药物浓度下 484 个无效转录因子 (TFs) 的烟曲霉文库，鉴定并表征了参与 CPE 应答的转录因子。我们确定了 11 个 TFs，这些 TFs 减少了 CPE，编码的蛋白参与了 RNA 聚合酶 II 起始位点的基础调节、钙代谢和细胞壁重塑。这些 TFs 之一，FhdA，对线粒体呼吸功能和铁代谢很重要。Δ fhda 突变体在暴露于刚果红或高温时表现出生长下降。转录组测序 (RNA-seq) 分析和进一步的实验验证表明，Δ fhda 突变体表现出呼吸能力减弱，可能影响了与卡泊芬净耐受性和耐药性相关的多个通路。我们的结果为理解对卡泊芬净耐受和耐药重要的信号通路提供了基础。烟曲霉是人类最重要的致病性真菌之一，可引起曲霉病，是一组具有广泛临床表现的异质性疾病。侵袭性肺曲霉病是患者预后和治疗中最严重的病理，主要影响免疫功能低下患者的死亡率为 50% ~ 95%。唑类药物作为治疗和预防侵袭性曲霉菌病的主要抗真菌药物已使用多年。然而，在过去十年中，有几篇关于临床唑类药物耐药性演变的报道。卡泊芬净是一种非竞争性 β-1,3-葡聚糖合成酶抑制剂，已用于抗烟曲霉，但具有抑菌作用，被推荐为侵袭性曲霉菌病的二线治疗药物。更多关于卡泊芬净耐受性和耐药性的信息是必要的，以便完善针对真菌细胞壁的抗真菌策略。在此，我们为能介导卡泊芬净耐受和抗性的 TFs 筛选了一个转录因子 (TF) 缺失文库。我们已经确定了 11 个对卡泊芬净敏感性和/或卡泊芬净矛盾效应 (CPE) 很重要的 TFs。这些 TFs 编码的蛋白参与 RNA 聚合酶 II 起始位点的基础调节、钙代谢或细胞壁重塑以及线粒体呼吸功能。这项工作中确定的那些由 TFs 调控的基因的研究将提供对卡泊芬净耐受和抗性重要的信号通路的更好理解。
METHODS:BACKGROUND AND PURPOSE:A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD). EXPERIMENTAL APPROACH:C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed. KEY RESULTS:Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K2 , and S1P receptors (S1P2 and S1P3 ) has been detected in the lung of smoking mice. Sphingosine kinases inhibition reversed the increased cholinergic response in airways of smoking mice. CONCLUSIONS AND IMPLICATIONS:S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.
METHODS::The interim results from this 90-day multi-dose, inhalation toxicology study with life-time post-exposure observation has shown an important fundamental difference in persistence and pathological response in the lung between brake dust derived from brake-pads manufactured with chrysotile, TiO2 or chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos. In the brake dust exposure groups no significant pathological response was observed at any time. Slight macrophage accumulation of particles was noted. Wagner-scores, were from 1 to 2 (1 = air-control group) and were similar to the TiO2 group. Chrysotile being biodegradable, shows a weakening of its matrix and breaking into short fibers & particles that can be cleared by alveolar macrophages and continued dissolution. In the chrysotile exposure groups, particle laden macrophage accumulation was noted leading to a slight interstitial inflammatory response (Wagner-score 1-3). There was no peribronchiolar inflammation and occasional very slight interstitial fibrosis. The histopathology and the confocal analyses clearly differentiate the pathological response from amphibole asbestos, crocidolite and amosite, compared to that from the brake dust and chrysotile. Both crocidolite and amosite induced persistent inflammation, microgranulomas, and fibrosis (Wagner-scores 4), which persisted through the post exposure period. The confocal microscopy of the lung and snap-frozen chestwalls quantified the extensive inflammatory response and collagen development in the lung and on the visceral and parietal surfaces. The interim results reported here, provide a clear basis for differentiating the effects from brake dust exposure from those following amphibole asbestos exposure. The subsequent results through life-time post-exposure will follow.
METHODS::The respiratory tract is lined by a pseudo-stratified epithelium from the nose to terminal bronchioles. This first line of defense of the lung against external stress includes five main cell types: basal, suprabasal, club, goblet and multiciliated cells, as well as rare cells such as ionocytes, neuroendocrine and tuft/brush cells. At homeostasis, this epithelium self-renews at low rate but is able of fast regeneration upon damage. Airway epithelial cell lineages during regeneration have been investigated in the mouse by genetic labeling, mainly after injuring the epithelium with noxious agents. From these approaches, basal cells have been identified as progenitors of club, goblet and multiciliated cells, but also of ionocytes and neuroendocrine cells. Single-cell RNA sequencing, coupled to lineage inference algorithms, has independently allowed the establishment of comprehensive pictures of cell lineage relationships in both mouse and human. In line with genetic tracing experiments in mouse trachea, studies using single-cell RNA sequencing (RNAseq) have shown that basal cells first differentiate into club cells, which in turn mature into goblet cells or differentiate into multiciliated cells. In the human airway epithelium, single-cell RNAseq has identified novel intermediate populations such as deuterosomal cells, 'hybrid' mucous-multiciliated cells and progenitors of rare cells. Novel differentiation dynamics, such as a transition from goblet to multiciliated cells have also been discovered. The future of cell lineage relationships in the respiratory tract now resides in the combination of genetic labeling approaches with single-cell RNAseq to establish, in a definitive manner, the hallmarks of cellular lineages in normal and pathological situations.