烟曲霉黑色素的研究进展

烟曲霉(Aspergillus fumigatus)是一种分布于世界各地的腐生真菌,属于人类临床常见的三大机会性致病真菌之一,是侵袭性曲霉菌病的主要病原菌。烟曲霉可以产生DHN-黑色素(dihydroxynaphthalene melanin)和脓黑素(pyomelanin)这2种类型黑色素。本综述介绍烟曲霉黑色素产生的遗传代谢途径、功能以及与宿主免疫系统相互作用的最新认识,有助于更好地理解烟曲霉的病理生理特征,为烟曲霉感染快速诊断技术和新型抗真菌药物的研发提供理论依据。     

Airborne Aspergillus fumigatus conidia: a risk factor for aspergillosis

Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis, a usually fatal infection. The disease has risen in prominence in recent years due to the increasing numbers of severely immunocompromised patients becoming infected. The fungus is ubiquitous in the environment, producing large numbers of conidia that are dispersed in the air. Humans inhale numerous conidia everyday, but infections are not seen in healthy individuals. As inhalation of conidia is the main route of infection, considerable efforts are required to prevent infection in susceptible patients. This review summarises the current knowledge on airborne concentrations of A. fumigatus conidia, their background levels in outdoor air and seasonal distribution patterns. New and established methods of air sampling for airborne A. fumigatus conidia are discussed. Common environmental sources of the fungus are reviewed, including its presence in compost heaps. Finally, the lack of stringent guidelines on the monitoring and control of airborne A. fumigatus concentrations in hospitals is discussed.     

Distinct Innate Immune Phagocyte Responses to Aspergillus fumigatus Conidia and Hyphae in Zebrafish Larvae

Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA.     

The hypoxia‐induced dehydrogenase HorA is required for coenzyme Q10 biosynthesis,azole sensitivity and virulence of Aspergillus fumigatus

Aspergillus fumigatus is the predominant airborne pathogenic fungus causing invasive aspergillosis in immunocompromised patients. During infection A. fumigatus has to adapt to oxygen‐limiting conditions in inflammatory or necrotic tissue. Previously, we identified a mitochondrial protein to be highly up‐regulated during hypoxic adaptation. Here, this protein was found to represent the novel oxidoreductase HorA. In Saccharomyces cerevisiae a homologue was shown to play a role in biosynthesis of coenzyme Q. Consistently, reduced coenzyme Q content in the generated ΔhorA mutant indicated a respective function in A. fumigatus. Since coenzyme Q is involved in cellular respiration and maintaining cellular redox homeostasis, the strain ΔhorA displayed an impaired response to both oxidative and reductive stress, a delay in germination and an accumulation of NADH. Moreover, an increased resistance against antifungal drugs was observed. All phenotypes were completely reversed by the addition of the synthetic electron carrier menadione. The deletion strain ΔhorA showed significantly attenuated virulence in two murine infection models of invasive pulmonary aspergillosis. Therefore, the biosynthesis of coenzyme Q and, particularly, the fungal‐specific protein HorA play a crucial role in virulence of A. fumigatus. Due to its absence in mammals, HorA might represent a novel therapeutic target against fungal infections.     

SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.     

The innate immune response to Aspergillus fumigatus

Despite the development of new treatments, the mortality due to invasive pulmonary aspergillosis remains above 50%, reaching 95% in certain situations. The battle against Aspergillus fumigatus involves several components of the pulmonary innate immune system: cells, mediators, and natural antifungal molecules involved in the recognition and elimination of the fungus, thereby preventing colonization of the respiratory system.With the 10,000–15,000 l of air we inhale each day, the lungs are constantly exposed to a wide range of microorganisms, such as A. fumigatus. This fungus is ubiquitous in the environment and can release large numbers of spores able, due to their small size, to penetrate the respiratory tract. The spores of A. fumigatus, like any other pathogen, are then confronted with the innate immune system, a constitutive defense system that is permanently active and tightly regulated. The various elements of the pulmonary innate immune system—physical and cellular barriers and soluble mediators—are involved in the recognition and elimination of pathogens, thereby preventing colonization of the respiratory system. Consequently, the presence of spores in immunocompetent hosts is completely innocuous, because these spores are normally eliminated. However, changes in one of the components of the defense system may lead to the development of pulmonary infections. Thus, in immunocompromised individuals, the spores are able to develop and cause pulmonary mycoses. These mycoses, known as aspergillosis, are highly variable, with the range of presentations extending from an allergy-type illness, allergic bronchopulmonary aspergilloses, to a very serious generalized and frequently fatal infection: invasive pulmonary aspergillosis (IPA).     

High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence

Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo‐dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and β‐1,3‐glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus.     

Aspergillus fumigatus MedA governs adherence,host cell interactions and virulence

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro‐inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.     

Exploring the Phospholipid Biosynthetic Pathways of Aspergillus fumigatus by Computational Genome Analysis

Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systematic diseases (invasive aspergillosis) with high mortality rates. In recent years, considerable progress in the genome sequencing of this fungus has been made by an international consortium, which includes the Wellcome Trust Sanger Institute (UK) and the Institute for Genome Research (USA). A tenfold whole genome shotgun sequence assembly of A. fumigatus has been made publicly available. In this study, it was attempted to identify the genes related to the phospholipid biosynthesis from the A. fumigatus genome by a gene prediction program (GlimmerM) and to reconstruct the metabolic pathway for phospholipids of A. fumigatus. Fifteen genes related to phospholipid pathway were identified in the A. fumigatus genomic sequence. The open reading frames predicted by GlimmerM showed a high amino acid sequence similarity with the other fungal phospholipid biosynthetic genes and well‐conserved functional domains. The obtained results also demonstrated that the reconstructed pathway of A. fumigatus in phospholipid biosynthesis was very similar to that of other fungi such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, and Neurospora crassa. Therefore it is postulated that the antifungal drugs targeted for the biosynthesis of phospholipids could also be effective against A. fumigatus.     

Deletion of the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> lysine biosynthesis gene <Emphasis Type="Italic">lysF</Emphasis> encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and -aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5×10³ conidia per mouse. In this murine model, the lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.     

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

The ZrfC alkaline zinc transporter is required for Aspergillus fumigatus virulence and its growth in the presence of the Zn/Mn‐chelating protein calprotectin

Aspergillus fumigatus can invade the lungs of immunocompromised individuals causing a life‐threatening disease called invasive pulmonary aspergillosis (IPA). To grow in the lungs, A. fumigatus obtains from the host all nutrients, including zinc. In living tissues, however, most zinc is tightly bound to zinc‐binding proteins. Moreover, during infection the bioavailability of zinc can be further decreased by calprotectin, an antimicrobial Zn/Mn‐chelating protein that is released by neutrophils in abscesses. Nevertheless, A. fumigatus manages to uptake zinc from and grow within the lungs of susceptible individuals. Thus, in this study we investigated the role of the zrfA, zrfB and zrfC genes, encoding plasma membrane zinc transporters, in A. fumigatus virulence. We showed that zrfC is essential for virulence in the absence of zrfA and zrfB, which contribute to fungal pathogenesis to a lesser extent than zrfC and are dispensable for virulence in the presence of zrfC. The special ability of ZrfC to scavenge and uptake zinc efficiently from lungtissue depended on its N‐terminus, which is absent in the ZrfA and ZrfB transporters. In addition, under Zn‐ and/or Mn‐limiting conditions zrfC enables A. fumigatus to grow in the presence of calprotectin, which is detected in fungal abscesses of non‐leucopenic animals. This study extends our knowledge about the pathobiology of A. fumigatus and suggests that fungal zinc uptake could be a promising target for new antifungals.     

MybA,a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA‐sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane‐associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.     

In vivo bioluminescence imaging and histopathopathologic analysis reveal distinct roles for resident and recruited immune effector cells in defense against invasive aspergillosis

Background  

Invasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients. Studies on the pathogenesis of IA have been limited by the difficulty to monitor disease progression in real-time. For real-time monitoring of the infection, we recently engineered a bioluminescent A. fumigatus strain.     

Discovery of a Sexual Cycle in Aspergillus lentulus,a Close Relative of A. fumigatus

Aspergillus lentulus was described in 2005 as a new species within the A. fumigatus sensu lato complex. It is an opportunistic human pathogen causing invasive aspergillosis with high mortality rates, and it has been isolated from clinical and environmental sources. The species is morphologically nearly identical to A. fumigatus sensu stricto, and this similarity has resulted in their frequent misidentification. Comparative studies show that A. lentulus has some distinguishing growth features and decreased in vitro susceptibility to several antifungal agents, including amphotericin B and caspofungin. Similar to the once-presumed-asexual A. fumigatus, it has only been known to reproduce mitotically. However, we now show that A. lentulus has a heterothallic sexual breeding system. A PCR-based mating-type diagnostic detected isolates of either the MAT1-1 or MAT1-2 genotype, and examination of 26 worldwide clinical and environmental isolates revealed similar ratios of the two mating types (38% versus 62%, respectively). MAT1-1 and MAT1-2 idiomorph regions were analyzed, revealing the presence of characteristic alpha and high-mobility-group (HMG) domain genes, together with other more unusual features such as a MAT1-2-4 gene. We then demonstrated that A. lentulus possesses a functional sexual cycle with mature cleistothecia, containing heat-resistant ascospores, being produced after 3 weeks of incubation. Recombination was confirmed using molecular markers. However, isolates of A. lentulus failed to cross with highly fertile strains of A. fumigatus, demonstrating reproductive isolation between these sibling species. The discovery of the A. lentulus sexual stage has significant implications for the management of drug resistance and control of invasive aspergillosis associated with this emerging fungal pathogen.