Toll样受体对病原真菌的天然免疫识别

天然免疫系统是宿主抵御病原入侵的第一道防线,在机体抗感染免疫中发挥重要作用。Toll样受体(Toll-like receptors,TLRs)是天然免疫系统最重要的模式识别受体(pattern recognitionreceptors,PRRs)之一,通过识别病原真菌的病原相关分子模式(pathogen-associated molecularpatterns,PAMPs),招募特异接头蛋白,激活一系列信号级联反应,引发炎症因子、趋化因子等的释放和树突状细胞(dendritic cells,DCs)的成熟,发挥抗真菌感染作用。通过简要介绍宿主的TLRs及信号通路的研究进展,总结了目前TLRs对不同病原真菌PAMPs的天然免疫识别及信号通路研究现状,以期对进一步研究宿主天然免疫系统与病原真菌相互作用的分子机制提供参考。     

树突状细胞在抗真菌感染免疫中的作用

近年来,随着广谱抗生素、抗肿瘤药物和免疫抑制剂等药物的广泛使用,免疫功能降低患者数量的增加,侵袭性真菌感染性疾病的发病率逐年升高。树突状细胞(Dendritic Cells,DCs)是已知功能最强的专职抗原提呈细胞,作为宿主固有免疫和适应性免疫的联系枢纽,DCs在病原微生物抗原的识别与呈递过程中发挥核心作用。研究证明,DCs可通过其细胞表面的多种受体有效识别病原真菌的抗原,并在诱导宿主免疫应答过程中发挥重要作用。本文将对树突状细胞分类及其在抗真菌感染免疫中的识别作用进行系统叙述。     

真菌Hsp90研究进展

近年来,随着广谱抗生素、化疗以及器官移植技术的广泛应用,真菌感染日益严重,从分子水平揭示病原真菌的致病机制对真菌感染的防控、治疗至关重要。微生物适应宿主微环境压力的能力在其共生与感染过程中发挥着关键作用,heat shock protein 90（Hsp90）是真核生物参与压力应答响应的分子伴侣,它不仅参与胞内蛋白质的折叠,还与许多底物蛋白相互作用共同调节病原真菌的形态发育、生物被膜形成、有性生殖、毒力以及耐药性。本文从真菌Hsp90的活性调节、底物蛋白,以及Hsp90与病原真菌形态发生、有性生殖、耐药性调控等方面综述了近年来真菌Hsp90信号通路的研究进展。     

白色念珠菌生物被膜研究进展

难治性真菌感染的临床分析发现,病灶感染病原常以生物被膜的形态存在。生物被膜的形成可帮助真菌躲避宿主细胞免疫系统清除和药物的攻击,所造成的持续性感染严重威胁人类健康,因此,认识研究真菌生物被膜及其耐药机理对于防治临床真菌感染有着重大意义。白色念珠菌是一种临床感染常见的条件性致病菌,也是目前真菌生物被膜研究的主要研究模型。白色念珠菌生物被膜主要由多糖、蛋白质和DNA构成,其形成由微生物间的群体感应调控,并受到环境中营养成分及其附着物表面性质影响。研究发现,胞外基质的屏障作用、耐药基因的表达等机制与生物被膜耐药性的产生密切相关。本文就白色念珠菌生物被膜的形成过程、结构组成、形成的影响因素、现有研究模型、耐药机制和治疗策略等几个方面介绍近年来的研究进展。     

环境因子对一种病原真菌(Amoeboaphelidium sp.)感染微藻细胞能力的影响

油球藻(Graesiella sp.WBG-1)是一株适合于开放池规模化培养的产油微藻,在室外规模培养过程中常因一种病原真菌(Amoeboaphelidium sp.)的污染严重影响其生长和油脂积累,甚至导致培养失败。本文利用MTT染色法借助光学显微镜对被病原真菌感染的油球藻细胞计数并统计感染率,设置不同温度、光照强度、p H以及通气量等培养条件,研究环境因子对病原真菌感染能力的影响。结果表明:MTT染色法简单易行,可用于油球藻规模培养中病原真菌的检测;温度、光照强度、通气量和p H值均能够显著影响病原真菌的感染能力,高温、高光照强度、弱酸性环境和藻液静止等培养条件不利于病原真菌对油球藻的感染;在温度30℃、光强140μmol·m~(-2)·s~(-1)、通气量1.0 L·min~(-1)和p H 9.0±0.5的培养条件下成功建立了病原真菌感染油球藻的连续传代培养。本研究在实验室内模拟油球藻规模培养中被病原真菌感染的全过程,为深入研究该病原真菌对微藻细胞的感染行为及其感染机制提供了平台。     

真菌黑色素及其与巨噬细胞免疫研究进展

真菌黑色素在真菌抵抗外界理化损伤及宿主的免疫杀伤过程中起重要作用,是真菌重要的毒力因子。巨噬细胞是抗真菌固有免疫的一线细胞,探讨真菌黑色素与巨噬细胞的相互作用,可为阐明产黑色素真菌感染的发病机制奠定基础。本文重点从黑色素的生物学特性、真菌黑色素对巨噬细胞免疫的影响两个方面进行阐述。     

烟曲霉细胞壁的组分、组装及其功能概述

环境中普遍存在的腐生丝状真菌烟曲霉Aspergillus fumigatus在免疫功能低下或缺陷的人群中可引起多种急慢性疾病,包括致死率很高的侵袭性曲霉病。细胞壁作为真菌的细胞外骨架结构不仅起维持细胞形状、保护细胞抵抗外界压力等作用,在病原真菌极性生长、入侵新的生态域、启动宿主免疫反应中也起重要作用。细胞壁组分还是真菌感染的分子诊断基础和开发抗真菌药物的理想靶标。近几十年来烟曲霉细胞壁的遗传、生物化学及免疫学方向的研究使其成为研究真菌细胞壁的模式真菌。本文主要概述烟曲霉细胞壁的组分、分子组装机制及其在真菌生存和感染中的作用,并对未来研究方向提出了展望。     

分泌型蛋白介导白念珠菌与宿主相互作用分子机制的研究进展

白念珠菌是人类最常见的条件性致病真菌之一,主要定植于人体粘膜表面。在白念珠菌与宿主相互作用过程中,分泌型蛋白起着非常重要的作用。针对分泌蛋白功能及其作用机理的研究有助于阐明白念珠菌致病分子机制,并为诊断、预防和治疗真菌感染提供新的理论策略。本文综述了白念珠菌分泌型蛋白在介导病原与宿主相互作用分子机制方面的最新研究进展,概括了分泌蛋白在组织侵入损伤、营养获取、细胞壁维持以及免疫逃避等方面的功能,同时对未来值得重点关注的研究方向进行了探讨。     

白念珠菌生物被膜形成的遗传调控机制

白念珠菌是人体重要的条件性致病真菌。形态的多样性和可塑性是白念珠菌典型的生物学特征,这与它的致病性、宿主适应能力以及有性生殖过程密切相关。白念珠菌生物被膜(Biofilm)是由不同形态细胞(包括酵母型、菌丝和假菌丝)以及胞外基质组成的致密结构,也是毒性和耐药性形成的重要因子。生物被膜对抗真菌药物、宿主免疫系统和环境胁迫因子等都表现出较强的抵抗力和耐受性,是临床上病原真菌感染防治的重大挑战。随着基因表达谱和遗传操作技术的发展,白念珠菌生物被膜的形成及其耐药性的获得所依赖的遗传调控通路和分子调控机制越来越清楚。主要包括MAPK和cAMP介导的信号途径以及Bcr1和Tec1等因子介导的转录调控。此外,白念珠菌生物被膜的形成与形态转换和有性生殖之间存在密切的联系。文中综述了白念珠菌生物被膜形成的遗传调控机制,重点介绍了细胞壁相关蛋白、转录因子和交配型对该过程的调控以及生物被膜的耐药机制。     

病原微生物对抗菌肽抗性机制的研究进展

抗菌肽(antimicrobial peptides, AMPs)是生物先天免疫系统的重要组成部分,可帮助宿主有效应对病原细菌、真菌和病毒等微生物的胁迫,被认为是医疗、食品加工和农业领域最具前途和潜力的抗生素替代物。病原微生物在与抗菌肽的互作中进化出了多种有针对性的抗性机制,本文从病原微生物对AMPs的感应与基因调控、细胞壁/膜成份的修饰、分泌蛋白酶降解及利用外排泵排出等四个方面综述了国内外的研究进展,并对AMPs类制品的研究前景进行了讨论与展望。     

Extracellular proteases of Trichoderma species. A review

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.     

Evolution in the structure and function of aspartic proteases

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.     

Fungal Proteases and Their Pathophysiological Effects

Proteolytic enzymes play an important role in fungal physiology and development. External digestion of protein substrates by secreted proteases is required for survival and growth of both saprophytic and pathogenic species. Extracellular serine, aspartic, and metalloproteases are considered virulence factors of many pathogenic species. New findings focus on novel membrane-associated proteases such as yapsins and ADAMs and their role in pathology. Proteases from fungi induce inflammatory responses by altering the permeability of epithelial barrier and by induction of proinflammatory cytokines through protease-activated receptors. Many fungal allergens possess proteolytic activity that appears to be essential in eliciting Th2 responses. Allergenic fungal proteases can act as adjuvants, potentiating responses to other allergens. Proteolytic enzymes from fungi contribute to inflammation through interactions with the kinin system as well as the coagulation and fibrinolytic cascades. Their effect on the host protease–antiprotease balance results from activation of endogenous proteases and degradation of protease inhibitors. Recent studies of the role of fungi in human health point to the growing importance of proteases not only as pathogenic agents in fungal infections but also in asthma, allergy, and damp building related illnesses. Proteolytic enzymes from fungi are widely used in biotechnology, mainly in food, leather, and detergent industries, in ecological bioremediation processes and to produce therapeutic peptides. The involvement of fungal proteases in diverse pathological mechanisms makes them potential targets of therapeutic intervention and candidates for biomarkers of disease and exposure.     

Independent subtilases expansions in fungi associated with animals

Many socially important fungi encode an elevated number of subtilisin-like serine proteases, which have been shown to be involved in fungal mutualisms with grasses and in parasitism of insects, nematodes, plants, other fungi, and mammalian skin. These proteins have endopeptidase activities and constitute a significant part of fungal secretomes. Here, we use comparative genomics to investigate the relationship between the quality and quantity of serine proteases and the ability of fungi to cause disease in invertebrate and vertebrate animals. Our screen of previously unexamined fungi allowed us to annotate and identify nearly 1000 subtilisin-containing proteins and to describe six new categories of serine proteases. Architectures of predicted proteases reveal novel combinations of subtilisin domains with other, co-occurring domains. Phylogenetic analysis of the most common clade of fungal proteases, proteinase K, showed that gene family size changed independently in fungi, pathogenic to invertebrates (Hypocreales) and vertebrates (Onygenales). Interestingly, simultaneous expansions in the S8 and S53 families of subtilases in a single fungal species are rare. Our analysis finds that closely related systemic human pathogens may not show the same gene family expansions, and that related pathogens and nonpathogens may show the same type of gene family expansion. Therefore, the number of proteases does not appear to relate to pathogenicity. Instead, we hypothesize that the number of fungal serine proteases in a species is related to the use of the animal as a food source, whether it is dead or alive.     

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.     

The autolysis of industrial filamentous fungi

Fungal autolysis is the natural process of self-digestion of aged hyphal cultures, occurring as a result of hydrolase activity, causing vacuolation and disruption of organelle and cell wall structure. Previously, authors have considered individual aspects of fungal lysis, in terms of either an enzyme, a process or an organism. This review considers both the physiology and morphology of fungal autolysis, with an emphasis on correlations between enzymological profiles and the morphological changes occurring during culture degeneration. The involvement of the main groups of autolytic hydrolases is examined (i.e., proteases, glucanases, and chitinases), in addition to the effects of autolysis on the morphology and products of industrial bioprocesses. We call for a concerted approach to the study of autolysis, as this will be fundamental for research to progress in this field. Increased understanding will allow for greater control of the prevention, or induction of fungal autolysis. Such advances will be applicable in the development of antifungal medicines and enable increased productivity and yields in industrial bioprocesses. Using paradigms in existing model systems, including mammalian cell death and aging in yeast, areas for future study are suggested in order to advance the study of fungal cell death.     

食线虫菌物胞外蛋白酶基因工程研究进展

在病原线虫的生物防治中 ,利用食线虫菌物在侵染过程中分泌的胞外蛋白酶 (重要毒力因子 )固定线虫并降解线虫体壁显示出巨大的潜力。综述了近年来食线虫菌物胞外蛋白酶的研究概况、目前开展蛋白酶基因工程研究存在的问题及解决策略;对真菌胞外蛋白酶应用于线虫生防和生物医药领域中的前景也进行了评述。     

Proteases Released by Entomopathogenic Fungi Impair Phagocytic Activity, Attachment and Spreading of Plasmatocytes Isolated from Haemolymph of the Greater Wax Moth Galleria mellonella

Reliable assays for the in vitro quantification of the attachment and spreading of isolated Galleria mellonella plasmatocytes have been established. The effects of extracellular proteases released by the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana on the phagocytic activity, spreading, attachment and cytoskeleton formation of isolated plasmatocytes of G. mellonella were examined to elucidate their putative role in the suppression of cellular immune responses in infected insects. In addition, the influence of different commercially available proteases on isolated plasmatocytes was studied. Among the proteases tested, the metalloprotease thermolysin produced the strongest inhibitory activity on plasmatocytes. The results obtained support the conclusion that invading fungal cells could interfere with the insect immune system via the release of proteases which affect cellular defence reactions. Isolated G. mellonella plasmatocytes incubated with fungal proteases had an impaired ability to ingest yeast cells and exhibited alterations in morphology and cytoskeleton formation. The effects were similar to those observed in plasmatocytes from infected larvae. The role of extracellular fungal proteases in the interactions of entomopathogens with the insect immune system is discussed.     

Pathogenesis of Dermatophytosis

Despite the superficial localization of most dermatophytosis, host-fungus relationship in these infections is complex and still poorly elucidated. Though many efforts have been accomplished to characterize secreted dermatophytic proteases at the molecular level, only punctual insights have been afforded into other aspects of the pathogenesis of dermatophytosis, such as fungal adhesion, regulation of gene expression during the infection process, and immunomodulation by fungal factors. However, new genetic tools were recently developed, allowing a more rapid and high-throughput functional investigation of dermatophyte genes and the identification of new putative virulence factors. In addition, sophisticated in vitro infection models are now used and will open the way to a more comprehensive view of the interactions between these fungi and host epidermal cells, especially keratinocytes.