Bidirectional gene transfer between Aspergillus fumigatus and Aspergillus nidulans

Abstract The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes β-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased β-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli .     

烟曲霉感染时内在化Dectin-1介导巨噬细胞自噬活化

目的探究在烟曲霉感染时Dectin-1是否内在化表达并介导了巨噬细胞的自噬活化,初步明确其作用机制。方法采用Western blot法和免疫荧光技术,观察经β-1,3-D-葡聚糖酶消化前后的烟曲霉孢子刺激下,RAW264.7细胞内Dectin-1与LC3Ⅱ的表达与定位,通过DCFH-DA探针检测消化β-葡聚糖对活性氧(ROS)生成的影响。结果烟曲霉孢子刺激后RAW264.7细胞的Dectin-1与LC3Ⅱ表达水平显著升高,同时二者呈斑点状聚集并共定位于烟曲霉孢子表面;消化β-葡聚糖后Dectin-1与LC3Ⅱ表达量降低,荧光斑点消失,并且ROS的生成受到抑制。结论烟曲霉感染时Dectin-1提高自身内在化表达并诱导了巨噬细胞自噬功能的活化。     

The interplay between iron and zinc metabolism in Aspergillus fumigatus

Zinc plays a critical role in a diverse array of biochemical processes. However, excess of zinc is deleterious to cells. Therefore, cells require finely tuned homeostatic mechanisms to balance uptake and storage of zinc. Here we show that iron starvation affects zinc metabolism by downregulating expression of the plasma membrane zinc importer encoding zrfB and upregulating the putative vacuolar zinc transporter-encoding zrcA in Aspergillus fumigatus. Nevertheless, the zinc content of iron-starved mycelia exceeded that of iron replete mycelia, possibly due to unspecific metal uptake induced by iron starvation. In agreement with increased zinc excess and zinc toxicity during iron starvation, deficiency in siderophore-mediated high-affinity iron uptake caused hypersensitivity to zinc. Moreover, an increase of zinc uptake by conditional overexpression of zrfB was more toxic under iron depleted compared to iron replete conditions. This deregulated zinc uptake under iron starvation caused a decrease in heme production and an increase in protoporphyrin IX accumulation. Furthermore, zinc excess impaired production of the extracellular siderophore triacetylfusarinine C but not the intracellular siderophore ferricrocin. Taken together, these data demonstrate a fine tuned coordination of zinc and iron metabolism in A. fumigatus.     

Glycosylinositolphosphoceramides in Aspergillus fumigatus

Fungal glycosylinositolphosphoceramides (GIPCs) are involved in cell growth and fungal-host interactions. In this study, six GIPCs from the mycelium of the human pathogen Aspergillus fumigatus were purified and characterized using Q-TOF mass spectrometry and 1H, 13C, and 31P NMR. All structures have the same inositolphosphoceramide moiety with the presence of a C(18:0)-phytosphingosine conjugated to a 2-hydroxylated saturated fatty acid (2-hydroxy-lignoceric acid). The carbohydrate moiety defines two types of GIPC. The first, a mannosylated zwitterionic glycosphingolipid contains a glucosamine residue linked in alpha1-2 to an inositol ring that has been described in only two other fungal pathogens. The second type of GIPC presents an alpha-Manp-(1-->3)-alpha-Manp-(1-->2)-IPC common core. A galactofuranose residue is found in four GIPC structures, mainly at the terminal position via a beta1-2 linkage. Interestingly, this galactofuranose residue could be substituted by a choline-phosphate group, as observed only in the GIPC of Acremonium sp., a plant pathogen.     

青蒿琥酯治疗肺烟曲霉感染自噬机制

目的探究青蒿琥酯在鼠肺感染烟曲霉后,对鼠肺自噬相关蛋白的表达影响。方法用一定量的烟曲霉的活化孢子感染鼠肺,建立肺烟曲霉病模型。同时用青蒿琥酯注射一组鼠肺,两性霉素B注射一组鼠肺,剩余的作为感染组。24h后,取各组鼠肺进行病理切片染色,观察各组病理情况;取各组鼠肺的组织并匀浆,收集各组的孢子,用真菌活性检测试剂盒检测孢子活性并将肺巨噬细胞进行分离、裂解,离心取上清,用Western-blot法检测上清液中的Dectin-1、ROS、LC3Ⅱ的表达水平。结果青蒿琥酯注射组的鼠肺病理切片中的孢子比感染组数量少且聚集在一起,未长出菌丝,而体外分离的真菌孢子,青蒿琥酯组的活性明显受到抑制。经Western-blot显示青蒿琥酯具有增强肺巨噬细胞Dectin-1、ROS、LC3Ⅱ的表达。结论青蒿琥酯可通过抑制烟曲霉的活性和增强鼠肺巨噬细胞的自噬水平,对抗鼠肺烟曲霉感染。     

不同形态的烟曲霉对巨噬细胞自噬水平的影响

目的探究烟曲霉静息孢子、膨胀孢子以及菌丝对巨噬细胞自噬水平的影响。方法培养烟曲霉并收获静息孢子,在沙保弱液体培养基中振荡不同时间获得膨胀孢子及菌丝。以3种形态的烟曲霉分组处理RAW264.7细胞,免疫印迹法检测LC3BⅡ蛋白的表达量,逆转录PCR检测自噬相关蛋白Atg5、Atg7、Atg12 mRNA的转录水平。观察不同形态的烟曲霉刺激GFP-LC3B-RAW264.7细胞后GFP-LC3B的表达与定位。结果膨胀的烟曲霉孢子及菌丝刺激巨噬细胞后LC3BⅡ表达水平升高;Atg5与Atg12 mRNA的转录均明显增高,Atg7转录水平无显著变化;膨胀孢子诱导LC3B呈斑点样聚集并与之共定位,烟曲霉菌丝刺激后自噬体增多。结论烟曲霉膨胀孢子与菌丝能显著提高巨噬细胞自噬水平,静息的分生孢子不能引起巨噬细胞自噬功能的应答。     

A molecular link between autophagy and circadian rhythm in plants

Extremely high or low autophagy levels disrupt plant survival under nutrient starvation. Recently, autophagy has been reported to display rhythms in animals. However, the mechanism of circadian regulation of autophagy is still unclear. Here, we observed that autophagy has a robust rhythm and that various autophagy-related genes (ATGs) are rhythmically expressed in Arabidopsis. Chromatin immunoprecipitation (ChIP) and dual-luciferase (LUC) analyses showed that the core oscillator gene TIMING OF CAB EXPRESSION 1 (TOC1) directly binds to the promoters of ATG (ATG1a, ATG2, and ATG8d) and negatively regulates autophagy activities under nutritional stress. Furthermore, autophagy defects might affect endogenous rhythms by reducing the rhythm amplitude of TOC1 and shortening the rhythm period of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Autophagy is essential for the circadian clock pattern in seedling development and plant sensitivity to nutritional deficiencies. Taken together, our studies reveal a plant strategy in which the TOC1-ATG axis involved in autophagy-rhythm crosstalk to fine-tune the intensity of autophagy.     

Molecular genetics in Aspergillus fumigatus

Manipulation of the genome of the human pathogen Aspergillus fumigatus is not well developed. Approaches and data from related model organisms are being used to develop molecular genetic systems in A. fumigatus; for example, the molecular typing of strains during infection. A genome-sequencing programme has begun and will form the basis for future development.     

A novel link between autophagy and the ubiquitin-proteasome system

Autophagy and the ubiquitin-proteasome system (UPS) are the major routes for intracellular protein degradation. These two pathways were previously thought to be largely distinct. Here we summarize our recent work that demonstrates that long-term autophagy inhibition slows the clearance of short-lived UPS-specific substrates, like p53. This is caused by the accumulation of p62 after autophagy inhibition. These data suggest that the ramifications of a block in autophagy may be much wider than what was previously thought. Rather than simply decreasing clearance of autophagic substrates, while UPS flux is undisturbed, the cell will have to contend with a decrease in clearance by both major routes.     

Unexpected link between insect innexins and apoptosis of HeLa cells

Little is known about how mammalian cells respond to the expression of innexins (Inxs), which are known to mediate cell‐to‐cell communication that causes apoptosis in the cells of the insect Spodoptera litura. The mammalian expression system, p3xFLAG tag protein, containing the CMV promoter, allowed us to construct two C‐terminally elongated innexins (Cte‐Inxs), SpliInx2 (Inx2‐FLAG), and SpliInx3 (Inx3‐FLAG), which were predicted to have the same secondary topological structures as the native SpliInx2 and SpliInx3. Here, we found that only the mRNAs of the two Cte‐Inxs were expressed under the control of the CMV promoter in HeLa cells. Unexpectedly, mRNA expression of the two Cte‐Inxs enhanced apoptosis of HeLa cells. The two Cte‐Inx mRNAs were associated with a significant decrease in Akt phosphorylation in HeLa cells undergoing apoptosis. Furthermore, Inx3‐FLAG mRNA expression in nonapoptotic HCT116 cells was also associated with a significant decrease in the levels of phosphorylated Akt. Intriguingly, expression of the mRNAs of the two Cte‐Inxs did not activate caspase 3, but it markedly reduced Bid levels in HeLa cells undergoing apoptosis. These results suggest that mRNA expression of the two Cte‐Inxs may activate a Bid‐dependent apoptotic pathway in HeLa cells. Our study demonstrates that invertebrate gap junction mRNAs can function in vertebrate cancer cells as tumor suppressors.     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

维生素D对烟曲霉感染的肺巨噬细胞 自噬机制的初步研究

目的探究维生素D在鼠肺细胞感染烟曲霉后对细胞自噬的影响。方法用一定量的烟曲霉活化孢子感染细胞后,一组细胞加入维生素D(维生素D组),一组细胞加入生理盐水,感染一定时间后用溶酶体探针检测自噬相关分子的表达;收集各组细胞并裂解细胞,离心取上清,用Western-blot法检测上清液中的LC3BII、Dectin-1及ROS的表达水平。结果活化的孢子感染肺巨噬细胞后,维生素D组自噬体与溶酶体共定位减少、吞噬孢子的速率在减少以及ROS水平降低,其对应的胞内Dectin-1、ROS、LC3BII减少且差异有统计学意义。结论烟曲霉感染肺巨噬细胞后,维生素D可通过减弱细胞自噬体与溶酶体的融合并下调自噬信号通路蛋白的表达以达到抵抗烟曲霉感染的作用。     

Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.     

In vitro interaction between glabridin and voriconazole against Aspergillus fumigatus isolates

BackgroundVoriconazole (VRC) is widely recommended as the first-line therapy for invasive aspergillosis. However, surveillance studies have demonstrated that there is an increase in the frequency of azole resistance among Aspergillus fumigates isolates. In recent years, more studies on effective synergisms between natural agents and antifungal drugs have been published.AimsTo evaluate the synergistic antifungal effect of glabridin (Gla) and VRC against A. fumigatus isolates.MethodsPotential interactions between Gla and VRC were studied by using a microdilution checkerboard method based on the CLSI reference technique. To assess the interaction of drugs the fractional inhibitory concentration index (FICI) was calculated based on the Loewe Additivity model.ResultsThe minimum inhibitory concentrations (MIC) obtained with Gla alone were relatively high (MIC₅₀ 16 μg/ml). However, our results showed synergistic interaction between Gla and VRC against A. fumigatus strains, with FICI range values between 0.15 and 0.5.ConclusionsSynergistic activity of Gla and VRC against both VRC-sensitive and -resistant A. fumigatus isolates may lead to design new antifungal agents, especially for inhibiting those azole-resistant strains.     

Impaired autophagy: a link between neurodegenerative and neuropsychiatric diseases

Protein misfolding, and subsequent aggregation have been proven as the leading cause of most known dementias. Many of these, in addition to neurodegeneration, show profound changes in behaviour and thinking, thus, psychiatric symptoms. On the basis of the observation that progressive myoclonic epilepsies and neurodegenerative diseases share some common features of neurodegeneration, we proposed autophagy as a possible common impairment in these diseases. Here, we argue along similar lines for some neuropsychiatric conditions, among them depression and schizophrenia. We propose that existing and new therapies for these seemingly different diseases could be augmented with drugs used for neurodegenerative or neuropsychiatric diseases, respectively, among them some which modulate or augment autophagy.