Research Of Pathogenic Mechanism In Aspergillus Fumigatus Based On Proteomics

Aspergillus fumigatus (A. fumigatus)is an conditioned pathogenic fungi. In recent years, because many reasons make the number of low immune function patients increasing gradually, and cause the infection rate and mortality rises unceasingly, the mortality has reached 50%. Furthermore, lacking of the high-efficient and low-poison medicine, fast and specific diagnostic methods and effective vaccine, the research to the pathogenesis mechanism of A. fumigatus is made to be the base of the clinical diagnosis, treatment and prevention.The research with the methods of physiology, biochemical, pathology, immunology, and morphology has found some virulence factors which maybe related to the A. fumigatus infection. But it is difficult to make a correct conclusion because we lack good research methods. Most recently, proteomics is gradually applied to the research in pathogenesis mechanism of the pathogen bacteria(such as Bacillus tuberculosis,enteropathogenic E.coli) .People have made many progresses and gained more complete informations about the virulent factors. Based on that, People continue studying the interaction between virulent factor and organism, and hold a new hope for the reach on the pathogenic mechanism. Compared with the pathogen bacteria, the research on the pathogenic mechanism of fungi is still backward. The investigations in internal and abroad are almost focus on the yeast fungi, such as Candida glabrata (C. glabrata), Cryptococcus neoformans(C.neoformans) and so on. Conversely, the research on filamentous fungus is just at the beginning. There is no report on the research of pathogenic mechanism of A. fumigatus with proteomics. Not all the A. fumigatus are pathogenic strains, and there are virulent and low virulent strains. This research analyze the differences between the proteomics of the virulent and low virulent strains of A. fumigatus, including the different expression abundance proteins and the specific proteins with the comparative proteomics. Based on the informations from the protein, we will approach the function, the action and the control mechanism in the expression of the virulent factor and make a multi-factor analysis of the pathogenic mechanism of A. fumigatus. Furthermore, we analyze proteomics graph of A. fumigatus and Candida..The obtained results are as followings:1. Constructed the infection animal model of A. fumigatus and observed the parameters of the animals infected with the different strains such as the survive days, mortality and the change of weight.2. Constructed the extraction methods of the filamentous fungus (A. fumigatus) and yeast fungi (Monilia guilliermondi(iM. guilliermondii)) and improved the condition of 2-DE. The results showed well-distinction, clear, high sensitivity and good repeatability.3. The analysis of comparative proteomics to the virulent strain and low virulent strain of A. fumigatus proved that there are some different proteins. Based on the analysis, We chose 39 spots to be identified with Matrix-assisted laser desorption ionization-time of flight mass spectrometry ( MALDI- TOF-MS) and identified 6 proteins with different construction and function.The remained spots may have much information, so it needs to improve the methods to identify the spots.(1) There are 2 proteins with high expression which are considered to have the relationship with pathogenesis. They are Cat1P and AspF3. Cat1P is considered to be one of the possible virulent factor of A. fumigatus, but there is not enough evidence to afford it. The Cat1P expression quantitation in virulent strain was high, but there was no expression in the weak one. The analysis of CAT1 which was the related gene of Cat1P has been done, the expression in virulent strains was 200 times higher than the low one by the detection of real-time PCR. The increasing was identical to the protein. It was proved that the different expression mainly happened in the process of transcription. This research proved the different expression of Cat1P in the virulent and low virulent strains with the molecular and protein standard and provided an evidence that Cat1P is a virulent factor. The allergen of A. fumigatus is a factor in ABPA, but the important one is still unknown. This study also founded that the expression of AspF3 in virulent strains is 4 times higher than the low one by the method of proteomics, and is 6 times higher than the gene expression analysis. It showed that there is an equal increasing between the proteomics and genome method and AspF3 is a virulent factor.It plays an important role in many allergens.(2) There were four proteins in the low virulent strains, including guanine nucleotide- binding beta subunit like protein ,actin, NADP linked protein and orotidine 5`-phosphate decarboxylase. Their functions are as follows: guanine nucleotide- binding beta subunit like protein participats in the process of the cell signal transmission and is related to many cell functions. Actin is the component of cytoskeleton microfilament, maintains cell phenotype and funtion and participates in cell division, providing base of cell texture. NADP linked glutamate dehydrogenase participates in glutamic acid synthesis. Orotidine 5`-phosphate decarboxylase is the key enzyme of pyrimidine synthesis.These suggest that the change of cell signal transmission path may influence cell division, metabolism and synthesis. Multi-factors make the strain's virulence decrease. The mechanism is to be studied.4. Compared the 2-DE of A. fumigatusz with Candida albicans(C. albicans), C. glabrata and M. guilliermondii, the important high expression quantitation spots are not in the same region. This suggests that there are significant differences between the virulent and low virulent strains in protein. It provided the base to find the same or similar function protein related to pathogenesis among different fungus.This research used proteomics method combining to molecular methods to study the pathogenic mechanism of A. fumigatus for the first time and provided important rationale and data of the investigation of the pathogenesis mechanism of A. fumigatus, and the clue for the research of the anti-fungi medicine and vaccine.