摘要
本研究以临床重要病原真菌烟曲霉(Aspergillus fumigatus)为材料,通过连续传代的方法,获得表型和致病力均发生明显变化的突变株。对野生株和突变株的胞内蛋白质、分泌蛋白质进行比较蛋白质组学研究,以及野生株的温度敏感性与致病性关系的研究,亦进行了不同菌株间胞内蛋白质的比较。胞内蛋白质图谱分析显示,有42个差异蛋白质点,通过质谱分析鉴定出9种。其中5种属于热休克蛋白家族(heat shock protein,Hsps),分别是Hsp90、Hsp88、Hsp70、HscA和Sti1。其他4种蛋白质分别为真核翻译起始因子EIF 4、核糖体60S亚基P0和2种肌动蛋白VIP 1和Act1。应用实时荧光定量分析技术对Hsp90、Hsp88、Sti1和EIF 4在基因水平的变化进行探讨,发现了3种热休克蛋白的mRNA水平和蛋白水平的表达一致。分泌蛋白质的研究中发现,野生株中有57种表达上调,突变株仅有11种上调。在不同温度(25℃和37℃)下,野生株胞内蛋白质有55个差异点,鉴定出6种:蛋白酶体调节亚单位Rpt3、热休克蛋白(Hsp82和Hsp BiP/Kar2)和3种“假想”蛋白质。上述实验发现热休克蛋白可能与致病性和温度变化有关。另外,还比较了来自临床和环境的6株烟曲霉的致病力差异,并对强毒株和弱毒株的胞内蛋白质进行了比较,选择其中49个差异点,鉴定出2种:菌丝体过氧化氢酶(CatB)和ATP合酶-β亚单位(ATP synthase F1,beta subunit)。并得到1种疑似Hsp70同系物。研究结果表明,热休克蛋白家族和过氧化氢酶、一些参与蛋白质合成、能量合成的蛋白质可能与烟曲霉的致病性相关。
In recent years,the deuteromycete Aspergillus fumigatus has gone from being a saprophytic fungus of minor interest to scientists to becoming one of the most important fungal pathogens. The main reason for this phenomenon lies in the steady increase in the number of immunocompromised individuals, which are the main risk group for such infections. Invasive pulmonary aspergillosis (IPA) has emerged as an important cause of mortality in high-risk patients such as neutropenia syndrome, Hematopoietic stem cell transplantation, the use of long-term high-dose hormonal therapy and patients with blood borne cancers, etc. It's a matter of great concern that their research, as the high incidence of IPA, the rapid progression and high mortality (60% -90%) and lack of effective drugs.
In this research, we made serial subculture for the clinically important pathogenic fungi Aspergillus fumigatus and got the 60th generation of the strain. During the subculture, we found that phenotype of the strain had changed, such as the decrease of growth of hypha, the reducing of germination to the conidium and more fragile of conidium. The wild strain(P1) and mutant strain were used to infect the immune suppression mice and we found that the survival rates of mice infected by mutant strain were higher than wild strain, and the extent of the decreasing of mean body weight were little. The infectious symptom of the lung were gently too. All these results suggested that the mutant strain became less virulent than wild strain. Two dimensional gel electrophoresis technology was used to separate the intracellular proteins from wild strain and mutant strain, and the differential proteins spots were compared and analyzed between the two strains. In the wild strain, we detect 1254±36 spots, while in the mutant strain, we detect 1346±23 spots. We analyzed 42 differential spots (8 spots upregulated in wild strain, 34 spots upregulated in mutant strain) ,and 9 proteins were umbiguously identified via MALDI-TOF MS, all of them were upregulated in mutant strain. The function of these proteins were related to molecular chaperones, protein synthesis and cytoskeleton. real-time PCR analytical technique was applied to assess the change of the gene level of Hsp90, Hsp88, Sti1 and EIF4. We found that the level of gene expression and protein have the same tendency in the 3 Hsps. While the level of gene expression didn’t coincide with the level ofEIF4. To protein, expression level in mutant strain was 2.37 times higher than wild strain, but to gene, expression level in wild strain was 5 times lower than mutant strain.
In our study, comparative proteomics technology was used to analyze the diffence of secretion proteins between wild strain and mutant strain. we detected 861±12 spots in wild strain and we detected 767±13 spots in mutant strain,. By analysis, we found that 57 protein spots were upregulated in wild strain and 11 protein spots were upregulated in mutant strain. In our study, the quantity of upregulated secretion proteins and extent of the upregulation was little. Statistical analysis indicated that sopts with vol% value above were 2 in wild strain, and spots with vol% value between 5 and 10 were 8, and spots with vol% value between 3 and 5 were 28. While in mutant strain, vol% value of most protein spots were between 2 and 4.
In addition, we compared the expression of intracellular protein of wild strain cultured in 25℃and 37℃. 1303±20 spots were detected in wild strain cultured in 25℃, while 1286±18 spots were detected in wild strain cultured in 37℃. In our study, 55 differential expression spots were picked and 6 proteins were identified, which were upregulated in mutant strain. The function of these proteins were involved in molecular chaperones and protein degradation. We also compared the difference of pathogenicity of strains isolated from clinical sample and environment for screening the more virulent strain and less virulent strain. By analyzing the differential expression of proteins between the two strains, we detected 1463±27 spots in more virulent strain and 1389±26 spots in less virulent strain. Then 42 differential spots, of which 23 spots were upregulated in virulent strain and 19 spots were upregulated in less virulent strain, and 2 proteins were identified. The function of these proteins were associated with oxidative stress and ATP synthesis, and we also found another protein presume to be Hsp70 homolog.
The achievement we have got:
1 The mutant strain of Aspergillus fumigatus with obviously change of phenotype were got by serial subculture, and pathogenicity of the mutant strain decreased, which is tested by animal experiment.
2 The differential expression of the intracellular proteins were found between wild strain and mutant strain. 42 differential spots were picked and 9 proteins were identified, among them, 5 proteins belong to heat shock protein family, which are sp90、Hsp88、Hsp70、Hsp70 chaperone(HscA)and heat shock protein(Sti1), the other 4 proteins are eukaryotic translation initiation factor 4, 60S ribosomal proteinP0, actin cytoskeleton protein(VIP 1)and actin Act1. The function of these identified proteins are involved in molecular chaperones, cytoskeleton and protein synthesis/ translation. Real-time PCR were used to analyze the expression leve of gene and protein of Hsp90、Hsp88、heat shock protein(Sti1)and ETIF4, and we found that the expression level of gene and protein have the same tendency in the 3 hsp, and found that hps may be related to the pathogenicity of Aspergillus fumigatus.
3 Differential expression of the secretion protein between wild strain and mutant strain were found in present study. 57 protein spots were upregulated in wild strain and 11 protein spots were upregulated in mutant strain.
4 Differential expression of intracellular proteins in wild strain cultured under different temperature were found. 55 differential expression protein spots were picked and 6 proteins were identified , including roteasome regulatory partical subunit Rpt3 ,hsp82 , Hsp70 chaperone BiP/Kar2 and 3 hypothetical proteins. Furthermore, Hsp70 was found to be involved in adaptation to the change of temperature.
5 Differential expression of intracellular proteins between virulent strain and less virulent strain were found, and 49 differential spots were picked, of which 2 proteins were identified: CatB and ATP synthase F1,beta subunit. We also found another protein which might be Hsp70 homolog. These results suggested that heat shock proteins and catalase may be involved in the pathogenicity of Aspergillus fumigatus.
6 Separation method of secretion protein and cell wall proteins from Aspergillus fumigatus were initially established.
In this research, we use the comparative proteomics technology to analyze the differential expression of intracellular proteins and secretion proteins in wild strain and mutant strain, and differential expression of intracellular proteins in wild strain cultured in different cultivation temperature, and differential expression of intracellular proteins in different strains. We found several proteins correlation to pathogenicity, which provide new point for the research of pathogenic mechanism of Aspergillus fumigatus. .
引文
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