红色毛癣菌蛋白质组学研究
摘要
红色毛癣菌(Trichophyton Rubrum)感染所致的浅部真菌病在世界各地广泛流行,红色毛癣菌感染难以治愈,且抗真菌药物停止治疗后频繁复发。本研究首次从整体水平对静止期红色毛癣菌分生孢子的蛋白质组进行全面的分析。
本研究首先构建了红色毛癣菌8个时相的cDNA文库,建立了EST编码蛋白数据库。然后采用液氮研磨、蜗牛酶消化后样品预分级,同时结合多种酶消化,酶切后得到的肽片段混合物通过二维液相色谱分离,采用鸟枪法对蛋白进行鉴定。结果获得5304个不同的肽段序列,总计1026个蛋白得到鉴定。我们建立蜗牛酶消化破壁,结合样品预分级,然后胰酶和蛋白酶K进行酶解的蛋白提取分级分离方法,可以显著提高鉴定的蛋白数量。
我们对所鉴定的1026个蛋白分别用NCBI non-redundant (NR)蛋白数据库, eukaryotic orthologous groups (KOG)数据库和gene ontology (GO)数据库进行检索,所鉴定的蛋白涵盖了所有主要的细胞器,如细胞核、线粒体、细胞膜、高尔基体、内质网,所鉴定的蛋白涉及到几乎所有的生物生化过程。本研究鉴定出红色毛癣菌分生孢子一些重要的蛋白,包括细胞壁结构蛋白、细胞壁生物合成相关蛋白,胞内蛋白质合成相关蛋白,静止期分生孢子代谢特征蛋白,信号传导蛋白以及环境压力耐受蛋白等等,并从蛋白分子水平上对其结构功能和机制进行了详细的讨论,红色毛癣菌分生孢子蛋白质组的分析结果证明了维持孢子的休眠状态是相当复杂和精细的过程,涉及到一系列的蛋白翻译、信号传导、环境压力耐受机制等,对将来真菌感染的预防、诊断,治疗和致病机制的研究有重要的提示作用。
Infections caused by dermatophytes are widespread, are increasing in prevalence on a global scale, and have been considered a major public health concern in some areas of the world. In diseases caused by fungi, superficial fungi are the most common causative organisms, while at least 69.5% of all infections of this type is due to Trichophyton rubrum in human. In addition to the well-known superficial infections caused by this organism, such as tinea capitis, tinea corporis, tinea inguinalis, tinea manus, tinea unguium, and tinea pedis, T.rubrum is also responsible for deep dermal invasion in immunocompromised patients . T. rubrum infections are often intractable, and relapse frequently occurs after cessation of antifungal therapy .The prevalence of T. rubrum infections and the anthrophilic nature of this species make it a good model for the study of pathogenic superficial fungi. Conidia are a primary means of dispersion and provide a safe house for the T.rubrum genome during adverse environmental conditions. We report the first comprehensive profile of the dormant T. rubrum conidia proteome.
In this report we describe a proteomic approach which provides a platform for parallel analysis of filamentous fungi proteome. We used sample prefractionation method to enrich T. rubrum conidia proteins and subsequently digested them using two enzyme (trypsin, proteinase k). The recovered peptide mixture was further separated using strong cation exchange column and RPLC, then ionized and detected with ESI-MS/MS. As a result, 5304 peptide hits were obtained and 1026 T. rubrum proteins were unambiguously identified. The resultant estimated false-positive rate for the proteins was 1.55%. we used multiple enzymatic digestion ways to detect overlapping but distinct sets of rubrum conidia proteins. 686 proteins were identified using trypsin and 795 proteins were identified using proteinase K. The number of proteins specifically identified by two different digestions was 571, accounting for 56% of the 1026 identified proteins. This shows that different enzymatic digestion can significantly improve proteome coverage, resulting in a more detailed map of the conidia proteome in this case. The prefractionation can effectively reduce sample complexity thereby increasing detection sensitivity, which is necessary for identifying low abundance proteins. Here, we adopted centrifugal ultrafiltration in order to divide the conidia proteome into three fractions based on molecular weight (MW). Of the 686 proteins identified by tryptic digestion, each fraction specifically contributed 191, 140, and 172 proteins respectively.This collection of proteins accounted for 73% (503/686) of total proteins identified by tryptic digestion. Similarly, proteins specifically identified in the three individual fractions accounted for 71% (571/795) of all proteins identified by proteinase K digestion. These results suggest that prefractionation by centrifugal ultrafiltration increases sensitivity.
To obtain as much functional annotation information as possible, the identified proteins were compared to those in the NCBI non-redundant (NR) protein database, the eukaryotic orthologous groups (KOG) database, and the gene ontology (GO) database The comparison with the NR database enabled us to assign putative functions to or find homologies from other organisms for 857 (83%) of the proteins. The remaining 169 (17%) proteins were only weakly similar or not similar to known sequences (E≥1E-05). Comparison with the GO database provided more information on the cellular components and the biological processes of the identified proteins. 630 (61%) of identified proteins obtained annotation information through GO database comparison. It is clear that the identified proteins are predicted to cover all main organelles such as nucleus, mitochondrion, plasma membrane, Golgi apparatus, and endoplasmic reticulum, indicating a reflective sampling of the T. rubrum conidia proteome. Proteins were sorted into main functional classes based on the KOG database comparison. 645 (63%) of the identified proteins fell into various functional categories, while 381 (37%) proteins had no related KOG based function. almost all categories and major biological processes are represented in identified proteins. This indicates a complex profile of the T. rubrum conidia proteome. Furthermore, the complexity of the conidia protein profile suggests that these dormant bodies are not merely warehouse of reserving the genome and that the maintenance of conidial dormancy may be a considerably intricate and elaborated process. Besides probably related to maintaining quiescent status of conidia, these proteins may also be involved in such processes as conidiation and conidial germination. Some important proteins of T. rubrum Conidia were identified in our study, including Cell wall proteins and wall biosynthesis related proteins, synthesis related protein synthesis, metabolism related proteins, Signaling transduction proteins, Environmental stress resistance proteins,and discuss theirs structure and function in detail. In dormant T. rubrum conidia, we identified 22 proteins that are components of cell wall or are related to cell wall biosynthesis or remodeling based on NR and KOG annotations.The presence of the 1,3-β-glucan synthase catalytic subunit, chitin synthase, and mannosyltransferases in conidia suggests that considerable cell wall synthesis or reorganization may be occurring during sporulation in T. rubrum. It is implied that the cell wall of T.rubrum conidia, similar to other filamentous fungi, is composed of 1,3-β-glucan, chitin, and mannoproteins.
Protein S11555 exhibits high homology to the protein sequence predicted from Ecm33 (E-value, 2.04E-71) which is a glycosylphosphatidylinositol-anchored cell wall organization protein. Ecm33 is found in all fungal genomes sequenced to date, and in yeast orthologs are essential for sporulation . A recent study showed that Ecm33 influenced conidial cell wall biosynthesis in Aspergillus fumigatus . Disruption of Ecm33 resulted in several morphogenetic aberrations, including: (1) a defect in conidia separation, (2) an increase in diameter of conidia associated with an increase in cell wall chitin concentration and (3) conidia that were sensitive to the absence of aeration during long-term storage. These results suggest that in T.rubrum, Ecm33 may also play some roles in conidiation, conidial cell wall biosynthesis, and viability of conidia in adverse environmental conditions.
The surface of many fungal conidia is covered by a thin layer of regularly arranged rodlets which are mainly proteinaceous, and favor air buoyancy and dispersion of the conidia by air currents. The proteins responsible for this rodlet configuration are hydrophobins and the rodlet layer of the conidia of Neurospora crassa, Beauveria bassiana, and Magnaporthe grisea only contain a single type of hydrophobin.Aspergillers nidulans and A. fumigatus have two conidial hydrophobins, which are rodA and dewA, rodA and rodB, respectively . In T.rubrum conidia, we identified a protein (S11382) that is homologous to conidial hydrophobin RodB (E-value, 5.82E-07), but did not find any protein corresponding to RodA. Based on these results, we hypothesize that the surface of T. rubrum conidia should have a hydropbobic rodlet layer which facilitates its dispersal by air.we hypothesize that the surface of T. rubrum conidia should have a hydropbobic rodlet layer which facilitates its dispersal by air.
Previous biochemical and genetic evidences suggested that protein synthesis is essential for spore germination in fungi and conidial germination cannot take place when protein synthesis is blocked. Translation is one of the first measurable effects (<20 min) during conidial germination. In dormant N.crassa conidia, high levels of free ribosomes are observed, which in the presence of a carbon source, associate with a preexisting pool of mRNA within 15 min to form polysomes.
In our result, 101 of proteins are assigned to“translation”based on KOG classification, and account for 10% of total identified proteins in T.rubrum conidia. This suggests that proteins involved in translation are relatively abundant in dormant T. rubrum conidia. Of these 101 proteins, 56 (more than 50%) were assigned as structural components of cytosolic ribosome. As mentioned above, a pool of mRNAs are preserved in dormant T.rubrum conidia. When encountering appropriate conditions, such as the presence of a carbon source, these ribosomes could rapidly associated with preserved mRNAs to produce proteins required for conidial germination. Aside from the structural components of cytosolic ribosomes, we also identified translational initiation factors, elongation factors, and aminoyl-tRNA synthases in the T. rubrum conidia proteome. In A.nidulans, sgdA, sgdB, and sgdC respectively encode translation initiation factor eIF3 subunit, seryl-, and cysteinyl-tRNA synthase and the phenotypes of these sgd mutants were characterized. At the restrictive temperature, there was no conidial germination in any of the sgd mutants.
In addition to proteins involved in cytoplasmic protein synthesis, we also identified some proteins implicated in mitochondrial protein synthesis in T.rubrum conidia, including nine structural components of the mitochondrial ribosome, one component of mitochondrial translation elongation factor, and two components of mitochondrial polypeptide chain release factor. Unlike the system of cytoplasmic protein synthesis, mitochondrial protein synthesis appears not to be essential for spore germination in some fungi.In N.crassa and Botryodiplodia, chloramphenicol and ethidium bromide (inhibitors of mitochondrial protein synthesis) did not block conidial germination. In T. rubrum, the necessity of mitochondrial protein synthesis for conidial germination has not been evaluated.
Although dormant conidia are in a quiescent state where metabolic activities and rates of oxygen consumption are low, in T. rubrum conidia we identified many proteins belonging to glycolysis system, subunits of the pyruvate dehydrogenase complex, the tricarboxylic acid cycle, and the oxidative phosphorylation machinery.These results indicate that proteins involved in energy production and carbohydrate metabolism are retained in dormant T. rubrum conidia. Early studies demonstrated that spore germination was dependent upon the function of the standard, cytochrome-mediated electron transport system in Botryodiplodia theobromae and Neurospora crassa. The presence of cyanide (an inhibitor of this pathway) resulted in a complete block of spore germination. In dormant T. rubrum conidia, some components of all five respiratory chain complexes are identified, including six subunits of complex I (NADH-CoQ reductase),one subunit of complex II (Succinate-CoQ reductase), five subunits of complex III (CoQ-Cytochrome C reductase), three subunits of complex IV (Cytochrome C oxidase), and 11 subunits of complex V (F1F0-type ATP synthase). This is consistent with dormant N. crassa conidia where all of the enzyme components of this standard pathway, such as cytochrome c oxidase and F1F0-type ATP synthase, appear to be assembled and preserved. These results indicate that the cytochrome-mediated electron transport system in T.rubrum conidia may be important for conidia germination, just as in Botryodiplodia and N. crassa. In fungal spores, trehalose may account for up to 15% of the dry mass and has been proposed to serve as a stress protectant. Systematic mobilization of the trehalose pool is one of the first measurable effects during spore germination, which suggests that it may also act as a reserve carbohydrate needed at the onset of this particular developmental stage. Two T. rubrum conidia proteins, S00116 and S04712, are involved in trehalose biosynthesis.They are annotated as trehalose-6-phosphate synthase component TPS1 and trehalose synthase ccg-9 respectively. In S. cerevisiae, glucose-6-phosphate plus UDP-D-glucose is converted toα,α-trehalose
-6-phosphate by TPS1, then latter converted to trehalose by TPS2.ccg-9 is trehalose synthase of N. crassa and its mRNA peaks at the time of initiation of conidiation. A ccg-9 null mutant produces morphologically abnormal spores, suggesting that ccg-9 is involved in developmental morphogenesis of the asexual conidiospores. These evidences imply that trehalose is also important for T. rubrum conidia and may be implicated in conidia morphogenesis, as is observed in N. crassa.
In T. rubrum conidia, we identified 32 proteins that are related to a variety of signal transduction components such as components of Ras-related GTPase pathway, the cAMP-PKA pathway, the calcium signaling pathway, the G-protein pathway, the two-component signal transduction system, and some serine/threonine protein kinase/phosphatases and signal histidine kinases . This reflects the potential complexity of signal transduction networks in T. rubrum conidia. Currently, three signaling transduction pathways (calcium signaling, Ras/MAPK pathway and cAMP-PKA pathway) have prompted intensive efforts focused on their roles in early spore germination. In various fungi, roles of these signaling events in spore germination seem to be conflicting and variable across species. Despite conflicting results obtained from previous studies, these signaling pathways are usually involved in mediating carbon source sensing,the transition from isotropic to polarized growth, activating metabolic activities, transcription,and protein synthesis once they are involved in spore germination . The study of the transcriptional pattern we present showed that the Ras-related GTPase and cAMP-PKA pathways may play roles in conidial germination and that the two-component signal transduction system may participate in response to changes in extracellular osmolarity during conidial germination in T. rubrum. The precise roles of these signal transduction pathways in maintaining dormant state of conidia in T. rubrum need to be further study.
Resistance to adverse environmental conditions,such as desiccation, heat, and osmotic stress, is one of the most impressive properties of dormant conidia. For example, conidia can be stored for long periods of time without loss of viability once dry, and more than 90% of dehydrated conidia can survive after being heated to 124℃for 3 min. These properties of conidia permit survival and dispersal in adverse environments. Consistent with these characteristics, we identified 58 proteins related to various environmental stresses in T. rubrum conidia according to GO annotation, including: desiccation, heat, cold, oxidative stress, osmotic stress, starvation, drug, salt stress, unfolded protein, metal ion, DNA damage and general stress proteins . The classification of proteins involved in response to this range of stresses is somewhat overlapping.
Of 62 proteins,S03304,S12066and S13929 are implicated in sporulation or conidium formation according to GO annotation.S03304 and S12066 are annotated as vacuolar protease B and ubiquitin-like protein by GO,corresponding to PRB1 and UBI4 of S.cerevisiae (E-value, 1.2E~(-115) and 5.0E~(-108)) respectively.PRB1 is one of the major proteolytic catalysts in yeast cell under cellular response to starvation and cells deficient in PRB1 show a considerably reduced sporulation frequency. UBI4 is required by yeast cells for resistance to stress conditions. Although ubi4/UBI4 diploids form viable spores initially, the spores lose viability extremely rapidly and ubi4/ubi4 diploids are sporulation defective.S13929 is the 22 homologue of catalase1 in N.crassa (E-value, 2.9E-75).
N.crassa possesses three catalases (catalase1-catalase3) which serve as hydrogen peroxide scavenging enzymes and are differentially expressed during the asexual life cycle.Catalase3 activity increases at the end of exponential growth,catalase2 activity rises transiently in the aerial hyphae, and catalase1 augments many times during formation of conidia.Catalase1 is found in conidia at a very high concentration and exhibits an unusual resistance to inactivation by temperature and various denaturants.These characteristics make catalase1 especially suitable for the spore and its survival. The comprehensive proteome of T. rubrum conidia has been profiled for the first time. Our results suggest that the proteome of T. rubrum conidia is considerably complex and identified proteins involved in nearly all biological processes are preserved in conidia. This data set provides the first global frame of reference for the dormant T. rubrum conidia proteome, Which could probably facilitate the design of novel prophylactic, diagnostic and therapeutic strategies against dermatophytes, furthermore a better Understanding pathogenic mechanism.
本研究首先构建了红色毛癣菌8个时相的cDNA文库,建立了EST编码蛋白数据库。然后采用液氮研磨、蜗牛酶消化后样品预分级,同时结合多种酶消化,酶切后得到的肽片段混合物通过二维液相色谱分离,采用鸟枪法对蛋白进行鉴定。结果获得5304个不同的肽段序列,总计1026个蛋白得到鉴定。我们建立蜗牛酶消化破壁,结合样品预分级,然后胰酶和蛋白酶K进行酶解的蛋白提取分级分离方法,可以显著提高鉴定的蛋白数量。
我们对所鉴定的1026个蛋白分别用NCBI non-redundant (NR)蛋白数据库, eukaryotic orthologous groups (KOG)数据库和gene ontology (GO)数据库进行检索,所鉴定的蛋白涵盖了所有主要的细胞器,如细胞核、线粒体、细胞膜、高尔基体、内质网,所鉴定的蛋白涉及到几乎所有的生物生化过程。本研究鉴定出红色毛癣菌分生孢子一些重要的蛋白,包括细胞壁结构蛋白、细胞壁生物合成相关蛋白,胞内蛋白质合成相关蛋白,静止期分生孢子代谢特征蛋白,信号传导蛋白以及环境压力耐受蛋白等等,并从蛋白分子水平上对其结构功能和机制进行了详细的讨论,红色毛癣菌分生孢子蛋白质组的分析结果证明了维持孢子的休眠状态是相当复杂和精细的过程,涉及到一系列的蛋白翻译、信号传导、环境压力耐受机制等,对将来真菌感染的预防、诊断,治疗和致病机制的研究有重要的提示作用。
Infections caused by dermatophytes are widespread, are increasing in prevalence on a global scale, and have been considered a major public health concern in some areas of the world. In diseases caused by fungi, superficial fungi are the most common causative organisms, while at least 69.5% of all infections of this type is due to Trichophyton rubrum in human. In addition to the well-known superficial infections caused by this organism, such as tinea capitis, tinea corporis, tinea inguinalis, tinea manus, tinea unguium, and tinea pedis, T.rubrum is also responsible for deep dermal invasion in immunocompromised patients . T. rubrum infections are often intractable, and relapse frequently occurs after cessation of antifungal therapy .The prevalence of T. rubrum infections and the anthrophilic nature of this species make it a good model for the study of pathogenic superficial fungi. Conidia are a primary means of dispersion and provide a safe house for the T.rubrum genome during adverse environmental conditions. We report the first comprehensive profile of the dormant T. rubrum conidia proteome.
In this report we describe a proteomic approach which provides a platform for parallel analysis of filamentous fungi proteome. We used sample prefractionation method to enrich T. rubrum conidia proteins and subsequently digested them using two enzyme (trypsin, proteinase k). The recovered peptide mixture was further separated using strong cation exchange column and RPLC, then ionized and detected with ESI-MS/MS. As a result, 5304 peptide hits were obtained and 1026 T. rubrum proteins were unambiguously identified. The resultant estimated false-positive rate for the proteins was 1.55%. we used multiple enzymatic digestion ways to detect overlapping but distinct sets of rubrum conidia proteins. 686 proteins were identified using trypsin and 795 proteins were identified using proteinase K. The number of proteins specifically identified by two different digestions was 571, accounting for 56% of the 1026 identified proteins. This shows that different enzymatic digestion can significantly improve proteome coverage, resulting in a more detailed map of the conidia proteome in this case. The prefractionation can effectively reduce sample complexity thereby increasing detection sensitivity, which is necessary for identifying low abundance proteins. Here, we adopted centrifugal ultrafiltration in order to divide the conidia proteome into three fractions based on molecular weight (MW). Of the 686 proteins identified by tryptic digestion, each fraction specifically contributed 191, 140, and 172 proteins respectively.This collection of proteins accounted for 73% (503/686) of total proteins identified by tryptic digestion. Similarly, proteins specifically identified in the three individual fractions accounted for 71% (571/795) of all proteins identified by proteinase K digestion. These results suggest that prefractionation by centrifugal ultrafiltration increases sensitivity.
To obtain as much functional annotation information as possible, the identified proteins were compared to those in the NCBI non-redundant (NR) protein database, the eukaryotic orthologous groups (KOG) database, and the gene ontology (GO) database The comparison with the NR database enabled us to assign putative functions to or find homologies from other organisms for 857 (83%) of the proteins. The remaining 169 (17%) proteins were only weakly similar or not similar to known sequences (E≥1E-05). Comparison with the GO database provided more information on the cellular components and the biological processes of the identified proteins. 630 (61%) of identified proteins obtained annotation information through GO database comparison. It is clear that the identified proteins are predicted to cover all main organelles such as nucleus, mitochondrion, plasma membrane, Golgi apparatus, and endoplasmic reticulum, indicating a reflective sampling of the T. rubrum conidia proteome. Proteins were sorted into main functional classes based on the KOG database comparison. 645 (63%) of the identified proteins fell into various functional categories, while 381 (37%) proteins had no related KOG based function. almost all categories and major biological processes are represented in identified proteins. This indicates a complex profile of the T. rubrum conidia proteome. Furthermore, the complexity of the conidia protein profile suggests that these dormant bodies are not merely warehouse of reserving the genome and that the maintenance of conidial dormancy may be a considerably intricate and elaborated process. Besides probably related to maintaining quiescent status of conidia, these proteins may also be involved in such processes as conidiation and conidial germination. Some important proteins of T. rubrum Conidia were identified in our study, including Cell wall proteins and wall biosynthesis related proteins, synthesis related protein synthesis, metabolism related proteins, Signaling transduction proteins, Environmental stress resistance proteins,and discuss theirs structure and function in detail. In dormant T. rubrum conidia, we identified 22 proteins that are components of cell wall or are related to cell wall biosynthesis or remodeling based on NR and KOG annotations.The presence of the 1,3-β-glucan synthase catalytic subunit, chitin synthase, and mannosyltransferases in conidia suggests that considerable cell wall synthesis or reorganization may be occurring during sporulation in T. rubrum. It is implied that the cell wall of T.rubrum conidia, similar to other filamentous fungi, is composed of 1,3-β-glucan, chitin, and mannoproteins.
Protein S11555 exhibits high homology to the protein sequence predicted from Ecm33 (E-value, 2.04E-71) which is a glycosylphosphatidylinositol-anchored cell wall organization protein. Ecm33 is found in all fungal genomes sequenced to date, and in yeast orthologs are essential for sporulation . A recent study showed that Ecm33 influenced conidial cell wall biosynthesis in Aspergillus fumigatus . Disruption of Ecm33 resulted in several morphogenetic aberrations, including: (1) a defect in conidia separation, (2) an increase in diameter of conidia associated with an increase in cell wall chitin concentration and (3) conidia that were sensitive to the absence of aeration during long-term storage. These results suggest that in T.rubrum, Ecm33 may also play some roles in conidiation, conidial cell wall biosynthesis, and viability of conidia in adverse environmental conditions.
The surface of many fungal conidia is covered by a thin layer of regularly arranged rodlets which are mainly proteinaceous, and favor air buoyancy and dispersion of the conidia by air currents. The proteins responsible for this rodlet configuration are hydrophobins and the rodlet layer of the conidia of Neurospora crassa, Beauveria bassiana, and Magnaporthe grisea only contain a single type of hydrophobin.Aspergillers nidulans and A. fumigatus have two conidial hydrophobins, which are rodA and dewA, rodA and rodB, respectively . In T.rubrum conidia, we identified a protein (S11382) that is homologous to conidial hydrophobin RodB (E-value, 5.82E-07), but did not find any protein corresponding to RodA. Based on these results, we hypothesize that the surface of T. rubrum conidia should have a hydropbobic rodlet layer which facilitates its dispersal by air.we hypothesize that the surface of T. rubrum conidia should have a hydropbobic rodlet layer which facilitates its dispersal by air.
Previous biochemical and genetic evidences suggested that protein synthesis is essential for spore germination in fungi and conidial germination cannot take place when protein synthesis is blocked. Translation is one of the first measurable effects (<20 min) during conidial germination. In dormant N.crassa conidia, high levels of free ribosomes are observed, which in the presence of a carbon source, associate with a preexisting pool of mRNA within 15 min to form polysomes.
In our result, 101 of proteins are assigned to“translation”based on KOG classification, and account for 10% of total identified proteins in T.rubrum conidia. This suggests that proteins involved in translation are relatively abundant in dormant T. rubrum conidia. Of these 101 proteins, 56 (more than 50%) were assigned as structural components of cytosolic ribosome. As mentioned above, a pool of mRNAs are preserved in dormant T.rubrum conidia. When encountering appropriate conditions, such as the presence of a carbon source, these ribosomes could rapidly associated with preserved mRNAs to produce proteins required for conidial germination. Aside from the structural components of cytosolic ribosomes, we also identified translational initiation factors, elongation factors, and aminoyl-tRNA synthases in the T. rubrum conidia proteome. In A.nidulans, sgdA, sgdB, and sgdC respectively encode translation initiation factor eIF3 subunit, seryl-, and cysteinyl-tRNA synthase and the phenotypes of these sgd mutants were characterized. At the restrictive temperature, there was no conidial germination in any of the sgd mutants.
In addition to proteins involved in cytoplasmic protein synthesis, we also identified some proteins implicated in mitochondrial protein synthesis in T.rubrum conidia, including nine structural components of the mitochondrial ribosome, one component of mitochondrial translation elongation factor, and two components of mitochondrial polypeptide chain release factor. Unlike the system of cytoplasmic protein synthesis, mitochondrial protein synthesis appears not to be essential for spore germination in some fungi.In N.crassa and Botryodiplodia, chloramphenicol and ethidium bromide (inhibitors of mitochondrial protein synthesis) did not block conidial germination. In T. rubrum, the necessity of mitochondrial protein synthesis for conidial germination has not been evaluated.
Although dormant conidia are in a quiescent state where metabolic activities and rates of oxygen consumption are low, in T. rubrum conidia we identified many proteins belonging to glycolysis system, subunits of the pyruvate dehydrogenase complex, the tricarboxylic acid cycle, and the oxidative phosphorylation machinery.These results indicate that proteins involved in energy production and carbohydrate metabolism are retained in dormant T. rubrum conidia. Early studies demonstrated that spore germination was dependent upon the function of the standard, cytochrome-mediated electron transport system in Botryodiplodia theobromae and Neurospora crassa. The presence of cyanide (an inhibitor of this pathway) resulted in a complete block of spore germination. In dormant T. rubrum conidia, some components of all five respiratory chain complexes are identified, including six subunits of complex I (NADH-CoQ reductase),one subunit of complex II (Succinate-CoQ reductase), five subunits of complex III (CoQ-Cytochrome C reductase), three subunits of complex IV (Cytochrome C oxidase), and 11 subunits of complex V (F1F0-type ATP synthase). This is consistent with dormant N. crassa conidia where all of the enzyme components of this standard pathway, such as cytochrome c oxidase and F1F0-type ATP synthase, appear to be assembled and preserved. These results indicate that the cytochrome-mediated electron transport system in T.rubrum conidia may be important for conidia germination, just as in Botryodiplodia and N. crassa. In fungal spores, trehalose may account for up to 15% of the dry mass and has been proposed to serve as a stress protectant. Systematic mobilization of the trehalose pool is one of the first measurable effects during spore germination, which suggests that it may also act as a reserve carbohydrate needed at the onset of this particular developmental stage. Two T. rubrum conidia proteins, S00116 and S04712, are involved in trehalose biosynthesis.They are annotated as trehalose-6-phosphate synthase component TPS1 and trehalose synthase ccg-9 respectively. In S. cerevisiae, glucose-6-phosphate plus UDP-D-glucose is converted toα,α-trehalose
-6-phosphate by TPS1, then latter converted to trehalose by TPS2.ccg-9 is trehalose synthase of N. crassa and its mRNA peaks at the time of initiation of conidiation. A ccg-9 null mutant produces morphologically abnormal spores, suggesting that ccg-9 is involved in developmental morphogenesis of the asexual conidiospores. These evidences imply that trehalose is also important for T. rubrum conidia and may be implicated in conidia morphogenesis, as is observed in N. crassa.
In T. rubrum conidia, we identified 32 proteins that are related to a variety of signal transduction components such as components of Ras-related GTPase pathway, the cAMP-PKA pathway, the calcium signaling pathway, the G-protein pathway, the two-component signal transduction system, and some serine/threonine protein kinase/phosphatases and signal histidine kinases . This reflects the potential complexity of signal transduction networks in T. rubrum conidia. Currently, three signaling transduction pathways (calcium signaling, Ras/MAPK pathway and cAMP-PKA pathway) have prompted intensive efforts focused on their roles in early spore germination. In various fungi, roles of these signaling events in spore germination seem to be conflicting and variable across species. Despite conflicting results obtained from previous studies, these signaling pathways are usually involved in mediating carbon source sensing,the transition from isotropic to polarized growth, activating metabolic activities, transcription,and protein synthesis once they are involved in spore germination . The study of the transcriptional pattern we present showed that the Ras-related GTPase and cAMP-PKA pathways may play roles in conidial germination and that the two-component signal transduction system may participate in response to changes in extracellular osmolarity during conidial germination in T. rubrum. The precise roles of these signal transduction pathways in maintaining dormant state of conidia in T. rubrum need to be further study.
Resistance to adverse environmental conditions,such as desiccation, heat, and osmotic stress, is one of the most impressive properties of dormant conidia. For example, conidia can be stored for long periods of time without loss of viability once dry, and more than 90% of dehydrated conidia can survive after being heated to 124℃for 3 min. These properties of conidia permit survival and dispersal in adverse environments. Consistent with these characteristics, we identified 58 proteins related to various environmental stresses in T. rubrum conidia according to GO annotation, including: desiccation, heat, cold, oxidative stress, osmotic stress, starvation, drug, salt stress, unfolded protein, metal ion, DNA damage and general stress proteins . The classification of proteins involved in response to this range of stresses is somewhat overlapping.
Of 62 proteins,S03304,S12066and S13929 are implicated in sporulation or conidium formation according to GO annotation.S03304 and S12066 are annotated as vacuolar protease B and ubiquitin-like protein by GO,corresponding to PRB1 and UBI4 of S.cerevisiae (E-value, 1.2E~(-115) and 5.0E~(-108)) respectively.PRB1 is one of the major proteolytic catalysts in yeast cell under cellular response to starvation and cells deficient in PRB1 show a considerably reduced sporulation frequency. UBI4 is required by yeast cells for resistance to stress conditions. Although ubi4/UBI4 diploids form viable spores initially, the spores lose viability extremely rapidly and ubi4/ubi4 diploids are sporulation defective.S13929 is the 22 homologue of catalase1 in N.crassa (E-value, 2.9E-75).
N.crassa possesses three catalases (catalase1-catalase3) which serve as hydrogen peroxide scavenging enzymes and are differentially expressed during the asexual life cycle.Catalase3 activity increases at the end of exponential growth,catalase2 activity rises transiently in the aerial hyphae, and catalase1 augments many times during formation of conidia.Catalase1 is found in conidia at a very high concentration and exhibits an unusual resistance to inactivation by temperature and various denaturants.These characteristics make catalase1 especially suitable for the spore and its survival. The comprehensive proteome of T. rubrum conidia has been profiled for the first time. Our results suggest that the proteome of T. rubrum conidia is considerably complex and identified proteins involved in nearly all biological processes are preserved in conidia. This data set provides the first global frame of reference for the dormant T. rubrum conidia proteome, Which could probably facilitate the design of novel prophylactic, diagnostic and therapeutic strategies against dermatophytes, furthermore a better Understanding pathogenic mechanism.
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