Verification of Ribosomal Proteins of &lt;i&gt;Aspergillus fumigatus&lt;/i&gt; for Use as Biomarkers in MALDI-TOF MS Identification

Nakamura, Sayaka; Sato, Hiroaki; Tanaka, Reiko; Yaguchi, Takashi

doi:10.5702/massspectrometry.a0049

Cited by 12 publications

(15 citation statements)

References 29 publications

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“…Functional annotation of the predicted genes was obtained from MycoCosm as described previously (5), which, in the case of the differentially expressed genes, was manually approved by examining the GO and KEGG terms and by blastp search. However, ribosomal subunits were named by homology with S. cerevisiae, as proposed by Nakamura et al (45).…”

Section: Methodsmentioning

confidence: 99%

FlbA-Regulated Gene rpnR Is Involved in Stress Resistance and Impacts Protein Secretion when Aspergillus niger Is Grown on Xylose

Aerts

Bergh

Post

et al. 2019

Appl Environ Microbiol

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Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene flbA results in mycelial colonies that secrete proteins throughout the colony. However, ΔflbA strain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which, rpnR, was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the ΔrpnR deletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when A. niger was grown on xylose. Moreover, the ΔrpnR strain showed decreased resistance to H 2 O 2 and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when A. niger is grown on xylose. IMPORTANCE Aspergillus niger secretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of A. niger are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in A. niger by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production in A. niger.

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Section: Methodsmentioning

confidence: 99%

FlbA-Regulated Gene rpnR Is Involved in Stress Resistance and Impacts Protein Secretion when Aspergillus niger Is Grown on Xylose

Aerts

Bergh

Post

et al. 2019

Appl Environ Microbiol

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“…As the first step, we have investigated the actual state of information of RSPs of fungi registered in public protein databases through the characterization of ribosomal protein fractions extracted from genome-sequenced A. fumigatus strains as a model [ 28 ]. In our previous paper [ 28 ], we revealed that more than half of the amino acid sequences of RSPs registered in the public databases were incorrect, due chiefly to mis-annotation of exon/intron structures. We were able to successfully correct the sequence errors using a combination of in silico inspection by sequence homology analysis and MALDI–TOF MS measurements.…”

Section: Introductionmentioning

confidence: 99%

Ribosomal subunit protein typing using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification and discrimination of Aspergillus species

et al. 2017

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BackgroundAccurate identification of Aspergillus species is a very important subject. Mass spectral fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is generally employed for the rapid identification of fungal isolates. However, the results are based on simple mass spectral pattern-matching, with no peak assignment and no taxonomic input. We propose here a ribosomal subunit protein (RSP) typing technique using MALDI-TOF MS for the identification and discrimination of Aspergillus species. The results are concluded to be phylogenetic in that they reflect the molecular evolution of housekeeping RSPs.ResultsThe amino acid sequences of RSPs of genome-sequenced strains of Aspergillus species were first verified and compared to compile a reliable biomarker list for the identification of Aspergillus species. In this process, we revealed that many amino acid sequences of RSPs (about 10–60%, depending on strain) registered in the public protein databases needed to be corrected or newly added. The verified RSPs were allocated to RSP types based on their mass. Peak assignments of RSPs of each sample strain as observed by MALDI-TOF MS were then performed to set RSP type profiles, which were then further processed by means of cluster analysis. The resulting dendrogram based on RSP types showed a relatively good concordance with the tree based on β-tubulin gene sequences. RSP typing was able to further discriminate the strains belonging to Aspergillus section Fumigati.ConclusionsThe RSP typing method could be applied to identify Aspergillus species, even for species within section Fumigati. The discrimination power of RSP typing appears to be comparable to conventional β-tubulin gene analysis. This method would therefore be suitable for species identification and discrimination at the strain to species level. Because RSP typing can characterize the strains within section Fumigati, this method has potential as a powerful and reliable tool in the field of clinical microbiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-1009-3) contains supplementary material, which is available to authorized users.

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“…Nakamura et al selected and verified 26 kinds of ribosomal subunit proteins (RSPs) whose molecular weights were under 16000 Da that could be used as biomarkers for A. fumigatus strains. 19 , 29 In conjunction with his study, we observed 20 kinds of RSPs that could be used as biomarkers when VITEK MS was used to identify our strains. Table 4 shows the RSP types selected in this study.…”

Section: Discussionmentioning

confidence: 53%

Performance of VITEK mass spectrometry V3.0 for rapid identification of clinical <em>Aspergillus fumigatus</em> in different culture conditions based on ribosomal proteins

Zhou¹,

Chen²,

Xu³

2017

IDR

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Fast and accurate discrimination of Aspergillus fumigatus is significant, since misidentification may lead to inappropriate clinical therapy. This study assessed VITEK mass spectrometry (MS) V3.0 for A. fumigatus identification using extracted fungal ribosomal proteins. A total of 52 isolates preliminarily identified as A. fumigatus by traditional morphological methods were inoculated in three different culture media and cultured at two different temperatures. The specific spectral fingerprints of different culture time points (48, 72, 96, and 120 h) were obtained. Of all strains, 88.5% (46/52) were discriminated as A. fumigatus, while the remaining 11.5% (6/52) produced results inconsistent with morphological analysis. Molecular sequencing, as a reference method for species identification, was used to validate the morphological analysis and matrix-assisted laser desorption/ionization time of flight MS. Chi-square tests (χ2 test, P=0.05) demonstrated that the culture medium and incubation temperature had no effects on identification accuracy; however, identification accuracy of the strains in the 48-h group was lower than that in other groups. In addition, we found that ribosomal proteins extracted from A. fumigatus can be stored in different environments for at least 1 week, with their profiles remaining stable and strain identification results showing no change. This is beneficial for medical institutions with no mass spectrometer at hand. Overall, this study showed the powerful ability of VITEK MS V 3.0 in identifying A. fumigatus.

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Verification of Ribosomal Proteins of <i>Aspergillus fumigatus</i> for Use as Biomarkers in MALDI-TOF MS Identification

Cited by 12 publications

References 29 publications

FlbA-Regulated Gene rpnR Is Involved in Stress Resistance and Impacts Protein Secretion when Aspergillus niger Is Grown on Xylose

FlbA-Regulated Gene rpnR Is Involved in Stress Resistance and Impacts Protein Secretion when Aspergillus niger Is Grown on Xylose

Ribosomal subunit protein typing using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification and discrimination of Aspergillus species

Performance of VITEK mass spectrometry V3.0 for rapid identification of clinical <em>Aspergillus fumigatus</em> in different culture conditions based on ribosomal proteins

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