Nutritional Heterogeneity Among Aspergillus fumigatus Strains Has Consequences for Virulence in a Strain- and Host-Dependent Manner

Acquisition and subsequent metabolism of different carbon and nitrogen sources have been shown to play an important role in virulence attributes of the fungal pathogen Aspergillus fumigatus, such as the secretion of host tissue-damaging proteases and fungal cell wall integrity. We examined the relationship between the metabolic processes of carbon catabolite repression (CCR), nitrogen catabolite repression (NCR) and virulence in a variety of A. fumigatus clinical isolates. A considerable amount of heterogeneity with respect to the degree of CCR and NCR was observed and a positive correlation between NCR and virulence in a neutropenic mouse model of pulmonary aspergillosis (PA) was found. Isolate Afs35 was selected for further analysis and compared to the reference strain A1163, with both strains presenting the same degree of virulence in a neutropenic mouse model of PA. Afs35 metabolome analysis in physiological-relevant carbon sources indicated an accumulation of intracellular sugars that also serve as cell wall polysaccharide precursors. Genome analysis showed an accumulation of missense substitutions in the regulator of protease secretion and in genes encoding enzymes required for cell wall sugar metabolism. Based on these results, the virulence of strains Afs35 and A1163 was assessed in a triamcinolone murine model of PA and found to be significantly different, confirming the known importance of using different mouse models to assess strain-specific pathogenicity. These results highlight the importance of nitrogen metabolism for virulence and provide a detailed example of the heterogeneity that exists between A. fumigatus isolates with consequences for virulence in a strain-specific and host-dependent manner

RNA Polymerase Trafficking in Bacillus subtilis Cells ▿ †

To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the Bacillus subtilis genome, we analyzed the distribution of the σA and β′ subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells using chromatin affinity precipitation coupled with gene chip mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RΝΑP is evenly distributed from the promoter to the coding sequences. This finding suggests that, in general, B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex and proceeds without intragenic transcription attenuation. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which can be identified as transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior between E. coli and B. subtilis during initiation and elongation steps might result in distinct strategies for postinitiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis

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

Abstract Background Accurate 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. Results The 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. Conclusions The 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

Global gene expression reveals stress-responsive genes in Aspergillus fumigatus mycelia

Abstract Background Aspergillus fumigatus is a human fungal pathogen that causes aspergillosis in immunocompromised hosts. A. fumigatus is believed to be exposed to diverse environmental stresses in the host cells. The adaptation mechanisms are critical for infections in human bodies. Transcriptional networks in response to diverse environmental challenges remain to be elucidated. To gain insights into the adaptation to environmental stresses in A. fumigatus mycelia, we conducted time series transcriptome analyses. Results With the aid of RNA-seq, we explored the global gene expression profiles of mycelia in A. fumigatus upon exposure to diverse environmental changes, including heat, superoxide, and osmotic stresses. From the perspective of global transcriptomes, transient responses to superoxide and osmotic stresses were observed while responses to heat stresses were gradual. We identified the stress-responsive genes for particular stresses, and the 266 genes whose expression levels drastically fluctuated upon exposure to all tested stresses. Among these, the 77 environmental stress response genes are conserved in S. cerevisiae, suggesting that these genes might be more general prerequisites for adaptation to environmental stresses. Finally, we revealed the strong correlations among expression profiles of genes related to ‘rRNA processing’. Conclusions The time series transcriptome analysis revealed the stress-responsive genes underlying the adaptation mechanisms in A. fumigatus mycelia. These results will shed light on the regulatory networks underpinning the adaptation of the filamentous fungi

Non-cyp51A Azole-Resistant Aspergillus fumigatus Isolates with Mutation in HMG-CoA Reductase

The recent increase in azole-resistant Aspergillus fumigatus is a global concern. Identifying the mutations that confer azole resistance is essential for developing novel methods for prompt diagnosis and effective drug treatment. We screened A. fumigatus clinical isolates for novel mutations conferring azole resistance. We compared the genomic sequences of susceptible and resistant isolates without mutations in cyp51A (non-cyp51A) and found mutations in hmg1 and erg6 involved in ergosterol biosynthesis. We also found the novel mutations in these genes in azole-resistant isolates with different genetic backgrounds. The resistant isolates with mutations in hmg1 showed increased intracellular ergosterol levels compared with susceptible isolates. This finding supports the concept that the ergosterol level is a determinant for resistance to any class of azoles. Multiple isolates with increased resistance to azole possessed a mutation in hmg1, indicating that this mutation is widely present in non-cyp51A azole-resistant A. fumigatus

Model of Allergic Asthma and Evaluation of the Parameters of Respiratory Functions

Quantitative real-time RT-PCR analyses of dehydrin-like genes in A. fumgiatus. Expression profiles of the dprA, dprB, and dprC in the resting conidia of Afs35 (WT) and ΔatfA were analyzed. Each value represents the expression ratio relative to that of actin. Data presented are averages of three replicates, and the bar indicates standard deviation. (PPTX 55 kb

Additional file 1: Table SI-1. of 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

The accession number (TrEMBL) of ribosomal protein biomarkers of genome-sequenced strains used in this study. (DOCX 22 kb

Additional file 7: Table S4. of Global gene expression reveals stress-responsive genes in Aspergillus fumigatus mycelia

log2 transformed ratio values of the 266 genes. (XLSX 289 kb