Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus.
ABSTRACT: Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37 degrees C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a DeltacgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the DeltacgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although DeltacgrA conidia germinated normally at 22 degrees C, they swelled excessively when incubated at 37 degrees C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.
Project description:Aspergillus fumigatus CgrA is the ortholog of a yeast nucleolar protein that functions in ribosome synthesis. To determine how CgrA contributes to the virulence of A. fumigatus, a Delta cgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The Delta cgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37 degrees C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the Delta cgrA mutant was unable to increase its growth rate. The absence of cgrA function caused a delay in both the onset and rate of germination at 37 degrees C but had little effect on germination at room temperature. The Delta cgrA mutant was significantly less virulent than the wild-type or reconstituted strain in immunosuppressed mice and was associated with smaller fungal colonies in lung tissue. However, this difference was less pronounced in a Drosophila infection model at 25 degrees C, which correlated with the comparable growth rates of the two strains at this temperature. To determine the intracellular localization of CgrA, the protein was tagged at the C terminus with green fluorescent protein, and costaining with propidium iodide revealed a predominantly nucleolar localization of the fusion protein in living hyphae. Together, these findings establish the intracellular localization of CgrA in A. fumigatus and demonstrate that cgrA is required for thermotolerant growth and wild-type virulence of the organism.
Project description:Translational regulation can be studied on a global scale by integrating polysome fractionation of mRNAs with microarray hybridization. This approach is based on the fact that translationally quiescent mRNAs are sequestered within messenger ribonucleoprotein (mRNP) particles or associated with single ribosomes (monosomes), whereas actively translated mRNAs are associated with multiple ribosomes (polysomes). The mRNAs associated within these fractions are then used to interrogate microarrays, providing insight into how the translational state of individual mRNAs is modified by environmental cues. In this study, we coupled polysome fractionation with microarray detection in order to identify changes in the translation state of the A. fumigatus transcriptome under conditions that perturb ER homeostasis such as chemical stress (DTT, tunicamycin) or thermal stress (shift from 25 degrees celsius to 37 degrees celsius).
Project description:A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55 degrees C but not at 10 degrees C, N. udagawae is able to grow at 10 degrees C but fails to grow at >42 degrees C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37 degrees C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91(phox-/-) mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.
Project description:BACKGROUND: Establishment of aspergillosis is depending upon the exit from dormancy and germination of the conidia of Aspergillus fumigatus in the lung. To gain an understanding of the molecular mechanisms underlying the early steps of conidial germination, we undertook a transcriptomic analysis using macroarrays constructed with PCR fragments from > 3,000 genes (around one third of the annotated A. fumigatus genome). RESULTS: Major results of this analysis are the following: (i) conidia stored pre-packaged mRNAs transcripts (27% of genes have transcripts in the resting conidia; (ii) incubation at 37 degrees C in a nutritive medium induced up- and down-regulation of genes: 19% of the total number of genes deposited on the array were up-regulated whereas 22% of the genes with pre-packaged mRNA in the resting conidia were down-regulated; (iii) most modifications were seen during the first 30 min of germination whereas very little modification of gene expression occurred during the following hour; (iv) one-year old conidia and one-week old conidia behaved similarly at transcriptional level. CONCLUSION: Transcriptomic data indicate that the exit from dormancy is associated with a shift from a fermentative metabolism to a respiratory metabolism as well as a trend toward immediate protein synthesis.
Project description:Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.
Project description:Translation is a costly, but inevitable, cell maintenance process. To reduce unnecessary ATP consumption in cells, a fine-tuning mechanism is needed for both ribosome biogenesis and translation. Previous studies have suggested that the ribosome functions as a hub for many cellular signals such as ribotoxic stress response, mammalian target of rapamycin (mTOR), and ribosomal S6 kinase (RSK) signaling. Therefore, we investigated the relationship between ribosomes and mitogen-activated protein kinase (MAPK) activation under ribotoxic stress conditions and found that the activation of c-Jun N-terminal kinases (JNKs) was suppressed by ribosomal protein knockdown but that of p38 was not. In addition, we found that JNK activation is driven by the association of inactive JNK in the 80S monosomes rather than the polysomes. Overall, these data suggest that the activation of JNKs by ribotoxic stress is attributable to 80S monosomes. These 80S monosomes are active ribosomes that are ready to initiate protein translation, rather than polysomes that are already acting ribosomes involved in translation elongation. [BMB Reports 2019; 52(8): 502-507].
Project description:The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.
Project description:The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (?fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ?fleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.
Project description:Background and Purpose:Aspergillus fumigatus is one of the most common opportunistic fungus, which causes infection in immunocompromised and neutropenic patients. The current guidelines recommend voriconazole as the initial therapeutic and prophylactic agent for almost all cases, especially in patients with organ transplants, which leads to increased medication resistance in A. fumigatus. The aim of the present study was to evaluate the antifungal activity and effect of kombucha as a natural compound on A. fumigatus growth, as well as on the expression of cgrA and cyp51A genes. Materials and Methods:A panel of 15 A. fumigatus strains with two quality controls of CM237 and CM2627 as susceptible and resistant strains were obtained from Tehran Medical Mycology Laboratory, Tehran,Iran(TMML).Antifungal susceptibility testing assay was performed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. Moreover, the mycelial dry weight of the fungus was calculated before and after being treated with kombucha. In addition, the quantitative changes in the expression of cgrA and cyp51A genes were analyzed by real-time polymerase chain reaction (real-time PCR) technique. Results:In the present study, the minimum inhibitory concentration ranges of kombucha were measured at 6,170 and 12,300 ?g/mL for ten A. fumigatus azole-susceptible strains and 24,700 ?g/mL for five A. fumigatus resistant strains. Moreover, changes in mycelial dry weight under kombucha treatment conditions underwent a significant reduction (P?0.05). A coordinate down-regulation of expression in cgrA and cyp51A genes was observed in all azole-susceptible and -resistant A. fumigatus strains, after treating the fungus with different concentrations of kombucha (P?0.05). Conclusion:According to the obtained results, kombucha as a natural antioxidant , can exert inhibitory effects against the growth and expression of some genes in A. fumigatusstrains.