Dataset Information

Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen Talaromyces marneffei.

ABSTRACT: Fungal infections are an increasing public health problem, particularly in immunocompromised individuals. While these pathogenic fungi show polyphyletic origins with closely related non-pathogenic species, many undergo morphological transitions to produce pathogenic cell types that are associated with increased virulence. However, the characteristics of these pathogenic cells that contribute to virulence are poorly defined. Talaromyces marneffei grows as a non-pathogenic hyphal form at 25°C but undergoes a dimorphic transition to a pathogenic yeast form at 37°C in vitro and following inhalation of asexual conidia by a host. Here we show that this transition is associated with major changes in central carbon metabolism, and that these changes are correlated with increased virulence of the yeast form. Comprehensive metabolite profiling and ¹³C-labeling studies showed that hyphal cells exhibited very active glycolytic metabolism and contain low levels of internal carbohydrate reserves. In contrast, yeast cells fully catabolized glucose in the mitochondrial TCA cycle, and store excess glucose in large intracellular pools of trehalose and mannitol. Inhibition of the yeast TCA cycle inhibited replication in culture and in host cells. Yeast, but not hyphae, were also able to use myo-inositol and amino acids as secondary carbon sources, which may support their survival in host macrophages. These analyses suggest that T. marneffei yeast cells exhibit a more efficient oxidative metabolism and are capable of utilizing a diverse range of carbon sources, which contributes to their virulence in animal tissues, highlighting the importance of dimorphic switching in pathogenic yeast.

SUBMITTER: Pasricha S

PROVIDER: S-EPMC5563070 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen <i>Talaromyces marneffei</i>.

Pasricha Shivani S MacRae James I JI Chua Hwa H HH Chambers Jenny J Boyce Kylie J KJ McConville Malcolm J MJ Andrianopoulos Alex A

Frontiers in cellular and infection microbiology 20170817

Fungal infections are an increasing public health problem, particularly in immunocompromised individuals. While these pathogenic fungi show polyphyletic origins with closely related non-pathogenic species, many undergo morphological transitions to produce pathogenic cell types that are associated with increased virulence. However, the characteristics of these pathogenic cells that contribute to virulence are poorly defined. <i>Talaromyces marneffei</i> grows as a non-pathogenic hyphal form at 25 ...[more]

PMID: 28861398

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Project description:Background: Talaromyces marneffei (T marneffei), known as a significant pathogen in patients with AIDS in Southeast Asia, is a dimorphic fungus, which can cause deadly systematic infection in immunocompromised hosts. What is more, the dimorphic phase transition has been reported as a conspicuous process linked with virulence. Interestingly, the yeast form was found in infected individuals, representing the pathogenic phase. However, few researches were found to study the mechanism of dimorphic transition. Thus, a diverse insight into the dimorphic switch mechanism, is urgently needed and we are the first one to research the mechanism of dimorphism.Methods: Firstly, we investigated the microarray of T. marneffei in the Gene Expression Omnibus database (GEO) for differentially expressed genes (DEGs). Then Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 was employed to analyze the underlying enrichment and pathway in biological process of DEGs. Meanwhile, protein-protein interaction (PPI) network was constructed using STRING database. On the strength of the theory that similar amino acid sequences share similar structures, which play a decisive role on the function of protein, three dimensional structures of hub-genes were predicted to further investigate the likely function of hub-genes.Results: GSE51109 was elected as the eligible series for the purpose of our research, including GSM1238923 (GSM23), GSM1238924 (GSM24), and GSM1238925 (GSM25). PMAA_012920, PMAA_028730, PMAA_068140, PMAA_092900, PMAA_032350 were the most remarkable genes in all of the three PPI networks, thus, were viewed as hub-genes. With regard to the three-dimensional construction, except that there was no significant prediction structure of PMAA_092900 with the criterion seq identify >?30%, GMQE: 0-1, QMEAN4: -4-0, the parallel templates for four structures were Crystal structure of Saccharomyces cerevesiae mitochondrial NADP(+)-dependent isocitrate dehydrogenase in complex with isocitrate, Organellar two-pore channels (TPCs), Yeast Isocitrate Dehydrogenase (Apo Form) and Crystal Structure Of ATP-Dependent Phosphoenolpyruvate Carboxykinase From Thermus thermophilus HB8 in order.Conclusion: The dimorphic transition of T. marneffei was viewed as a pathogenic factor and DEGs were observed. In-depth study of the function and pathway of DEGs revealed that PMAA_012920, PMAA_028730, PMAA_068140, PMAA_092900, PMAA_032350 were most likely acting as the hub-genes and were likely taking effect through regulating energy metabolism.

Project description:BackgroundPenicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37 degrees C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei.ResultsWhole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous Aspergillus proteins.ConclusionThis study clearly demonstrates the utility of proteomic approaches to studying dimorphism in P. marneffei. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in P. marneffei.

Project description:The opportunistic human pathogen Talaromyces marneffei exhibits a temperature-dependent dimorphic transition, which is closely related with its pathogenicity. This species grows as multinucleate mycelia that produce infectious conidia at 25°C, while undergoes a dimorphic transition to generate uninucleate yeast form cells at 37°C. The mechanisms of phenotype switching are not fully understood. The transcription factor madsA gene is a member of the MADS-box gene family. Previously, it was found that overexpression of madsA gene resulted in mycelial growth instead of yeast form at 37°C. In the current study, the madsA deletion mutant (ΔmadsA) and complemented strain (CMA) were constructed by genetic manipulation. We compared the phenotypes, growth, conidiation, conidial germination and susceptibility to stresses (including osmotic and oxidative) of the ΔmadsA with the wild-type (WT) and CMA strains. The results showed that the ΔmadsA displayed a faster process of the yeast-to-mycelium transition than the WT and CMA. In addition, the deletion of madsA led to a delay in conidia production and conidial germination. The tolerance of ΔmadsA conidia to hydrogen peroxide was better than that of the WT and CMA strains. Then, RNA-seq was performed to identify differences in gene expression between the ΔmadsA mutant and WT strain during the yeast phase, mycelium phase, yeast-to-mycelium transition and mycelium-to-yeast transition, respectively. Gene ontology functional enrichment analyses indicated that some important processes such as transmembrane transport, oxidation-reduction process, protein catabolic process and response to oxidative stress were affected by the madsA deletion. Together, our results suggest that madsA functions as a global regulator involved in the conidiation and germination, especially in the dimorphic transition of T. marneffei. Its roles in the survival, pathogenicity and transmission of T. marneffei require further investigation.

Project description:Talaromycosis is a fungal infection caused by an opportunistic dimorphic fungus Talaromyces marneffei. During infection, T. marneffei resides inside phagosomes of human host macrophages where the fungus encounters nutrient scarcities and host-derived oxidative stressors. Previously, we showed that the deletion of acuK, a gene encoding Zn(2)Cys(6) transcription factor, caused a decreased ability for T. marneffei to defend against macrophages, as well as a growth impairment in T. marneffei on both low iron-containing medium and gluconeogenic substrate-containing medium. In this study, a paralogous gene acuM was deleted and characterized. The ΔacuM mutant showed similar defects with the ΔacuK mutant, suggesting their common role in gluconeogenesis and iron homeostasis. Unlike the pathogenic mold Aspergillus fumigatus, the ΔacuK and ΔacuM mutants unexpectedly exhibited normal siderophore production and did not show lower expression levels of genes involved in iron uptake and siderophore synthesis. To identify additional target genes of AcuK and AcuM, RNA-sequencing analysis was performed in the ΔacuK and ΔacuM strains growing in a synthetic dextrose medium with 1% glucose at 25 °C for 36 hours. Downregulated genes in both mutants participated in iron-consuming processes, especially in mitochondrial metabolism and anti-oxidative stress. Importantly, the ΔacuM mutant was sensitive to the oxidative stressors menadione and hydrogen peroxide while the ΔacuK mutant was sensitive to only hydrogen peroxide. The yeast form of both mutants demonstrated a more severe defect in antioxidant properties than the mold form. Moreover, ribosomal and ribosomal biogenesis genes were expressed at significantly lower levels in both mutants, suggesting that AcuK and AcuM could affect the protein translation process in T. marneffei. Our study highlighted the role of AcuK and AcuM as global regulators that control multiple cellular adaptations under various harsh environmental conditions during host infection. These transcription factors could be potentially exploited as therapeutic targets for the treatment of this neglected infectious disease.

Project description:During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host's defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells.

Dataset Information

Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen Talaromyces marneffei.

Publications

Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen <i>Talaromyces marneffei</i>.

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