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The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei.

ABSTRACT: Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25 degrees C and unicellular yeast growth at 37 degrees C. P. marneffei also undergoes asexual development into differentiated multicellular conidiophores bearing uninucleate spores. These morphogenetic processes require regulated changes in cell polarity establishment, cell cycle dynamics, and nuclear migration. The RFX (regulatory factor X) proteins are a family of transcriptional regulators in eukaryotes. We sought to determine how the sole P. marneffei RFX protein, RfxA, contributes to the regulation of morphogenesis. Attempts to generate a haploid rfxA deletion strain were unsuccessful, but we did isolate an rfxA(+)/rfxADelta heterozygous diploid strain. The role of RfxA was assessed using conditional overexpression, RNA interference (RNAi), and the production of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses during growth at 25 degrees C and 37 degrees C. This was also observed for the heterozygous diploid strain during growth at 37 degrees C. In contrast, overexpression of rfxA caused growth arrest during conidial germination. The data show that rfxA must be precisely regulated for appropriate nuclear division and to maintain genome integrity. Perturbations in rfxA expression also caused defects in cellular proliferation and differentiation. The data suggest a role for RfxA in linking cellular division with morphogenesis, particularly during conidiation and yeast growth, where the uninucleate state of these cell types necessitates coupling of nuclear and cellular division tighter than that observed during multinucleate hyphal growth.

SUBMITTER: Bugeja HE

PROVIDER: S-EPMC2863407 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei.

Bugeja Hayley E HE Hynes Michael J MJ Andrianopoulos Alex A

Eukaryotic cell 20100129 4

Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25 degrees C and unicellular yeast growth at 37 degrees C. P. marneffei also undergoes asexual development into differentiated multicellular conidiophores bearing uninucleate spores. These morphogenetic processes require regulated changes in cell polarity establi ...[more]

PMID: 20118209

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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: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.

Project description:BackgroundTalaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors.Methodology/principal findingsWe examined the pathogenic roles of Mp1p and its homologs in a mouse model. All mice died 21 and 30 days after challenge with wild-type T. marneffei PM1 and MP1 complemented mutant respectively. None of the mice died 60 days after challenge with MP1 knockout mutant (P<0.0001). Seventy percent of mice died 60 days after challenge with MP1 knockdown mutant (P<0.0001). All mice died after challenge with MPLP1 to MPLP13 knockdown mutants, suggesting that only Mp1p plays a significant role in virulence. The mean fungal loads of PM1 and MP1 complemented mutant in the liver, lung, kidney and spleen were significantly higher than those of the MP1 knockout mutant. Similarly, the mean load of PM1 in the liver, lung and spleen were significantly higher than that of the MP1 knockdown mutant. Histopathological studies showed an abundance of yeast in the kidney, spleen, liver and lung with more marked hepatic and splenic necrosis in mice challenged with PM1 compared to MP1 knockout and MP1 knockdown mutants. Likewise, a higher abundance of yeast was observed in the liver and spleen of mice challenged with MP1 complemented mutant compared to MP1 knockout mutant. PM1 and MP1 complemented mutant survived significantly better than MP1 knockout mutant in macrophages at 48 hours (P<0.01) post-infection. The mean fungal counts of Pichia pastoris GS115-MP1 in the liver (P<0.001) and spleen (P<0.05) of mice were significantly higher than those of GS115 at 24 hours post-challenge.Conclusions/significanceMp1p is a key virulence factor of T. marneffei. Mp1p mediates virulence by improving the survival of T. marneffei in macrophages.

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The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei.

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The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei.

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