Morphogenetic Circuitry Regulating Growth and Development in the Dimorphic Pathogen Penicillium marneffei

ABSTRACTPenicillium marneffeiis an emerging human-pathogenic fungus endemic to Southeast Asia. Like a number of other fungal pathogens,P. marneffeiexhibits temperature-dependent dimorphic growth and grows in two distinct cellular morphologies, hyphae at 25°C and yeast cells at 37°C. Hyphae can differentiate to produce the infectious agents, asexual spores (conidia), which are inhaled into the host lung, where they are phagocytosed by pulmonary alveolar macrophages. Within macrophages, conidia germinate into unicellular yeast cells, which divide by fission. This minireview focuses on the current understanding of the genes required for the morphogenetic control of conidial germination, hyphal growth, asexual development, and yeast morphogenesis inP. marneffei.

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The CDC42 Homolog of the Dimorphic FungusPenicillium marneffei Is Required for Correct Cell Polarization during Growth but Not Development

Journal of Bacteriology ◽

10.1128/jb.183.11.3447-3457.2001 ◽

2001 ◽

Vol 183 (11) ◽

pp. 3447-3457 ◽

Cited By ~ 63

Author(s):

Kylie J. Boyce ◽

Michael J. Hynes ◽

Alex Andrianopoulos

Keyword(s):

Cell Shape ◽

Pathogenic Fungus ◽

Cell Polarization ◽

Dominant Negative ◽

Yeast Cells ◽

Polarized Growth ◽

Temperature Dependent ◽

Human Pathogenic Fungus ◽

Rho Family

ABSTRACT The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffeinamed cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflAfunction is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression ofcflA alleles in Aspergillus nidulansprevented conidiation.

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Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00128-12 ◽

2012 ◽

Vol 11 (8) ◽

pp. 1042-1054 ◽

Cited By ~ 34

Author(s):

Matthias Kretschmer ◽

Joyce Wang ◽

James W. Kronstad

Keyword(s):

Carbon Source ◽

Cryptococcus Neoformans ◽

Virulence Factor ◽

Fungal Pathogens ◽

Pathogenic Fungus ◽

Subsequent Work ◽

Content Type ◽

Capsule Production ◽

Human Pathogenic Fungus ◽

Host Tissues

ABSTRACTAn understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungusCryptococcus neoformansis important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth ofC. neoformanson fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor forC. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity forC. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence inC. neoformansby multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.

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The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

Eukaryotic Cell ◽

10.1128/ec.00227-10 ◽

2011 ◽

Vol 10 (3) ◽

pp. 343-351 ◽

Cited By ~ 87

Author(s):

Milene C. Vallejo ◽

Alisson L. Matsuo ◽

Luciane Ganiko ◽

Lia C. Soares Medeiros ◽

Kildare Miranda ◽

...

Keyword(s):

Extracellular Vesicles ◽

Fungal Pathogens ◽

Histoplasma Capsulatum ◽

Paracoccidioides Brasiliensis ◽

Pathogenic Fungus ◽

Yeast Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Dimorphic Fungus ◽

Content Type

ABSTRACTExosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such asCryptococcus neoformansandHistoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes inParacoccidioides brasiliensis. P. brasiliensisis a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ×g. P. brasiliensisantigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, withMarasmius oreadesagglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. InP. brasiliensiscells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role playedin vivoby vesicle components and α-galactosyl epitopes.

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Comprehensive transcription analysis of human pathogenic fungus Penicillium marneffei in mycelial and yeast cells

Medical Mycology ◽

10.3109/13693786.2012.678398 ◽

2012 ◽

Vol 50 (8) ◽

pp. 835-842 ◽

Cited By ~ 7

Author(s):

Xinyu Lin ◽

Yuping Ran ◽

Lantu Gou ◽

Fei He ◽

Ruifeng Zhang ◽

...

Keyword(s):

Pathogenic Fungus ◽

Penicillium Marneffei ◽

Yeast Cells ◽

Transcription Analysis ◽

Human Pathogenic Fungus

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Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00250-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 2-9 ◽

Cited By ~ 58

Author(s):

Frans M. Klis ◽

Chris G. de Koster ◽

Stanley Brul

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Candida Albicans ◽

Cell Size ◽

Pathogenic Fungus ◽

Turgor Pressure ◽

Hyphal Growth ◽

Biological Research ◽

Content Type ◽

Starting Point

ABSTRACTBionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeastSaccharomyces cerevisiaeand the polymorphic, pathogenic fungusCandida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation ofin vivovalues. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allowsC. albicansto cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.

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The Putative APSES Transcription Factor RgdA Governs Growth, Development, Toxigenesis, and Virulence in Aspergillus fumigatus

mSphere ◽

10.1128/msphere.00998-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Sang-Cheol Jun ◽

Yong-Ho Choi ◽

Min-Woo Lee ◽

Jae-Hyuk Yu ◽

Kwang-Soo Shin

Keyword(s):

Transcription Factor ◽

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Pathogenic Fungus ◽

Mrna Levels ◽

Transcript Levels ◽

Content Type ◽

Asexual Sporulation ◽

Human Pathogenic Fungus ◽

Growth Development

ABSTRACT The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus. The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA, brlA, and wetA were decreased in the ΔrgdA mutant compared to those in the wild type (WT). Moreover, ΔrgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the ΔrgdA mutant. Comparative transcriptomic analyses revealed that the ΔrgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the ΔrgdA mutant exhibited elevated production of GT, ΔrgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the ΔrgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling. IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus. This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus. Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.

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Unraveling the Molecular Basis of Temperature-Dependent Genetic Regulation in Penicillium marneffei

Eukaryotic Cell ◽

10.1128/ec.00159-13 ◽

2013 ◽

Vol 12 (9) ◽

pp. 1214-1224 ◽

Cited By ~ 16

Author(s):

Ence Yang ◽

Gang Wang ◽

Patrick C. Y. Woo ◽

Susanna K. P. Lau ◽

Wang-Ngai Chow ◽

...

Keyword(s):

High Throughput Sequencing ◽

Current Knowledge ◽

Genetic Regulation ◽

Gene Clusters ◽

Penicillium Marneffei ◽

Integrated Analysis ◽

Temperature Dependent ◽

Mycelial Form ◽

Content Type ◽

Heat Response

ABSTRACTPenicillium marneffeiis an opportunistic fungal pathogen endemic in Southeast Asia, causing lethal systemic infections in immunocompromised patients.P. marneffeigrows in a mycelial form at the ambient temperature of 25°C and transitions to a yeast form at 37°C. The ability to alternate between the mycelial and yeast forms at different temperatures, namely, thermal dimorphism, has long been considered critical for the pathogenicity ofP. marneffei, yet the underlying genetic mechanisms remain elusive. Here we employed high-throughput sequencing to unravel global transcriptional profiles ofP. marneffeiPM1 grown at 25 and 37°C. Among ∼11,000 protein-coding genes, 1,447 were overexpressed and 1,414 were underexpressed at 37°C. Counterintuitively, heat-responsive genes, predicted inP. marneffeithrough sequence comparison, did not tend to be overexpressed at 37°C. These results suggest thatP. marneffeimay take a distinct strategy of genetic regulation at the elevated temperature; the current knowledge concerning fungal heat response, based on studies of model fungal organisms, may not be applicable toP. marneffei. Our results further showed that the tandem repeat sequences (TRSs) are overrepresented in coding regions ofP. marneffeigenes, and TRS-containing genes tend to be overexpressed at 37°C. Furthermore, genomic sequences and expression data were integrated to characterize gene clusters, multigene families, and species-specific genes ofP. marneffei. In sum, we present an integrated analysis and a comprehensive resource toward a better understanding of temperature-dependent genetic regulation inP. marneffei.

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Identification of an Aminothiazole with Antifungal Activity against Intracellular Histoplasma capsulatum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00459-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4349-4359 ◽

Cited By ~ 13

Author(s):

Jessica A. Edwards ◽

Megan M. Kemski ◽

Chad A. Rappleye

Keyword(s):

Drug Targets ◽

Fungal Pathogens ◽

Histoplasma Capsulatum ◽

Host Cells ◽

Yeast Cells ◽

Aromatic Substituent ◽

Content Type ◽

Fungistatic Activity ◽

Phenotypic Screen ◽

Cell Components

ABSTRACTAs eukaryotes, fungi possess relatively few molecules sufficiently unique from mammalian cell components to be used as drug targets. Consequently, most current antifungals have significant host cell toxicity. Primary fungal pathogens (e.g.,Histoplasma) are of particular concern, as few antifungals are effective in treating them. To identify additional antifungal candidates for the treatment of histoplasmosis, we developed a high-throughput platform for monitoringHistoplasmagrowth and employed it in a phenotypic screen of 3,600 commercially available compounds. Seven hit compounds that inhibitedHistoplasmayeast growth were identified. Compound 41F5 has fungistatic activity againstHistoplasmayeast at micromolar concentrations, with a 50% inhibitory concentration (IC50) of 0.87 μM, and has the greatest selectivity for yeast (at least 62-fold) relative to host cells. Structurally, 41F5 consists of an aminothiazole core with an alicyclic substituent at the 2-position and an aromatic substituent at the 5-position. 41F5 inhibitsHistoplasmagrowth in liquid culture and similarly inhibits yeast cells within macrophages, the actual host environment of this fungal pathogen during infection. Importantly, 41F5 protects infected host cells fromHistoplasma-induced macrophage death, making this aminothiazole hit compound an excellent candidate for development as an antifungal forHistoplasmainfections.

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G-Protein Signaling Mediates Asexual Development at 25°C but Has No Effect on Yeast-Like Growth at 37°C in the Dimorphic Fungus Penicillium marneffei

Eukaryotic Cell ◽

10.1128/ec.1.3.440-447.2002 ◽

2002 ◽

Vol 1 (3) ◽

pp. 440-447 ◽

Cited By ~ 39

Author(s):

Sophie Zuber ◽

Michael J. Hynes ◽

Alex Andrianopoulos

Keyword(s):

G Protein ◽

Regulatory Gene ◽

Penicillium Marneffei ◽

Yeast Cells ◽

Asexual Development ◽

Protein Signaling ◽

Dimorphic Fungus ◽

Gene Encoding ◽

Opportunistic Human Pathogen

ABSTRACT The ascomycete Penicillium marneffei is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25°C, P. marneffei grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37°C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a Gα subunit of a heterotrimeric G protein from P. marneffei named gasA with high similarity to fadA in Aspergillus nidulans. Through the characterization of a ΔgasA strain and mutants carrying a dominant activating or a dominant interfering gasA allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth. A dominant activating gasA mutant whose mutation results in a G42-to-R change (gasA G42R) does not express brlA, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering gasA G203R mutant shows inappropriate brlA expression and conidiation. Interestingly, the gasA mutants have no apparent defect in dimorphic switching or yeast-like growth at 37°C. Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway.

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Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

Eukaryotic Cell ◽

10.1128/ec.00375-08 ◽

2009 ◽

Vol 8 (3) ◽

pp. 315-326 ◽

Cited By ~ 63

Author(s):

Alexander Idnurm ◽

Felicia J. Walton ◽

Anna Floyd ◽

Jennifer L. Reedy ◽

Joseph Heitman

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Insertional Mutagenesis ◽

Pathogenic Fungus ◽

Mammalian Host ◽

New Genes ◽

Strain Collection ◽

Mutant Strains ◽

Human Pathogenic Fungus

ABSTRACT A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

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