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Cell-type-specific transcriptional profiles of the dimorphic pathogen Penicillium marneffei reflect distinct reproductive, morphological, and environmental demands.

Pasricha S, Payne M, Canovas D, Pase L, Ngaosuwankul N, Beard S, Oshlack A, Smyth GK, Chaiyaroj SC, Boyce KJ, Andrianopoulos A - G3 (Bethesda) (2013)

Bottom Line: Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B.The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late.Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Melbourne, Victoria 3010, Australia.

ABSTRACT
Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify "phase or cell-state-specific" gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase-encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

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Cell-state–specific differential gene expression in P. marneffei. Microarray analysis was conducted with wild-type P. marneffei under three growth conditions, 25° on ANM plates for asexual development (4 d), agitated in liquid BHI for hyphal growth at 25° (2 d), and yeast growth at 37° (4 d of growth followed by 10 ml transferred to fresh medium for an additional 2 d). The data set represents the top 500 differentially expressed genes. Points closest to the top corner represent genes with maximum log expression in the yeast phase. Points in the bottom right corner are genes most highly expressed during conidiation, and points at the bottom left corner of the triangle represent genes differentially expressed highest in the hyphal phase. Points between two corners, along the sides of the triangle, depict clones upregulated in two conditions with respect to the one in the opposite corner. Probes expressed evenly among the three states were omitted from the data set used to generate this representation, accounting for the absence of spots in the center of the triangle.
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fig1: Cell-state–specific differential gene expression in P. marneffei. Microarray analysis was conducted with wild-type P. marneffei under three growth conditions, 25° on ANM plates for asexual development (4 d), agitated in liquid BHI for hyphal growth at 25° (2 d), and yeast growth at 37° (4 d of growth followed by 10 ml transferred to fresh medium for an additional 2 d). The data set represents the top 500 differentially expressed genes. Points closest to the top corner represent genes with maximum log expression in the yeast phase. Points in the bottom right corner are genes most highly expressed during conidiation, and points at the bottom left corner of the triangle represent genes differentially expressed highest in the hyphal phase. Points between two corners, along the sides of the triangle, depict clones upregulated in two conditions with respect to the one in the opposite corner. Probes expressed evenly among the three states were omitted from the data set used to generate this representation, accounting for the absence of spots in the center of the triangle.

Mentions: One of the goals of this study was to identify a set of genes that represent cell-type–specific markers to be used for genetic screens and studies of cell function. A second goal was to identify potentially interesting genes for further characterization. Random probes from the libraries (5376) along with 38 probes for 34 previously characterized genes, spotted in duplicate, were arrayed and used in these experiments. More than 38% of the microarray probes were found to show statistically significant differences in expression levels between the three cell states (FDR < 0.05). The 500 most significant clones were selected for further study. Of the top 500 probes, 31.2% were found to be hyphal-specific, 18.2% were conidiation-specific, and 20.2% were yeast-specific (Figure 1). Here, we define hyphal-specific probes to be those with at least 50% higher expression in hyphal cells compared to either of the other two states (similarly for conidiation-specific and yeast-specific probes). The data successfully show dynamic changes in gene expression profiles among the three states and clearly identify probes expressed predominantly in one cell state. Such probes correspond to genes with possible roles in cell-type maintenance, whether they are morphological or physiological.


Cell-type-specific transcriptional profiles of the dimorphic pathogen Penicillium marneffei reflect distinct reproductive, morphological, and environmental demands.

Pasricha S, Payne M, Canovas D, Pase L, Ngaosuwankul N, Beard S, Oshlack A, Smyth GK, Chaiyaroj SC, Boyce KJ, Andrianopoulos A - G3 (Bethesda) (2013)

Cell-state–specific differential gene expression in P. marneffei. Microarray analysis was conducted with wild-type P. marneffei under three growth conditions, 25° on ANM plates for asexual development (4 d), agitated in liquid BHI for hyphal growth at 25° (2 d), and yeast growth at 37° (4 d of growth followed by 10 ml transferred to fresh medium for an additional 2 d). The data set represents the top 500 differentially expressed genes. Points closest to the top corner represent genes with maximum log expression in the yeast phase. Points in the bottom right corner are genes most highly expressed during conidiation, and points at the bottom left corner of the triangle represent genes differentially expressed highest in the hyphal phase. Points between two corners, along the sides of the triangle, depict clones upregulated in two conditions with respect to the one in the opposite corner. Probes expressed evenly among the three states were omitted from the data set used to generate this representation, accounting for the absence of spots in the center of the triangle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815061&req=5

fig1: Cell-state–specific differential gene expression in P. marneffei. Microarray analysis was conducted with wild-type P. marneffei under three growth conditions, 25° on ANM plates for asexual development (4 d), agitated in liquid BHI for hyphal growth at 25° (2 d), and yeast growth at 37° (4 d of growth followed by 10 ml transferred to fresh medium for an additional 2 d). The data set represents the top 500 differentially expressed genes. Points closest to the top corner represent genes with maximum log expression in the yeast phase. Points in the bottom right corner are genes most highly expressed during conidiation, and points at the bottom left corner of the triangle represent genes differentially expressed highest in the hyphal phase. Points between two corners, along the sides of the triangle, depict clones upregulated in two conditions with respect to the one in the opposite corner. Probes expressed evenly among the three states were omitted from the data set used to generate this representation, accounting for the absence of spots in the center of the triangle.
Mentions: One of the goals of this study was to identify a set of genes that represent cell-type–specific markers to be used for genetic screens and studies of cell function. A second goal was to identify potentially interesting genes for further characterization. Random probes from the libraries (5376) along with 38 probes for 34 previously characterized genes, spotted in duplicate, were arrayed and used in these experiments. More than 38% of the microarray probes were found to show statistically significant differences in expression levels between the three cell states (FDR < 0.05). The 500 most significant clones were selected for further study. Of the top 500 probes, 31.2% were found to be hyphal-specific, 18.2% were conidiation-specific, and 20.2% were yeast-specific (Figure 1). Here, we define hyphal-specific probes to be those with at least 50% higher expression in hyphal cells compared to either of the other two states (similarly for conidiation-specific and yeast-specific probes). The data successfully show dynamic changes in gene expression profiles among the three states and clearly identify probes expressed predominantly in one cell state. Such probes correspond to genes with possible roles in cell-type maintenance, whether they are morphological or physiological.

Bottom Line: Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B.The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late.Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Melbourne, Victoria 3010, Australia.

ABSTRACT
Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify "phase or cell-state-specific" gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase-encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

Show MeSH
Related in: MedlinePlus