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Genes differentially expressed in conidia and hyphae of Aspergillus fumigatus upon exposure to human neutrophils.

Sugui JA, Kim HS, Zarember KA, Chang YC, Gallin JI, Nierman WC, Kwon-Chung KJ - PLoS ONE (2008)

Bottom Line: Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells.Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione.This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.

Methodology/principal findings: Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.

Conclusion: This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.

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Related in: MedlinePlus

Expression level of genes differentially regulated between conidia and hyphae.The relative fold-change in the mRNA levels of the genes fdh encoding an NAD-dependent formate dehydrogenase (A), icl encoding an isocitrate lyase (B) and pex11 encoding a peroxisomal biogenesis factor (C) were obtained by qRT-PCR. The assays were carried out with RNA isolated from hyphae and conidia exposed to neutrophils from two normal (grey bars) or two CGD (white bars) donors. Each bar represents a replicate carried out with neutrophils from a single donor (±SD of each qRT-PCR replicate). The relative fold-change represents the log2 ratio between fungal cells exposed to neutrophils and fungal cells without neutrophil challenge.
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pone-0002655-g003: Expression level of genes differentially regulated between conidia and hyphae.The relative fold-change in the mRNA levels of the genes fdh encoding an NAD-dependent formate dehydrogenase (A), icl encoding an isocitrate lyase (B) and pex11 encoding a peroxisomal biogenesis factor (C) were obtained by qRT-PCR. The assays were carried out with RNA isolated from hyphae and conidia exposed to neutrophils from two normal (grey bars) or two CGD (white bars) donors. Each bar represents a replicate carried out with neutrophils from a single donor (±SD of each qRT-PCR replicate). The relative fold-change represents the log2 ratio between fungal cells exposed to neutrophils and fungal cells without neutrophil challenge.

Mentions: Since CGD neutrophils inhibited conidial growth as efficiently as normal neutrophils, we first focused on the identification of common genes that showed alterations of expression in the presence of either normal or CGD neutrophils. Conidia were exposed to neutrophils from normal or CGD donors and microarray analysis was carried out. To analyze the microarray data, genes were grouped based on their expression vectors using the k-means algorithm. The selected groups of genes were again organized for similar expression patterns using Euclidean distance and hierarchical clustering with the average linkage clustering method of JCVI MEV (see Materials and Methods). One of the most significant findings from the Clustering analysis was the identification of 244 genes up-regulated in conidia upon exposure to neutrophils (Fig. 2A, Table S1). Since most of these genes were up-regulated in conidia but not in hyphae, it is likely that these genes are part of a conidial-specific response. A general classification, based on the annotated functions, showed that the major groups consisted of genes involved in transport, regulation of transcription, metabolism of molecules with 1–3 carbons and peroxisomal proteins (Fig. 2B). Many of these genes are involved in the beta-oxidation of fatty acids, acetate metabolism, glyoxylate cycle and peroxisome biogenesis, suggesting a reprogramming of conidial metabolic pathways in response to neutrophil exposure (Table 3). In order to confirm changes in expression levels, we chose three of the up-regulated genes, fdh, icl, pex11, and analyzed their expression levels by qRT-PCR. The fdh gene, encoding an NAD-dependent formate dehydrogenase, is one of the genes that showed the highest increases in mRNA levels (average of 4-fold increase compared to control). The icl and pex11 genes encode isocitrate lyase and the peroxisome biogenesis factor, respectively, which are putatively involved in fatty acid catabolism (Table 3). The qRT-PCR data confirmed that these genes were highly up-regulated only in conidia upon exposure to either normal or CGD neutrophils, (Fig. 3A–C).


Genes differentially expressed in conidia and hyphae of Aspergillus fumigatus upon exposure to human neutrophils.

Sugui JA, Kim HS, Zarember KA, Chang YC, Gallin JI, Nierman WC, Kwon-Chung KJ - PLoS ONE (2008)

Expression level of genes differentially regulated between conidia and hyphae.The relative fold-change in the mRNA levels of the genes fdh encoding an NAD-dependent formate dehydrogenase (A), icl encoding an isocitrate lyase (B) and pex11 encoding a peroxisomal biogenesis factor (C) were obtained by qRT-PCR. The assays were carried out with RNA isolated from hyphae and conidia exposed to neutrophils from two normal (grey bars) or two CGD (white bars) donors. Each bar represents a replicate carried out with neutrophils from a single donor (±SD of each qRT-PCR replicate). The relative fold-change represents the log2 ratio between fungal cells exposed to neutrophils and fungal cells without neutrophil challenge.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481287&req=5

pone-0002655-g003: Expression level of genes differentially regulated between conidia and hyphae.The relative fold-change in the mRNA levels of the genes fdh encoding an NAD-dependent formate dehydrogenase (A), icl encoding an isocitrate lyase (B) and pex11 encoding a peroxisomal biogenesis factor (C) were obtained by qRT-PCR. The assays were carried out with RNA isolated from hyphae and conidia exposed to neutrophils from two normal (grey bars) or two CGD (white bars) donors. Each bar represents a replicate carried out with neutrophils from a single donor (±SD of each qRT-PCR replicate). The relative fold-change represents the log2 ratio between fungal cells exposed to neutrophils and fungal cells without neutrophil challenge.
Mentions: Since CGD neutrophils inhibited conidial growth as efficiently as normal neutrophils, we first focused on the identification of common genes that showed alterations of expression in the presence of either normal or CGD neutrophils. Conidia were exposed to neutrophils from normal or CGD donors and microarray analysis was carried out. To analyze the microarray data, genes were grouped based on their expression vectors using the k-means algorithm. The selected groups of genes were again organized for similar expression patterns using Euclidean distance and hierarchical clustering with the average linkage clustering method of JCVI MEV (see Materials and Methods). One of the most significant findings from the Clustering analysis was the identification of 244 genes up-regulated in conidia upon exposure to neutrophils (Fig. 2A, Table S1). Since most of these genes were up-regulated in conidia but not in hyphae, it is likely that these genes are part of a conidial-specific response. A general classification, based on the annotated functions, showed that the major groups consisted of genes involved in transport, regulation of transcription, metabolism of molecules with 1–3 carbons and peroxisomal proteins (Fig. 2B). Many of these genes are involved in the beta-oxidation of fatty acids, acetate metabolism, glyoxylate cycle and peroxisome biogenesis, suggesting a reprogramming of conidial metabolic pathways in response to neutrophil exposure (Table 3). In order to confirm changes in expression levels, we chose three of the up-regulated genes, fdh, icl, pex11, and analyzed their expression levels by qRT-PCR. The fdh gene, encoding an NAD-dependent formate dehydrogenase, is one of the genes that showed the highest increases in mRNA levels (average of 4-fold increase compared to control). The icl and pex11 genes encode isocitrate lyase and the peroxisome biogenesis factor, respectively, which are putatively involved in fatty acid catabolism (Table 3). The qRT-PCR data confirmed that these genes were highly up-regulated only in conidia upon exposure to either normal or CGD neutrophils, (Fig. 3A–C).

Bottom Line: Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells.Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione.This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.

Methodology/principal findings: Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.

Conclusion: This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.

Show MeSH
Related in: MedlinePlus