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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

Effect of menadione on growth of the wild type strain B-5233 and the deletant strains T9 and T10.Conidia from B-5233 (A and D), T9 (B and E) and T10 (C and F) were pre-incubated for 8 h to allow germination before menadione was added. Samples were incubated for an additional 24 h and observed by bright-field microscopy. Panels A–C, no menadione added. Panels D–F, 40 µg/ml menadione. Magnification bar in F also applies to A–E.
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pone-0002655-g007: Effect of menadione on growth of the wild type strain B-5233 and the deletant strains T9 and T10.Conidia from B-5233 (A and D), T9 (B and E) and T10 (C and F) were pre-incubated for 8 h to allow germination before menadione was added. Samples were incubated for an additional 24 h and observed by bright-field microscopy. Panels A–C, no menadione added. Panels D–F, 40 µg/ml menadione. Magnification bar in F also applies to A–E.

Mentions: Interestingly, four genes that are predicted to be involved in iron/copper assimilation were also differentially regulated. These genes encode two copper transporters (Afu2g03730 and Afu6g020810), one metalloreductase (Afu6g02820) and one ferric-chelate reductase (Afu6g13750) (Table 4). The genes encoding the copper transporter (Afug6g02810) and the metalloreductase (Afug602820) are located on chromosome 6 adjacent to the gene encoding a putative GPI-anchored protein (Afu6g02800) that was also differentially regulated (Table 4). Using qRT-PCR, we confirmed that the mRNA levels of these five genes were higher in hyphae exposed to normal neutrophils compared to CGD neutrophils (Fig. 6A). To evaluate whether some of these genes were relevant during interaction with neutrophils, we chose to delete the three genes that are located adjacent to each other on chromosome 6 (Afu6g02800-02820). Two independent strains, T9 and T10, containing a deletion of the three genes were selected for the experiments. Since one of the hypotheses proposes that these genes are involved in the resistance to oxidative stress, T9 and T10 were assayed with the oxidizing agents hydrogen peroxide and menadione. Menadione, a chemical that generates superoxide ion through enzymatic redox cycling, has been previously used to assess oxidative stress response in A. fumigatus and Sacharomyces cerevisiae [28], [29]. The mutant strains did not show an increased susceptibility to hydrogen peroxide (data not shown). However, in the presence of menadione, T9 and T10 were no longer able to germinate and develop mycelia while B-5233 grew robustly (Fig. 7). The defective growth of T9 and T10 suggests that one or more of the deleted genes are likely to be involved in resistance to menadione. However, in the interaction assays with normal neutrophils the mutant strains did not show an increased susceptibility to the phagocytes (data not shown). Our findings suggest that although these three genes are important for the response to the oxidative agent menadione, they are not essential for resistance to neutrophils. Although we were able to identify, by the SAM method, the genes whose expression levels were significantly higher in hyphae exposed to normal neutrophils relative to CGD neutrophils, we did not detect any genes whose expression levels were significantly lower (fold-change ≥2) in hyphae exposed to normal neutrophils.


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)

Effect of menadione on growth of the wild type strain B-5233 and the deletant strains T9 and T10.Conidia from B-5233 (A and D), T9 (B and E) and T10 (C and F) were pre-incubated for 8 h to allow germination before menadione was added. Samples were incubated for an additional 24 h and observed by bright-field microscopy. Panels A–C, no menadione added. Panels D–F, 40 µg/ml menadione. Magnification bar in F also applies to A–E.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481287&req=5

pone-0002655-g007: Effect of menadione on growth of the wild type strain B-5233 and the deletant strains T9 and T10.Conidia from B-5233 (A and D), T9 (B and E) and T10 (C and F) were pre-incubated for 8 h to allow germination before menadione was added. Samples were incubated for an additional 24 h and observed by bright-field microscopy. Panels A–C, no menadione added. Panels D–F, 40 µg/ml menadione. Magnification bar in F also applies to A–E.
Mentions: Interestingly, four genes that are predicted to be involved in iron/copper assimilation were also differentially regulated. These genes encode two copper transporters (Afu2g03730 and Afu6g020810), one metalloreductase (Afu6g02820) and one ferric-chelate reductase (Afu6g13750) (Table 4). The genes encoding the copper transporter (Afug6g02810) and the metalloreductase (Afug602820) are located on chromosome 6 adjacent to the gene encoding a putative GPI-anchored protein (Afu6g02800) that was also differentially regulated (Table 4). Using qRT-PCR, we confirmed that the mRNA levels of these five genes were higher in hyphae exposed to normal neutrophils compared to CGD neutrophils (Fig. 6A). To evaluate whether some of these genes were relevant during interaction with neutrophils, we chose to delete the three genes that are located adjacent to each other on chromosome 6 (Afu6g02800-02820). Two independent strains, T9 and T10, containing a deletion of the three genes were selected for the experiments. Since one of the hypotheses proposes that these genes are involved in the resistance to oxidative stress, T9 and T10 were assayed with the oxidizing agents hydrogen peroxide and menadione. Menadione, a chemical that generates superoxide ion through enzymatic redox cycling, has been previously used to assess oxidative stress response in A. fumigatus and Sacharomyces cerevisiae [28], [29]. The mutant strains did not show an increased susceptibility to hydrogen peroxide (data not shown). However, in the presence of menadione, T9 and T10 were no longer able to germinate and develop mycelia while B-5233 grew robustly (Fig. 7). The defective growth of T9 and T10 suggests that one or more of the deleted genes are likely to be involved in resistance to menadione. However, in the interaction assays with normal neutrophils the mutant strains did not show an increased susceptibility to the phagocytes (data not shown). Our findings suggest that although these three genes are important for the response to the oxidative agent menadione, they are not essential for resistance to neutrophils. Although we were able to identify, by the SAM method, the genes whose expression levels were significantly higher in hyphae exposed to normal neutrophils relative to CGD neutrophils, we did not detect any genes whose expression levels were significantly lower (fold-change ≥2) in hyphae exposed to normal neutrophils.

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