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HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans.

Jung WH, Saikia S, Hu G, Wang J, Fung CK, D'Souza C, White R, Kronstad JW - PLoS Pathog. (2010)

Bottom Line: However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression.Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease.Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Chung-Ang University, Gyeonggi-Do, Republic of Korea.

ABSTRACT
The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37°C. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

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The hap3Δ, hap5Δ and hapXΔ mutants grow poorly on low-iron media supplemented with hemin.Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid YPD medium, solid YNB medium (control), low-iron medium (YNB+100 µM BPS), low-iron medium supplemented with 100 µM FeCl3, 10 µM Hemin or 10 µM Feroxamine. The bottom panel shows a comparison of the growth of the ferroxidase mutant cfo1Δ with double mutants lacking the CFO1 gene and the HAP3, HAP5 or HAPX gene. For the double mutants, two independent strains of each mutant were tested as indicated. The plates were incubated at 30°C for two days.
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ppat-1001209-g001: The hap3Δ, hap5Δ and hapXΔ mutants grow poorly on low-iron media supplemented with hemin.Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid YPD medium, solid YNB medium (control), low-iron medium (YNB+100 µM BPS), low-iron medium supplemented with 100 µM FeCl3, 10 µM Hemin or 10 µM Feroxamine. The bottom panel shows a comparison of the growth of the ferroxidase mutant cfo1Δ with double mutants lacking the CFO1 gene and the HAP3, HAP5 or HAPX gene. For the double mutants, two independent strains of each mutant were tested as indicated. The plates were incubated at 30°C for two days.

Mentions: As shown in Figure 1, the wild type (WT) and the reconstructed mutant strains all had weak growth on low-iron medium, with especially poor growth observed for the hapXΔ, cfo1Δ and double mutants. Growth was restored for all of the strains upon addition of the siderophore feroxamine; however, ferric chloride allowed growth of all of the single mutants but not the double mutants lacking the HAP genes and CFO1. The hap3Δ, hap5Δ and hapXΔ mutants each showed weaker growth on medium with hemin as the sole iron source, and complementation with the corresponding genes restored growth to the WT level. Similarly, the double mutants all failed to grow on hemin. The poor growth of the mutants with hemin was further confirmed with assays in liquid media (Figure S2). The sequence alignments, deletion constructs and confirmation of the mutant genotypes are shown in Figures S3 and S4. Taken together, these results indicated that Hap3, Hap5 and HapX all contribute to iron utilization from hemin (e.g., by regulating uptake and/or processing functions); this finding prompted a further examination of the impact of hap mutations on iron-related transcription and virulence.


HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans.

Jung WH, Saikia S, Hu G, Wang J, Fung CK, D'Souza C, White R, Kronstad JW - PLoS Pathog. (2010)

The hap3Δ, hap5Δ and hapXΔ mutants grow poorly on low-iron media supplemented with hemin.Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid YPD medium, solid YNB medium (control), low-iron medium (YNB+100 µM BPS), low-iron medium supplemented with 100 µM FeCl3, 10 µM Hemin or 10 µM Feroxamine. The bottom panel shows a comparison of the growth of the ferroxidase mutant cfo1Δ with double mutants lacking the CFO1 gene and the HAP3, HAP5 or HAPX gene. For the double mutants, two independent strains of each mutant were tested as indicated. The plates were incubated at 30°C for two days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991262&req=5

ppat-1001209-g001: The hap3Δ, hap5Δ and hapXΔ mutants grow poorly on low-iron media supplemented with hemin.Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid YPD medium, solid YNB medium (control), low-iron medium (YNB+100 µM BPS), low-iron medium supplemented with 100 µM FeCl3, 10 µM Hemin or 10 µM Feroxamine. The bottom panel shows a comparison of the growth of the ferroxidase mutant cfo1Δ with double mutants lacking the CFO1 gene and the HAP3, HAP5 or HAPX gene. For the double mutants, two independent strains of each mutant were tested as indicated. The plates were incubated at 30°C for two days.
Mentions: As shown in Figure 1, the wild type (WT) and the reconstructed mutant strains all had weak growth on low-iron medium, with especially poor growth observed for the hapXΔ, cfo1Δ and double mutants. Growth was restored for all of the strains upon addition of the siderophore feroxamine; however, ferric chloride allowed growth of all of the single mutants but not the double mutants lacking the HAP genes and CFO1. The hap3Δ, hap5Δ and hapXΔ mutants each showed weaker growth on medium with hemin as the sole iron source, and complementation with the corresponding genes restored growth to the WT level. Similarly, the double mutants all failed to grow on hemin. The poor growth of the mutants with hemin was further confirmed with assays in liquid media (Figure S2). The sequence alignments, deletion constructs and confirmation of the mutant genotypes are shown in Figures S3 and S4. Taken together, these results indicated that Hap3, Hap5 and HapX all contribute to iron utilization from hemin (e.g., by regulating uptake and/or processing functions); this finding prompted a further examination of the impact of hap mutations on iron-related transcription and virulence.

Bottom Line: However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression.Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease.Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Chung-Ang University, Gyeonggi-Do, Republic of Korea.

ABSTRACT
The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37°C. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

Show MeSH
Related in: MedlinePlus