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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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Related in: MedlinePlus

HapX positively regulates the levels of the CIR1 and RIM101 transcripts.Quantification of HAPX, CIR1 and RIM101 transcripts by quantitative RT-PCR in WT, hapXΔ and cir1Δ strains under low-iron (top) and iron-replete (bottom) conditions. The data were normalized using 18S rRNA as an internal control and are presented as relative expression in comparison to the WT value set at 1. The data are from three biological and two technical replicates, and the bars represent standard deviations.
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ppat-1001209-g004: HapX positively regulates the levels of the CIR1 and RIM101 transcripts.Quantification of HAPX, CIR1 and RIM101 transcripts by quantitative RT-PCR in WT, hapXΔ and cir1Δ strains under low-iron (top) and iron-replete (bottom) conditions. The data were normalized using 18S rRNA as an internal control and are presented as relative expression in comparison to the WT value set at 1. The data are from three biological and two technical replicates, and the bars represent standard deviations.

Mentions: Overall, the microarray data therefore indicated interconnected levels of regulation for the three transcription factors. To confirm the relationships between the transcription factors, we employed quantitative RT-PCR with RNA prepared from the cir1Δ and hapXΔ mutants grown under low-iron and iron-replete conditions. As shown in Figure 4, the quantitative RT-PCR analysis supports the conclusion that HapX has a positive regulatory influence on the transcript levels of both CIR1 and RIM101. We also noticed that a larger influence of HapX on the CIR1 transcript was found under the iron-replete condition by quantitative RT-PCR. This may reflect the greater sensitivity of this method relative to microarray analysis. We should also note that O'Meara et al. [27] performed a microarray comparison of the rim101 mutant with the WT parental strain and found that Rim101 positively influences the expression of HAPX (4.8-fold).


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)

HapX positively regulates the levels of the CIR1 and RIM101 transcripts.Quantification of HAPX, CIR1 and RIM101 transcripts by quantitative RT-PCR in WT, hapXΔ and cir1Δ strains under low-iron (top) and iron-replete (bottom) conditions. The data were normalized using 18S rRNA as an internal control and are presented as relative expression in comparison to the WT value set at 1. The data are from three biological and two technical replicates, and the bars represent standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001209-g004: HapX positively regulates the levels of the CIR1 and RIM101 transcripts.Quantification of HAPX, CIR1 and RIM101 transcripts by quantitative RT-PCR in WT, hapXΔ and cir1Δ strains under low-iron (top) and iron-replete (bottom) conditions. The data were normalized using 18S rRNA as an internal control and are presented as relative expression in comparison to the WT value set at 1. The data are from three biological and two technical replicates, and the bars represent standard deviations.
Mentions: Overall, the microarray data therefore indicated interconnected levels of regulation for the three transcription factors. To confirm the relationships between the transcription factors, we employed quantitative RT-PCR with RNA prepared from the cir1Δ and hapXΔ mutants grown under low-iron and iron-replete conditions. As shown in Figure 4, the quantitative RT-PCR analysis supports the conclusion that HapX has a positive regulatory influence on the transcript levels of both CIR1 and RIM101. We also noticed that a larger influence of HapX on the CIR1 transcript was found under the iron-replete condition by quantitative RT-PCR. This may reflect the greater sensitivity of this method relative to microarray analysis. We should also note that O'Meara et al. [27] performed a microarray comparison of the rim101 mutant with the WT parental strain and found that Rim101 positively influences the expression of HAPX (4.8-fold).

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