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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

The hap3Δ and hap5Δ mutants exhibit poor growth on acetate, ethanol and sucrose.Ten-fold serial dilutions of the WT, hap3Δ, hap5Δ and hapXΔ mutants and the reconstituted strains (starting at 104 cells) were spotted on YNB supplemented with the indicated carbon sources (all at 2%). Plates were incubated at 30°C for two (on glucose and sucrose) or three (on acetate and ethanol) days.
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ppat-1001209-g007: The hap3Δ and hap5Δ mutants exhibit poor growth on acetate, ethanol and sucrose.Ten-fold serial dilutions of the WT, hap3Δ, hap5Δ and hapXΔ mutants and the reconstituted strains (starting at 104 cells) were spotted on YNB supplemented with the indicated carbon sources (all at 2%). Plates were incubated at 30°C for two (on glucose and sucrose) or three (on acetate and ethanol) days.

Mentions: We also examined the carbon source requirements for the hap mutants because it is known that the HAP genes in other fungi regulate growth on non-fermentable carbon sources [29], [30]. The C. neoformans mutants were tested for growth on glucose, sucrose, acetate and ethanol, and it was found that the hap3Δ and hap5Δ mutants showed weaker growth on the latter three carbon sources (Figure 7). Unexpectedly, the hapXΔ mutant did not show these growth phenotypes even though the transcriptional profiling suggested that HapX and Hap3 both participate in the regulation of genes needed for respiratory growth (Figure 3). It is possible that the growth differences between the hap3Δ and hapXΔ mutants reflect differential influences on the expression of respiratory functions under the iron-replete conditions in the media. The addition of hemin did not rescue the growth defects of the hap3Δ and hap5Δ mutants suggesting that lack of this iron source is not responsible for the observed phenotypes on acetate and ethanol (data not shown). Overall, these analyses provided several examples of distinct phenotypes for the hapXΔ mutant relative to the hap3Δ and hap5Δ mutants. In a further example, we noted that the hap3Δ and hap5Δ mutants were more sensitive to growth in the presence of 0.01% SDS, compared with the hapXΔ mutant and the WT strain (data not shown). As described below, the distinct behavior of the hapXΔ mutant extends to 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 and hap5Δ mutants exhibit poor growth on acetate, ethanol and sucrose.Ten-fold serial dilutions of the WT, hap3Δ, hap5Δ and hapXΔ mutants and the reconstituted strains (starting at 104 cells) were spotted on YNB supplemented with the indicated carbon sources (all at 2%). Plates were incubated at 30°C for two (on glucose and sucrose) or three (on acetate and ethanol) days.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001209-g007: The hap3Δ and hap5Δ mutants exhibit poor growth on acetate, ethanol and sucrose.Ten-fold serial dilutions of the WT, hap3Δ, hap5Δ and hapXΔ mutants and the reconstituted strains (starting at 104 cells) were spotted on YNB supplemented with the indicated carbon sources (all at 2%). Plates were incubated at 30°C for two (on glucose and sucrose) or three (on acetate and ethanol) days.
Mentions: We also examined the carbon source requirements for the hap mutants because it is known that the HAP genes in other fungi regulate growth on non-fermentable carbon sources [29], [30]. The C. neoformans mutants were tested for growth on glucose, sucrose, acetate and ethanol, and it was found that the hap3Δ and hap5Δ mutants showed weaker growth on the latter three carbon sources (Figure 7). Unexpectedly, the hapXΔ mutant did not show these growth phenotypes even though the transcriptional profiling suggested that HapX and Hap3 both participate in the regulation of genes needed for respiratory growth (Figure 3). It is possible that the growth differences between the hap3Δ and hapXΔ mutants reflect differential influences on the expression of respiratory functions under the iron-replete conditions in the media. The addition of hemin did not rescue the growth defects of the hap3Δ and hap5Δ mutants suggesting that lack of this iron source is not responsible for the observed phenotypes on acetate and ethanol (data not shown). Overall, these analyses provided several examples of distinct phenotypes for the hapXΔ mutant relative to the hap3Δ and hap5Δ mutants. In a further example, we noted that the hap3Δ and hap5Δ mutants were more sensitive to growth in the presence of 0.01% SDS, compared with the hapXΔ mutant and the WT strain (data not shown). As described below, the distinct behavior of the hapXΔ mutant extends to 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