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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 hapXΔ mutant is modestly attenuated for virulence in a mouse inhalation model of cryptococcosis.A. Ten female BALB/c mice were infected intranasally with 5×104 cells of each of the strains indicated (H99 (WT), cfo1Δ, hap3Δ, hap3Δ::HAP3, and hap3Δ cfo1Δ) and the survival of the mice was monitored twice per day. Assessment of the p-values for survival did not reveal significant differences between WT-, hap3Δ::HAP3- and hap3Δ-infected mice, or for comparisons of survival for the cfo1Δ mutant versus the hap3Δ cfo1Δ double mutant. The WT and cfo1Δ strains show differences in virulence as previously reported [18]. B. Parallel virulence tests were performed for the hapXΔ, hapXΔ::HAPX, and hapXΔ cfo1Δ strains. Note that the same data for the WT (H99) strain and the cfo1Δ mutants are presented in both figures to allow comparisons with the hap mutants. Assessments of survival revealed significant differences between WT or the hapXΔ::HAPX strain and hapXΔ (p<0.05); however, no significant difference was found upon comparison of the survival for the cfo1Δ mutant versus the hapXΔ cfo1Δ double mutant.
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ppat-1001209-g008: The hapXΔ mutant is modestly attenuated for virulence in a mouse inhalation model of cryptococcosis.A. Ten female BALB/c mice were infected intranasally with 5×104 cells of each of the strains indicated (H99 (WT), cfo1Δ, hap3Δ, hap3Δ::HAP3, and hap3Δ cfo1Δ) and the survival of the mice was monitored twice per day. Assessment of the p-values for survival did not reveal significant differences between WT-, hap3Δ::HAP3- and hap3Δ-infected mice, or for comparisons of survival for the cfo1Δ mutant versus the hap3Δ cfo1Δ double mutant. The WT and cfo1Δ strains show differences in virulence as previously reported [18]. B. Parallel virulence tests were performed for the hapXΔ, hapXΔ::HAPX, and hapXΔ cfo1Δ strains. Note that the same data for the WT (H99) strain and the cfo1Δ mutants are presented in both figures to allow comparisons with the hap mutants. Assessments of survival revealed significant differences between WT or the hapXΔ::HAPX strain and hapXΔ (p<0.05); however, no significant difference was found upon comparison of the survival for the cfo1Δ mutant versus the hapXΔ cfo1Δ double mutant.

Mentions: The small capsule phenotype of the hap3Δ mutant, and the growth defects of the hap3Δ and hapXΔ mutants on hemin as the sole iron source (particularly when combined with the cfo1Δ mutation), prompted an investigation of the virulence of these mutants in a mouse inhalation model of cryptococcosis. We therefore inoculated mice with the WT strain, the cfo1Δ mutant and the hap3Δ or hapXΔ mutants. We also included the complemented mutants and the hap3Δ cfo1Δ and hapXΔ cfo1Δ double mutants. The rationale for including cfo1Δ in the analysis came from previous observations that loss of the Cfo1 ferroxidase or the iron permease Cft1 resulted in attenuated virulence [17], [18]. The Cfo1/Cft1 high-affinity iron uptake system is needed for the use of transferrin but not hemin in culture [17], [18]. We reasoned that loss of Cfo1 and the Hap proteins might interfere with the utilization of both transferrin and hemin, and further attenuate virulence beyond that observed for the cfo1Δ mutant alone. As seen in Figure 8A, deletion of HAP3 did not result in a virulence defect despite the growth defect on hemin (Figure 1) and the small capsule size observed in culture (Figure 5). Also, the double mutant, hap3Δ cfo1Δ, showed a delayed virulence phenotype similar to that of the cfo1Δ single mutant thus indicating no additive influence.


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 hapXΔ mutant is modestly attenuated for virulence in a mouse inhalation model of cryptococcosis.A. Ten female BALB/c mice were infected intranasally with 5×104 cells of each of the strains indicated (H99 (WT), cfo1Δ, hap3Δ, hap3Δ::HAP3, and hap3Δ cfo1Δ) and the survival of the mice was monitored twice per day. Assessment of the p-values for survival did not reveal significant differences between WT-, hap3Δ::HAP3- and hap3Δ-infected mice, or for comparisons of survival for the cfo1Δ mutant versus the hap3Δ cfo1Δ double mutant. The WT and cfo1Δ strains show differences in virulence as previously reported [18]. B. Parallel virulence tests were performed for the hapXΔ, hapXΔ::HAPX, and hapXΔ cfo1Δ strains. Note that the same data for the WT (H99) strain and the cfo1Δ mutants are presented in both figures to allow comparisons with the hap mutants. Assessments of survival revealed significant differences between WT or the hapXΔ::HAPX strain and hapXΔ (p<0.05); however, no significant difference was found upon comparison of the survival for the cfo1Δ mutant versus the hapXΔ cfo1Δ double mutant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991262&req=5

ppat-1001209-g008: The hapXΔ mutant is modestly attenuated for virulence in a mouse inhalation model of cryptococcosis.A. Ten female BALB/c mice were infected intranasally with 5×104 cells of each of the strains indicated (H99 (WT), cfo1Δ, hap3Δ, hap3Δ::HAP3, and hap3Δ cfo1Δ) and the survival of the mice was monitored twice per day. Assessment of the p-values for survival did not reveal significant differences between WT-, hap3Δ::HAP3- and hap3Δ-infected mice, or for comparisons of survival for the cfo1Δ mutant versus the hap3Δ cfo1Δ double mutant. The WT and cfo1Δ strains show differences in virulence as previously reported [18]. B. Parallel virulence tests were performed for the hapXΔ, hapXΔ::HAPX, and hapXΔ cfo1Δ strains. Note that the same data for the WT (H99) strain and the cfo1Δ mutants are presented in both figures to allow comparisons with the hap mutants. Assessments of survival revealed significant differences between WT or the hapXΔ::HAPX strain and hapXΔ (p<0.05); however, no significant difference was found upon comparison of the survival for the cfo1Δ mutant versus the hapXΔ cfo1Δ double mutant.
Mentions: The small capsule phenotype of the hap3Δ mutant, and the growth defects of the hap3Δ and hapXΔ mutants on hemin as the sole iron source (particularly when combined with the cfo1Δ mutation), prompted an investigation of the virulence of these mutants in a mouse inhalation model of cryptococcosis. We therefore inoculated mice with the WT strain, the cfo1Δ mutant and the hap3Δ or hapXΔ mutants. We also included the complemented mutants and the hap3Δ cfo1Δ and hapXΔ cfo1Δ double mutants. The rationale for including cfo1Δ in the analysis came from previous observations that loss of the Cfo1 ferroxidase or the iron permease Cft1 resulted in attenuated virulence [17], [18]. The Cfo1/Cft1 high-affinity iron uptake system is needed for the use of transferrin but not hemin in culture [17], [18]. We reasoned that loss of Cfo1 and the Hap proteins might interfere with the utilization of both transferrin and hemin, and further attenuate virulence beyond that observed for the cfo1Δ mutant alone. As seen in Figure 8A, deletion of HAP3 did not result in a virulence defect despite the growth defect on hemin (Figure 1) and the small capsule size observed in culture (Figure 5). Also, the double mutant, hap3Δ cfo1Δ, showed a delayed virulence phenotype similar to that of the cfo1Δ single mutant thus indicating no additive influence.

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