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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 and hap5Δ mutants have a smaller capsule than WT or hapXΔ cells.A. Cells were cultured in low-iron medium (LIM) at 30°C for 48 h. Capsule formation was assessed by India ink staining for the WT strain, the hap3Δ, hap5Δ, hapXΔ, cfo1Δ, hap3Δ cfo1Δ, hap5Δ cfo1Δ, and hapXΔ cfo1Δ mutants, as well as the reconstituted strains. Bar = 10 µ. B. Fifty cells from each strain were measured to determine cell diameter and capsule radius. The capsule sizes of hap3Δ, hap5Δ, hap3Δ cfo1Δ (both #1 and #2) and hap5Δ cfo1Δ (both #54 and #61) cells were statistically different (t-test, p<0.001) from the sizes for cells of the WT strain, the hapXΔ cfo1Δ mutants and the reconstituted strains. Each bar represents the average of 50 measurements with standard deviations.
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ppat-1001209-g005: The hap3Δ and hap5Δ mutants have a smaller capsule than WT or hapXΔ cells.A. Cells were cultured in low-iron medium (LIM) at 30°C for 48 h. Capsule formation was assessed by India ink staining for the WT strain, the hap3Δ, hap5Δ, hapXΔ, cfo1Δ, hap3Δ cfo1Δ, hap5Δ cfo1Δ, and hapXΔ cfo1Δ mutants, as well as the reconstituted strains. Bar = 10 µ. B. Fifty cells from each strain were measured to determine cell diameter and capsule radius. The capsule sizes of hap3Δ, hap5Δ, hap3Δ cfo1Δ (both #1 and #2) and hap5Δ cfo1Δ (both #54 and #61) cells were statistically different (t-test, p<0.001) from the sizes for cells of the WT strain, the hapXΔ cfo1Δ mutants and the reconstituted strains. Each bar represents the average of 50 measurements with standard deviations.

Mentions: We next examined the hap3Δ, hap5Δ, and hapXΔ mutants, and the double mutants lacking the HAP genes and CFO1, for expression of the three major virulence factors: capsule, melanin, and growth at 37°C. Initially, we found that the hap3Δ and hap5Δ mutants (and the double mutants for these genes and cfo1Δ) produced a smaller capsule than the WT strain or the hapX mutant (Figure 5). Complementation with the HAP3 and HAP5 genes restored normal capsule size. We also found that all of the mutants displayed WT levels of melanin formation when tested on medium containing the substrate L-DOPA (data not shown). This result was consistent with the lack of an influence of the hap3Δ or hapXΔ mutations on the transcript levels for the laccase gene LAC1 (gene ID: CNAG_03465).


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 have a smaller capsule than WT or hapXΔ cells.A. Cells were cultured in low-iron medium (LIM) at 30°C for 48 h. Capsule formation was assessed by India ink staining for the WT strain, the hap3Δ, hap5Δ, hapXΔ, cfo1Δ, hap3Δ cfo1Δ, hap5Δ cfo1Δ, and hapXΔ cfo1Δ mutants, as well as the reconstituted strains. Bar = 10 µ. B. Fifty cells from each strain were measured to determine cell diameter and capsule radius. The capsule sizes of hap3Δ, hap5Δ, hap3Δ cfo1Δ (both #1 and #2) and hap5Δ cfo1Δ (both #54 and #61) cells were statistically different (t-test, p<0.001) from the sizes for cells of the WT strain, the hapXΔ cfo1Δ mutants and the reconstituted strains. Each bar represents the average of 50 measurements with standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001209-g005: The hap3Δ and hap5Δ mutants have a smaller capsule than WT or hapXΔ cells.A. Cells were cultured in low-iron medium (LIM) at 30°C for 48 h. Capsule formation was assessed by India ink staining for the WT strain, the hap3Δ, hap5Δ, hapXΔ, cfo1Δ, hap3Δ cfo1Δ, hap5Δ cfo1Δ, and hapXΔ cfo1Δ mutants, as well as the reconstituted strains. Bar = 10 µ. B. Fifty cells from each strain were measured to determine cell diameter and capsule radius. The capsule sizes of hap3Δ, hap5Δ, hap3Δ cfo1Δ (both #1 and #2) and hap5Δ cfo1Δ (both #54 and #61) cells were statistically different (t-test, p<0.001) from the sizes for cells of the WT strain, the hapXΔ cfo1Δ mutants and the reconstituted strains. Each bar represents the average of 50 measurements with standard deviations.
Mentions: We next examined the hap3Δ, hap5Δ, and hapXΔ mutants, and the double mutants lacking the HAP genes and CFO1, for expression of the three major virulence factors: capsule, melanin, and growth at 37°C. Initially, we found that the hap3Δ and hap5Δ mutants (and the double mutants for these genes and cfo1Δ) produced a smaller capsule than the WT strain or the hapX mutant (Figure 5). Complementation with the HAP3 and HAP5 genes restored normal capsule size. We also found that all of the mutants displayed WT levels of melanin formation when tested on medium containing the substrate L-DOPA (data not shown). This result was consistent with the lack of an influence of the hap3Δ or hapXΔ mutations on the transcript levels for the laccase gene LAC1 (gene ID: CNAG_03465).

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