HapX Positively and Negatively Regulates the Transcriptional Response to Iron Deprivation in Cryptococcus neoformans

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.

Author Summary

Cryptococcus neoformans causes life-threatening central nervous system infections in immunocompromised people such as AIDS patients. The competition for iron between pathogens such as C. neoformans and mammalian hosts is a key aspect of disease outcome. We previously identified and characterized the major iron regulatory protein Cir1 in C. neoformans, as well as proteins for the transport of iron-binding molecules (siderophores) and for high-affinity iron uptake. In this study, we examined the roles of additional regulatory proteins (Hap proteins) in the response to low-iron conditions and the use of host iron sources such as heme and transferrin. We discovered that the HapX protein has a conserved regulatory function to repress iron-dependent functions during iron deprivation, as well as a positive regulatory role for the expression of putative siderophore transporters. A hapX mutant was defective in the use of heme as an iron source in culture but was only modestly attenuated for virulence in mice. This result suggests that additional mechanisms for iron uptake must be available to support C. neoformans proliferation in the host, and that HapX may play an important role in environmental iron acquisition.