In this study, we describe the function of six C. neoformans genes that share homology with those involved in the mammalian SREBP pathway. These include SRE1, SCP1, SFB2, STP1, KAP123, and GSK3. Involvement of SFB2, KAP123, and GSK3 in the SREBP pathway has not been reported in other fungi. In addition, we found a new gene, DAM1, whose mutation slightly affected transient accumulation of the nuclear form of Sre1 in C. neoformans. More importantly, mutations in these genes resulted in growth defects under low-oxygen conditions at 37°C. Mutations in SCP1 and STP1 caused a major decrease in the accumulation of both precursor and nuclear forms of Sre1. Concurrently, these mutants with a reduction in ergosterol content were hypersensitive to several chemical agents, including triazoles, CoCl2, and the agents generating ROS and RNS, and were less virulent. Table summarizes all the phenotypes of the mutants in this study. These findings confirm the importance of the Sre1 pathway as a link between sterol biosynthesis, oxygen sensing, CoCl2 sensitivity, and virulence in C. neoformans.
Summary of mutant phenotypes compared to wild type
The existence of a conserved SREBP pathway in C. neoformans
was demonstrated by the identification of genes whose mutations affected the maturation steps of Sre1 before and after production of the nuclear form of the protein. Since it is known that Sre1 regulates its own expression in mammals and in S. pombe
), it is possible that a reduced amount of the nuclear Sre1 form in C. neoformans
would also cause a reduction in the accumulated precursor as seen in scp1
mutants. However, it is not clear why the Sre1 precursor levels were not affected in sfb2
mutants despite low levels of the nuclear Sre1 form in sfb2
The other phenotypic defects, such as low ergosterol production or hypersensitivity to triazoles and CoCl2
, that resulted from mutations in genes that function in steps after rather than before generation of the nuclear Sre1 were less compelling. It is possible that once the mature Sre1 nuclear form is produced, its transport into the nucleus or quick turnover has less of an impact on the various functions affected by Sre1. Alternatively, other proteins functionally related to the mutated protein might compensate for the defects in the mutants. For example, C. neoformans
Kap123 shares similarity with karyopherin β, Kap123, and two other proteins of S. cerevisiae
(Kap104 and Pse1). In S. cerevisia
e, it has been proposed that in the absence of Kap123 these karyopherins are able to supplant the role of Kap123 in importing molecules (40
). Although we did not detect any gene closely related to KAP123
in C. neoformans
, other unidentified proteins may functionally substitute for Kap123 and reduce the severity of the phenotype under stressful conditions.
The present study has identified most of the key players of the SREBP pathway in C. neoformans
except for Insig and S1P. In fission yeast, deletion of the INSIG
, had no effect on Sre1 activation, and ins1+
is dedicated to regulation of the HMG-coenzyme A reductase, Hmg1 (2
). In budding yeast, a homolog of SREBP was not found, but the Insig homolog called Nsg1 acts as a chaperone and is involved in the regulation of sterol biosynthesis. It specifically stabilizes Hmg2, one of the two HMG-coenzyme A isoenzymes that catalyze the rate-limiting step in sterol biosynthesis (9
). It is possible that exit of Sre1/Scp1 from the ER is regulated by a different protein or mechanism in different fungal species. We were unable to delete the putative S1P
homolog, CNH01120, in a serotype D strain. In mice, homozygous germ line disruptions of S1P
were embryonically lethal (44
), suggesting that CNH01120 may also be an essential gene in serotype D strains. Thus, the function of CNH01120 in serotype D strains remains unclear. We successfully deleted the putative S1P
homolog, CNAG_05446.2, in the serotype A strain H99 (data not shown). This deletant was resistant to CoCl2
, which is inconsistent with the sensitive phenotype of all other Sre1 pathway mutants. Since Sre1 processing in the Golgi complex requires two sequential cleavage events by S1P and S2P in mammalian systems, it is possible that C. neoformans
uses different proteins or mechanisms to release the N-terminal transcriptional factor domain of Sre1 in the Golgi complex.
(CNF02850), the newly identified gene which slightly affected turnover of the Sre1 nuclear form, is annotated as a hypothetical gene in the C. neoformans
database and is absent in S. cerevisiae
. The BCAS2 family consists of several eukaryotic sequences of unknown function, and the human Dam1 is a putative spliceosome-associated protein (29
). It is unclear how Dam1 operates in the Sre1 pathway of C. neoformans
. Western blot analysis showed that mutations in both DAM1
affected the turnover rate of the nuclear Sre1 form, which suggests that Dam1 may function in the later steps of Sre1 processing.
In mammals, the central regulator of hypoxic gene expression is the heterodimeric transcription factor HIF (hypoxia-inducible factor) (for reviews, see references 13
). The degradation and activity of the HIF-1α subunit are both regulated by oxygen-dependent posttranslational hydroxyl modifications catalyzed by enzymes belonging to the 2-oxoglutarate-Fe(II) dioxygenase family (31
). In fission yeast, the Sre1 transcription factor is a principal regulator of low-oxygen gene expression (41
). Recently, a prolyl 4-hydroxylase-like 2-oxoglutarate-Fe(II) dioxygenase, Ofd1, that accelerates Sre1 degradation in the presence of oxygen was identified in S. pombe
). C. neoformans
contains a gene, CNA04020, that shares 42.8% similarity to Ofd1, and its expression is regulated by oxygen levels (H. Lee and Y. Chang, unpublished data). However, unlike in S. pombe
, deletion of CNA04020 did not affect Sre1 levels under 21% or 1% oxygen conditions. In the C. neoformans
genome, there are two more prolyl 4-hydroxylase α-subunit domain (P4Hc)-containing genes, CNF02010 and CNBK2780, but the similarity to Ofd1 is limited only to the P4Hc domain in these two genes. It remains to be elucidated if C. neoformans
utilizes oxygen-dependent posttranslational hydroxyl modifications to regulate Sre1 levels.
This study was undertaken to determine whether the ability to grow under low-oxygen conditions is an essential property in the pathobiology of C. neoformans. Five of the six low-oxygen-sensitive mutants for the SREBP pathway showed a significant reduction in virulence. However, the kap123 mutant, which is as sensitive to low oxygen as the stp1 mutant, showed virulence comparable to wild type. We also examined the virulence of six other low-oxygen-sensitive mutants from our collection that are not involved in the Sre1 pathway. Again, one of these mutants was as virulent as the wild-type strain, while the other five mutants showed a significant reduction in virulence (data not shown). Oxygen is a key requirement for many pathways, ranging from sterol to heme biosynthesis. Since sterol content of the kap123 mutant, one of the six that showed no reduction in virulence, was not significantly altered, it is possible that the kap123 mutation affects other cellular processes important for growth under low-oxygen conditions in vitro but can be circumvented in vivo. Therefore, the relationship between the ability to grow under low-oxygen conditions and virulence merits further study.
In other organisms, several of the genes we identified in the Sre1 pathway, such as SFB2
, and GSK3
, are known to be involved in many other physiological processes besides Sre1 processing. Moreover, the sfb2
mutants of C. neoformans
were not sensitive to low-oxygen conditions at 30°C (Fig. ). In fact, most of the mutants that we obtained by screening for a growth defect under low-oxygen conditions did not influence Sre1 production. This observation suggests that only a limited overlap exists between the Sre1 pathway and oxygen-sensing pathways in C. neoformans.
Interestingly, all the Sre1 pathway mutants were sensitive to CoCl2
and conversely, all the previously identified CoCl2
-sensitive mutants showed sensitivity to low-oxygen conditions (18
). Mutants sensitive to CoCl2
apparently are defective in multiple physiological processes that affect growth. Further studies of these mutants showed that mitochondria play a prominent role in CoCl2
sensitivity. Although most of the Sre1 pathway mutants displayed the CoCl2
-sensitive phenotype, their mitochondrial function was not significantly impacted as assessed by their mitochondrial membrane potential and respiration efficiency. In addition, most of the low-oxygen-sensitive mutants obtained from our screening were not sensitive to CoCl2
(data not shown). Collectively, these observations indicate that the Sre1 pathway plays an important role in the pathobiology of C
, and cryptococcal cells employ complex processes to overcome unfavorable environmental conditions, such as the low oxygen levels they encounter in the animal host.