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activity.

Gene profiling analysis however, does not explain the Pho4-dependent stress phenotypes identified from the QFA screen so we wondered if acid phosphatase activity was important for stress resistance. To identify C. albicans mutants with defective acid phosphatase activity, the C. albicans deletion libraries were screened under phosphate-rich and phosphate-deplete conditions. Cells were grown to

exponential phase in 96 well plates without shaking and then spot inoculated onto solid agar plates using a 96-pin tool. After overnight growth at 30°_{C, the agar plates}

were overlaid with soft agar containing phosphate substrate and dye. Experiment was performed alongside a wild-type C. albicans strain as positive control and the

pho4 mutant as the negative control. Screen identified 9 mutant strains with no

detectable phosphatase activity and 15 with a partial defect (Fig 4.10A; Table 4.1). Positive strains were confirmed by a more quantitative method where optical

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0.5 and 10- fold serial dilutions carried out (Fig 4.10B). Of the 24 acid phosphatase defective mutants identified, only the pho100 mutant is Pho4-dependent (Fig 4.10A; Table 4.1). Interestingly, several of the mutant strains with defect in phosphatase activity appear to be under the control of the iron-response regulator, the

transcription factor Hap43, strongly suggesting phosphate limitation has an impact on iron availability (Table 4.1). Perturbations in phosphate homeostasis in yeast has been shown to create iron starvation response supporting the above finding (Rosenfeld et al., 2010). In conclusion, the screen performed supports the role of Pho100 as an acid phosphatase required for phosphate scavenging and also supports a link exists between phosphate and iron homeostasis.

Fig. 4.10. Identification of genes with defective secreted acid phosphatase activity. Colorimetric plate phosphatase screen of C. albicans deletion libraries. Dark colour indicates phosphatase activity (A) Examples of scanned images of phosphatase plate assay demonstrating secreted acid phosphatase activity. Same method as Fig 4.2A (B) Quantitative validation of phosphatase activity defects. 2 ×103 cells, and 10-fold dilutions thereof, of exponentially-growing wild-type and mutant strains were spotted onto agar plates, with or without Pi. Plates were incubated at 30°C for 24 h following which colonies were overlaid with p- nitrophenylphosphate and fast blue salt B and incubated at 30°C for 30 mins.

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Defective acid phosphatase activity Partial acid phosphatase activity PHO4 - bHLH transcription factor of the myc-

family; required for growth in medium lacking phosphate and for resistance to copper and Phloxine B; induced by Mnl1 under weak acid stress

FPG1 - Formamidopyrimidine DNA glycosylase, involved in repair of gamma- irradiated DNA; Hap43p-repressed gene

DAL81 - Zn(II)2Cys6 transcription factor; ortholog of S. cerevisiae Dal81, involved in the regulation of nitrogen-degradation genes

ORF19.2838 - Protein of unknown function; mutation confers hypersensitivity to

amphotericin B; flow model biofilm induced ORF19.287 - Putative NADH-ubiquinone

oxidoreductase subunit; Hap43p-repressed gene; repressed by nitric oxide

ORF19.2850 - Protein of unknown function; induced by nitric oxide independent of Yhb1p

ORF19.1625 - Putative ubiquinone oxidoreductase; repressed by nitric oxide; Hap43p-repressed

SWI4 - Putative component of the SBF transcription complex involved in G1/S cell- cycle progression

ORF19.1710 - Putative NADH-ubiquinone oxidoreductase; in detergent-resistant membrane fraction (possible lipid raft

component); predicted N-terminal acetylation

PHO100 - Putative inducible acid

phosphatase; DTT-extractable and observed in culture supernatant in low-phosphate conditions; slight effect on murine virulence PWP1 - Putative rRNA processing protein;

Hap43-induced; repressed in core stress response

PHO15 - 4-nitrophenyl phosphatase, possible histone H2A phosphatase; involved in

regulation of white-opaque switch; hyphal repressed; induced in core stress response; induced by cadmium stress via Hog1 MCI4 - Putative NADH-ubiquinone

dehydrogenase; Hap43p-repressed gene

ORF19.2500 - Has domain(s) with predicted transferase activity

ORF19.4758 - Putative reductase or dehydrogenase; Hap43-repressed gene; alkaline repressed

ORF19.7590 - Putative NADH-ubiquinone oxidoreductase; identified in detergent- resistant membrane fraction

ORF19.5547 - Protein of unknown function; Hap43-repressed gene

GIN4 - Autophosphorylated kinase; role in pseudohyphal-hyphal switch and cytokinesis ORF19.3029 - Predicted 3-hydroxyisobutyryl- CoA hydrolase; mitochondrially localized CCN1- G1 cyclin; required for hyphal growth maintenance (not initiation); cell-cycle regulated transcription (G1/S); Cdc28p-

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Ccn1p initiates Cdc11p S394 phosphorylation on hyphal induction SAP5 - Secreted aspartyl proteinase; sap4,5,6 triple null defective in utilization of protein as N source; virulence role effected by URA3; expressed during infection COX4 - Putative cytochrome c oxidase subunit IV; Mig1-regulated;

macrophage/pseudohyphal-induced gene; macrophage-induced protein; repressed by nitric oxide; 5'-UTR intron; Hap43-repressed ASG1 - Gal4p family zinc-finger transcription factor with similarity to S. cerevisiae Asg1p ORF19.6607 - Ortholog(s) have role in mitochondrial respiratory chain complex I assembly

Table 4.1 C. albicans strains with defective acid phosphatase activity.

4.3 Discussion

While the response to phosphate limitation has been extensively characterised in the model yeast, S. cerevisiae, very little is known about phosphate response in the fungal pathogen, C. albicans. This part of the study established the role of CaPho4 in phosphate homeostasis and found similarities as well as deviations in the regulatory mechanism of Pho4 between S. cerevisiae and C. albicans.

The first deviation identified was in the sequence of CaPho4 which was found to diverge significantly from that of ScPho4. More significant was the observation that most of the phosphorylation sites of ScPho4, which regulates cellular localisation and DNA binding, are not conserved in CaPho4 (Fig 4.1). ScPho4 sequence has five Serine-Proline (SP1 to SP4 and SP6) sites phosphorylated by the CDK complex Pho80-Pho85 (O`Neill et al., 1996). Each site plays a distinct role in the regulation of Pho4 activity. Phosphorylation at SP2 and SP3 ensures Pho4 nuclear export while phosphorylation at SP4 promotes its import, and interaction with Pho2, the co-

transcription factor, during phosphate-rich growth is prevented by phosphorylation at SP6 (Komeili and O`Shea, 1999). In addition, it was noted that some of the Pho4- dependent genes did not have either of the Pho4 binding motifs, for example PHO84,

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suggesting additional factors dictate Pho4-dependency for activation other than the presence of the DNA-binding motif in the promoter of the gene. In S. cerevisiae, Pho4 transcriptional specificity for PHO genes is regulated at the promoter level by the presence of nucleosomes and another transcription factor that recognises the same binding motif as Pho4 (Zhou and O`Shea, 2011). During growth in phosphate- rich environment, nucleosomes prevent Pho4 binding and at sites where there are no nucleosomes Cbf1, which is more abundantly present as phosphorylated Pho4 would be exported out of the nucleus, outcompetes Pho4 for these nucleosome-free sites (Zhou and O`Shea, 2011). During low-phosphate nuclear levels of unphosphorylated Pho4 increase so Pho4 can now outcompete Cbf1 (Zhou and O`Shea, 2011). This ensures Pho4 only binds to PHO genes and only during phosphate-limiting

conditions.

Based on the sequence divergence observed, the regulation and localisation of CaPho4 in wild-type cells in response to phosphate concentration was then

examined. In a manner similar to that of ScPho4, the Pho81 CDK inhibitor, necessary for Pho4 activation in S. cerevisiae, is also induced in C. albicans under phosphate- limiting conditions which indicates this part of the regulatory mechanism is

conserved. In addition, under phosphate-limiting conditions, CaPho4 was found to accumulate in the nucleus (Fig 4.4A). An orthologue of Pho80 (C6_03810W_B), the cyclin-dependent protein kinase has been identified in C.albicans but its role in regulating Pho4 has not been validated. CaPho85 however, has been shown to complement a pho85Δ in S. cerevisiae suggesting the function of this protein kinase may be conserved in C. albicans (Miyakawa, 2000). The next objective then was to examine if CaPho4 activity during growth in minus or plus phosphate was also regulated by phosphorylation. In contrast to ScPho4, CaPho4 is phosphorylated under both phosphate replete and deplete conditions however, if phosphorylated sites change in response to phosphate concentrations could not be determined (Fig 4.5). On the other hand, it was discovered that an additional post translational

modification appears to regulate CaPho4 (Fig 4.5D). This extra PTM requires further investigation. Pho4 may be modified by ubiquitin-like modifier such as SUMO as sumoylation of certain transcription factors, for example Tec1 required for invasive growth in yeast cells (Wang et al., 2009) has been shown to regulate nuclear localisation.

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While in the nucleus, Pho4 activates the expression of key genes for example, secreted acid phosphatases. In S. cerevisiae, the expression of an acid

phosphatase, PHO5 which is Pho4-dependent, increases during growth in phosphate-limiting conditions in S. cerevisiae (Kaffman et al., 1994). Phosphate scavenging is facilitated by the action of secreted acid phosphatases which hydrolyse phosphate-containing compounds to release phosphate. Acid and alkaline

phosphatase activities were first demonstrated in clinical isolates of C. albicans and reported to be low or delayed however, a recent study confirmed phosphate

starvation triggers a robust induction of phosphatase activity (Chattaway et al., 1971; Smith et al., 1973; Romanowski et al., 2012). This robust response was found to be CaPho4 dependent. The role of Pho4 and phosphatase activity in response to phosphate limitation is further corroborated by findings from this study. No acid phosphatase activity was detected in cells lacking PHO4 under phosphate-limiting conditions (Fig 4.2A). RNA-seq experiments also revealed that under phosphate starvation cells respond by massive induction of various phosphatases including acid phosphatases, phospholipases, and glycerophosphocholine phosphodiesterases (Appendix 2; Fig 4.9). In S. cerevisiae, deleting PHO84 results in growth defects under low-phosphate conditions, little polyP synthesis as well as the constitutive expression of PHO response genes (Wykoff et al., 2007). Another key protein up- regulated during phosphate limitation as identified by the RNA-seq analysis, in C.

albicans was the high-affinity phosphate transporter, Pho84.

PolyP functions mainly as a phosphate reservoir in virtually all living cells with around 99% of the polymer found in the vacuole (Kornberg, 1999). The remaining fraction of polyP is found in the nucleus, cytoplasm, mitochondria, and cell wall (Secco et al., 2012). The proteins involved in synthesising polyP have been identified in yeast. Deleting the genes that encode for these proteins completely abolishes polyP synthesis (Ogawa et al., 2000). Genes required for polyP synthesis as well as mobilisation during growth in phosphate-limiting conditions are under the regulation of ScPho4 (Ault-Riche et al., 1998; Oshima, 1997; Ogawa et al., 2000). In most living cells, phosphate availability is ensured by the mobilisation of internal polyP stores during growth under conditions of limiting external phosphate. In this study this mechanism was found to be conserved in C. albicans (Fig 4.4A; Fig 4.4B).

Collectively these data strongly indicate the role of Pho4 in regulating the response to phosphate limitation is conserved in C. albicans. Therefore in C. albicans, Pho4 plays

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a crucial role in the acquisition and storage of phosphate essential for various cellular processes and growth. During growth in phosphate-rich conditions, Pho4 is

phosphorylated, possibly by the conserved Pho85-Pho80 protein kinases, and dispersed throughout the cell. Following phosphate depletion, Pho81 inhibits further phosphorylation of Pho4 thereby triggering its nuclear accumulation. In the nucleus, Pho4 activates the expression of genes involved in phosphate acquisition which include the acid phosphatases, PHO100, PHO112, and PHO113, the high-affinity phosphate transporter, PHO84 and genes involved in polyP hydrolysis, VTC1, VTC3,

VTC4, and PHM5. Also activated are the GIT genes, GIT3 and GDE3, to enable

phosphate acquisition from glycerophosphodiester. During phosphate uptake, genes involved in polyP synthesis, for example VTC1, are activated to synthesise polyP from phosphate to replenish depleted polyP stores in the vacuole.

Having established the role of Pho4 in phosphate homeostasis, the next aim of the project was to analyse whether this role of Pho4 was extended to mediating

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