Phosphate theft: a path to fungal pathogenic success

Inorganic phosphate/PO₄^3–/P_i is an essential and major constituent of numerous cellular components in all eukaryotes, including fungi. These components include nucleic acids, phospholipids and ATP. Despite its abundance in organic compounds, P_i is relatively scarce in its free form. To become successful pathogens, fungi must therefore acquire free P_i from the host environment via enzyme-mediated hydrolysis of P_i-containing molecules and/or via more efficient use of their own P_i. Fungal adaptation to a P_i-limited environment is governed by the phosphate (PHO) system, a cellular pathway consisting of P_i transporters, P_i mobilising enzymes and regulatory elements, such as kinases and transcription factors that respond to P_i levels. This system is well studied in the model non-pathogenic yeast, Saccharomyces cerevisiae, but not in fungal pathogens. In this review we present what is known about the PHO system in the model fungal pathogen, Cryptococcus neoformans, including our identification and characterisation of a secreted acid phosphatase, Aph1, which serves as a valuable reporter for identifying the less well-conserved PHO elements, including transcription factors.

Cryptococcus neoformans is a deadly fungal pathogen with a high rate of morbidity and mortality worldwide¹. Its pathogenicity is attributable to the production of a multitude of virulence factors, including a polysaccharide capsule, melanin and the enzymes phospholipase B/C and acid phosphatase (Aph1), which collectively promote host invasion, protection against host-derived stress, evasion of the host immune system and nutrient acquisition^2,3 (for review see Coelho et al.⁴). C. neoformans also provides a suitable model for understanding the PHO system in fungal pathogens. It has a fully sequenced genome that is highly amenable to manipulation and is currently being used by many laboratories around the world, including our own, to understand mechanisms of fungal virulence. C. neoformans, which infects the lungs and disseminates to the central nervous system via the blood stream and lymphatics, must obtain P_i from the host if it is to survive and become a successful pathogen, since mutant strains with reduced ability to either hydrolyse extracellular complexes containing P_i (acid phosphatase-deficient)⁵ or transport free P_i across the plasma membrane⁶ are hypovirulent in mice. Identifying the full repertoire of PHO responsive genes involved in the mobilisation of P_i from host and fungal sources, and their mode of regulation, is therefore of paramount importance. Knowledge of the PHO system in S. cerevisiae has led to the identification of some components of the PHO system in C. neoformans, including a secreted acid phosphatase Aph1, which is a useful reporter for identification of the less conserved elements, including transcription factors.

The PHO system was extensively studied in S. cerevisiae and includes the high affinity membrane transporters (Pho84 and Pho89) system, polyphosphate (polyP) metabolising enzymes predominantly located in vacuoles, elements of the PHO regulation machinery and a number of PHO responsive genes involved in acquisition and storage of P_i. PHO-responsive genes include the high affinity P_i transporters and the secreted acid phosphatases, Pho5, Pho11 and Pho12, with Pho5 being the major secreted acid phosphatase. Excess cellular P_i is stored in vacuoles in chains of tens to hundreds of inorganic P_i molecules (polyPs), and is the first P_i storage source to be accessed during P_i deprivation. PolyP stores are believed to be exhausted prior to activation of the PHO system.

The S. cerevisiae PHO regulation machinery consists of the cyclin/cyclin-dependent kinase (CDK) complex Pho80/Pho85, the CDK inhibitor Pho81 and the transcription factor Pho4. During P_i deprivation, Pho81 represses the Pho80-Pho85 complex, preventing phosphorylation of Pho4. Hypophosphorylated Pho4 activates the expression of PHO responsive gene. Lee et al demonstrated that activation of the PHO system during P_i limitation paradoxically requires highly phosphorylated inositol species known as inositol pyrophosphates (PP-IPs)^7,8. PP-IPs are comprised of inositol covalently linked to mono- and di-phosphate groups, and are derived from the mono-phosphorylated (IP) species. Exactly how PP-IPs are involved in PHO system regulation remains controversial. Lee et al established that the concentration of intracellular PP-IP₅ (IP₇) increased under P_i-deficient conditions and that IP₇ allosterically modulates the CDK inhibitor Pho81 leading to a conformational change in the Pho85-Pho80-Pho81 complex and reduced phosphorylation of Pho4^7,8. However, Lonetti et al. demonstrated the opposite, with intracellular PP-IP levels decreasing by 80% during P_i deprivation⁹. Despite extensive investigation of the PHO pathway in S. cerevisiae, the mechanism for sensing intracellular P_i concentrations and the identity of the P_i sensor remain to be elucidated.

Only a few components of the PHO system have been identified in C. neoformans on the basis of their homology to PHO components from S. cerevisiae and responsiveness to the intracellular P_i status. These include the high-affinity P_i transporters (Pho840, Pho84 and Pho89)⁶, a family of secreted and intracellular acid phosphatases (Aph1, Aph3 and Aph4) (Lev et al.⁵ and our unpublished observation) that hydrolyse complex organic P_i sources, and the polyP polymerase Vtc4, which synthesises vacuolar polyPs⁶.

P_i transporters: A cryptococcal mutant defective in P_i uptake, Δpho840Δpho84Δpho89, is significantly attenuated for virulence in a mouse model of cryptococcosis, confirming that C. neoformans must acquire P_i from the host environment to reach its full virulence potential⁶.

Extracellular acid phosphatase Aph1: Extracellular P_i is often complexed to organic molecules and must be released by secreted acid phosphatases. The liberated P_i is then taken up by the P_i transporters. Acid phosphatase activity has been detected in the secretions of a large majority of C. neoformans strains isolated from patients with AIDS including the clinical type strain H99¹⁰. In a proteomic analysis of the H99 secretome, we identified the classically secreted acid phosphatase, Aph1, and deleted its encoding gene, creating Δaph1⁵. Using a chromogenic enzyme assay which measures hydrolysis of the synthetic substrate, para-nitrophenol phosphate (pNPP), we found that the Δaph1 mutant was deficient in secreted acid phosphatase activity during P_i deprivation, confirming that Aph1 is the major secreted acid phosphatase in C. neoformans⁵. qPCR revealed that, similar to Pho5 from S. cerevisiae, Aph1 production during P_i deprivation is regulated at the transcriptional level. We also found that the concentration of P_i in a standard cell culture medium is sufficiently low to induce APH1 expression providing further evidence that C. neoformans encounters a low P_i environment during host infection. Δaph1 was less virulent in Galleria mellonella and mice, consistent with P_i mobilisation from complex sources being essential for virulence⁵. However, Aph1 deficiency had less of an impact on virulence than loss of the high affinity P_i transport system, suggesting that Aph1 hydrolyses only a proportion of the extracellular P_i sources available within the host. Other enzymes that potentially work in conjunction with Aph1 to achieve comprehensive mobilisation of P_i from complex sources include alkaline phosphatases, phosphodiesterases and serine/threonine/tryrosine phosphatases.

A role of intracellular Aph1 in P_i mobilisation: We found that Aph1 hydrolyses a broad range of substrates, including glucose-1-phosphate, β-glycerol phosphate, adenosine monophosphate (AMP) and mannose-6-phosphate and prefers the aromatic amino acid phosphotyrosine to phosphoserine/phosphothreonine⁵. By tagging Aph1 with the red fluorescent protein DsRed we observed its transport to vacuoles and the cell periphery via endosome-like structures (Figure 1A)⁵. Aph1-containing endosomes were highly mobile and were often observed transiently contacting the plasma membrane and vacuoles, reminiscent of the kiss-and-run mechanism observed for synaptic vesicle release¹¹ (Figure 1B). Acid phosphatases are often found inside acidic vacuoles, an environment conducive to the working pH of Aph1 (pH 5). The dual targeting of Aph1 to vacuoles and the extracellular environment, and the broad substrate specificity of Aph1 is consistent with a role for Aph1 in releasing P_i from a wide range of both extra- and intracellular sources.

Figure 1. Acid phosphatase (Aph1) is localised to the cell periphery, vacuoles and mobile endosome-like organelles in C. neoformans. (a) Fungal cells expressing Aph1-dsRed fusion protein were starved of P_i for 3 hours and visualised with a deconvolution microscope. Vacuolar Aph1 is indicated by red arrows. DIC, differential interference contrast. (b) An endosome-like organelle containing Aph1-dsRed (black arrows) was followed using time lapse photography. The organelle is first observed contacting the membrane, retracting from it and then re-making contact in a ‘kiss-and-run’ type movement. Membrane contact potentially allows release of Aph1 into the periplasmic space. The images are presented using thermal scale to indicate fluorescence intensity, with red being the most intense.

Vacuolar PolyP polymerases: Interestingly, the recruitment of P_i from polyPs is dispensable for fungal virulence since a VTC4 deletion mutant was as virulent as WT in a mouse infection model⁶.

Intracellular acid phosphatases: C. neoformans also produces three intracellular acid phosphatases (Aph2, Aph3 and Aph4). APH3 and APH4, but not APH2, are induced by low P_i (our unpublished observation). The decoupling of Aph2 from P_i regulation was unexpected given that Aph2 is most similar to Aph1. To test for potential up-regulation of other members of the APH family as a compensation for the loss of APH1, we measured APH 2, 3 and 4 mRNA in Δaph1, but found similar levels to WT (our unpublished observation), consistent with a lack of compensation. The contribution of each intracellular APH to cryptococcal virulence will be investigated by constructing single and combination deletion mutants.

Pho transcription regulatory machinery: Probable PHO regulatory components with similarity to the cyclin-dependent kinase Pho85, the cyclin Pho80 and the cyclin-dependent kinase inhibitor Pho81 have been identified in C. neoformans: (CNAG_07871), (CNAG_01922) and (CNAG_02541) respectively⁶. However their role in regulation of the PHO system remains to be determined. Interestingly, no Pho4 transcription factor homologue has been identified in C. neoformans. The Bahn laboratory (Yonsei University, Korea) has created a cryptococcal transcription factor knockout library. Use of Aph1 as a reporter for screening this library should determine the identity of transcription factor(s) regulating PHO gene expression in C. neoformans.

PP-IPs: Cryptococcal mutants deficient in IP₇ production also fail to induce APH1 expression and secrete Aph1 during P_i deprivation (our unpublished observation). Via gene deletion analysis, we recently characterised the entire inositol polyphosphate biosynthesis pathway in C. neoformans, including the kinase directly responsible for IP₇ synthesis (Kcs1). Using the full set of kinase deletion mutants we also observed that IP₇ is essential for fungal virulence and dissemination to the brain in a mouse model (Lev et al.¹² and our unpublished observation).

In summary, little is known about how P_i homeostasis is regulated in pathogenic fungi, as compared with S. cerevisiae. We have identified and extensively characterised the major secreted acid phosphatase in C. neoformans, Aph1, which will provide a valuable reporter for identifying PHO system regulators in this important fungal pathogen. As individual components of the PHO system have been demonstrated to play a role in virulence, the investigation of P_i homeostasis in C. neoformans may also provide unique opportunities for antifungal drug development.