Identification of Aph1, a Phosphate-Regulated, Secreted, and Vacuolar Acid Phosphatase in Cryptococcus neoformans

Abstract

Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the Δaph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a Δsec14-1 mutant. Secretions from the WT, but not from Δaph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, β-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment.

Virulence-Associated Enzymes of Cryptococcus neoformans

“…Acid phosphatase is encoded by the gene APH1 in C. neoformans. In both wax worm and murine models of cryptococcosis, aph1⌬ strain-infected animals survived longer than those in the wild-type-infected model (106), demonstrating the importance of this enzyme during infection.…”

Phosphate theft: a path to fungal pathogenic success

“…Galleria mellonella and mice, consistent with P i mobilisation from complex sources being essential for virulence 5 . 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.…”

“…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 polyPs 6 .…”

“…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 membrane 6 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.…”

Usefulness of silkworm as a host animal for understanding pathogenicity of <i>Cryptococcus neoformans </i>

“…mellonella is large moth which belong to Lepidoptera, same as silkworm. G. mellonella has been studied as infection models of fungi including C. neoformans (39)(40)(41)(42). G. mellonella is possible to perform infection experiments at 37˚C.…”