Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans

Bowring, Bethany; Sethiya, Pooja; Desmarini, Desmarini; Lev, Sophie; Le, Lisa Tran; Bahn, Yong-Sun; Lee, Seung-Heon; Toh‐e, Akio; Proschogo, Nicholas; Savage, Trevor N.; Djordjevic, Julianne T.

doi:10.1128/mbio.03551-22

Cited by 3 publications

(2 citation statements)

References 70 publications

(150 reference statements)

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“…This contrasts with Saccharomyces cerevisiae ( Sc ) , where the phosphate acquisition machinery is constitutively activated in the absence of IP 3-4 K ( 16 , 17 ). Of all the virulence phenotypes promoted by IP 3-4 K in C. neoformans , only phosphate homeostasis has been shown to depend on IP 3-4 K catalytic activity, where the distal Cn Arg1 product, IP 7 , stabilizes the PHO signaling machinery and allows C. neoformans to respond to low phosphate ( 16 ), and potentially to promote energy metabolism and stress-related calcineurin signaling ( 18 ).…”

Section: Introductionmentioning

confidence: 99%

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP _3-4 kinase activity

Desmarini,

Liu,

Jessen

et al. 2024

mBio

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Inositol tris/tetrakis phosphate kinases (IP 3-4 K) in the human fungal priority pathogens, Cryptococcus neoformans ( Cn Arg1) and Candida albicans ( Ca Ipk2), convey numerous virulence functions, yet it is not known whether the IP 3-4 K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) Cn Arg1 strain (dkArg1). We verified that, although dk ARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP 3-4 K activity. Using recombinant Cn Arg1 and Ca Ipk2 , we confirmed that, unlike the IP 3-4 K homologs in humans and Saccharomyces cerevisiae , Cn Arg1 and Ca Ipk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP 3 conversion is blocked in the dkArg1 and ARG1 deletion ( Cnarg1 Δ) strains and that 1-IP 7 and a recently discovered isomer (4/6-IP 7 ) are made by wild-type C. neoformans . Importantly, the dkArg1 and Cnarg1 Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP 3-4 K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since Cn Arg1 is dispensable for growth on different nitrogen sources. IP 3-4 K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens. IMPORTANCE The World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP 3-4 K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans , and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP 3-4 K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP 3-4 K catalytic activity of Cn Arg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion , reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP 3-4 K catalytic site, as a new antifungal drug class.

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Section: Introductionmentioning

confidence: 99%

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP _3-4 kinase activity

Desmarini,

Liu,

Jessen

et al. 2024

mBio

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“…In the pho81Δ mutant, phosphate (Pi), polyP, ATP and energy metabolism were reduced. Blocking the PHO pathway reduced fungal virulence in mouse infection models to a greater extent, and this is most likely attributable to depleted phosphate stores and ATP and compromised cellular bioenergetics [ 38 ].…”

Section: Introductionmentioning

confidence: 99%

The Extracellular Vesicles Containing Inorganic Polyphosphate of Candida Yeast upon Growth on Hexadecane

Zvonarev,

Trilisenko,

Farofonova

et al. 2023

JoX

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The cell wall of Candida yeast grown on presence of hexadecane as a sole carbon source undergoes structural and functional changes including the formation of specific supramolecular complexes—canals. The canals contain specific polysaccharides and enzymes that provide primary oxidization of alkanes. In addition, inorganic polyphosphate (polyP) was identified in Candida maltosa canals. The aim of the work was a comparative study of the features of cell walls and extracellular structures in yeast C. maltosa, C. albicans and C. tropicalis with special attention to inorganic polyphosphates as possible part of these structures when grown on the widely used xenobiotic hexadecane (diesel fuel). Fluorescence microscopy with DAPI has shown an unusual localization of polyP on the cell surface and in the exovesicles in the three yeast species, when growing on hexadecane. Electron-scanning microscopy showed that the exovesicles were associated with the cell wall and also presented in the external environment probably as biofilm components. Treatment of hexadecane-grown cells with purified Ppx1 polyphosphatase led to the release of phosphate into the incubation medium and the disappearance of polyP in vesicles and cell wall observed using microscopic methods. The results indicate the important role of polyP in the formation of extracellular structures in the Candida yeast when consuming hexadecane and are important for the design of xenobiotic destructors based on yeast or mixed cultures.

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Phosphate availability conditions caspofungin tolerance, capsule attachment and titan cell formation in Cryptococcus neoformans

Qu,

Bhalla,

Horianopoulos

et al. 2024

Front. Fungal Biol.

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There is an urgent need for new antifungal drugs to treat invasive fungal diseases. Unfortunately, the echinocandin drugs that are fungicidal against other important fungal pathogens are ineffective against Cryptococcus neoformans, the causative agent of life-threatening meningoencephalitis in immunocompromised people. Contributing mechanisms for echinocandin tolerance are emerging with connections to calcineurin signaling, the cell wall, and membrane composition. In this context, we discovered that a defect in phosphate uptake impairs the tolerance of C. neoformans to the echinocandin caspofungin. Our previous analysis of mutants lacking three high affinity phosphate transporters revealed reduced elaboration of the polysaccharide capsule and attenuated virulence in mice. We investigated the underlying mechanisms and found that loss of the transporters and altered phosphate availability influences the cell wall and membrane composition. These changes contribute to the shedding of capsule polysaccharide thus explaining the reduced size of capsules on mutants lacking the phosphate transporters. We also found an influence of the calcineurin pathway including calcium sensitivity and an involvement of the endoplasmic reticulum in the response to phosphate limitation. Furthermore, we identified membrane and lipid composition changes consistent with the role of phosphate in phospholipid biosynthesis and with previous studies implicating membrane integrity in caspofungin tolerance. Finally, we discovered a contribution of phosphate to titan cell formation, a cell type that displays modified cell wall and capsule composition. Overall, our analysis reinforces the importance of phosphate as a regulator of cell wall and membrane composition with implications for capsule attachment and antifungal drug susceptibility.

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Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans

Abstract: Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 of these deaths attributable to Cryptococcal meningitis. Despite the high mortality, treatment options are limited.

Cited by 3 publications

References 70 publications

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP _3-4 kinase activity

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP _3-4 kinase activity

The Extracellular Vesicles Containing Inorganic Polyphosphate of Candida Yeast upon Growth on Hexadecane

Phosphate availability conditions caspofungin tolerance, capsule attachment and titan cell formation in Cryptococcus neoformans

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Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans

Abstract: Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 of these deaths attributable to Cryptococcal meningitis. Despite the high mortality, treatment options are limited.

Cited by 3 publications

References 70 publications

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP 3-4 kinase activity

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP 3-4 kinase activity

The Extracellular Vesicles Containing Inorganic Polyphosphate of Candida Yeast upon Growth on Hexadecane

Phosphate availability conditions caspofungin tolerance, capsule attachment and titan cell formation in Cryptococcus neoformans

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Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP _3-4 kinase activity

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP _3-4 kinase activity