Inositol Polyphosphate Kinases, Fungal Virulence and Drug Discovery.
ABSTRACT: Opportunistic fungi are a major cause of morbidity and mortality world-wide, particularly in immunocompromised individuals. Developing new treatments to combat invasive fungal disease is challenging given that fungal and mammalian host cells are eukaryotic, with similar organization and physiology. Even therapies targeting unique fungal cell features have limitations and drug resistance is emerging. New approaches to the development of antifungal drugs are therefore needed urgently. Cryptococcus neoformans, the commonest cause of fungal meningitis worldwide, is an accepted model for studying fungal pathogenicity and driving drug discovery. We recently characterized a phospholipase C (Plc1)-dependent pathway in C. neoformans comprising of sequentially-acting inositol polyphosphate kinases (IPK), which are involved in synthesizing inositol polyphosphates (IP). We also showed that the pathway is essential for fungal cellular function and pathogenicity. The IP products of the pathway are structurally diverse, each consisting of an inositol ring, with phosphate (P) and pyrophosphate (PP) groups covalently attached at different positions. This review focuses on (1) the characterization of the Plc1/IPK pathway in C. neoformans; (2) the identification of PP-IP₅ (IP₇) as the most crucial IP species for fungal fitness and virulence in a mouse model of fungal infection; and (3) why IPK enzymes represent suitable candidates for drug development.
Project description:Phospholipase C (PLC) of Cryptococcus neoformans (CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP(3)). In Saccharomyces cerevisiae, Plc1-derived IP(3) is a substrate for the inositol polyphosphate kinase Arg82, which converts IP(3) to more complex inositol polyphosphates. In this study, we show that in C. neoformans, the enzyme encoded by ARG1 is the major IP(3) kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in the Galleria mellonella infection model. IP(3) content was reduced in the Cn?plc1 mutant and markedly increased in the Cn?arg1 mutant, while PIP(2) was increased in both mutants. The Cn?plc1 and Cn?arg1 mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to the S. cerevisiae ARG82 deletion mutant (Sc?arg82) strain, the Cn?arg1 mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP(3) causes Ca(2+) release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence of C. neoformans and further show that production of IP(3) as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1.
Project description:UNLABELLED:Inositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis. We investigate the cryptococcal kinases responsible for the production of PP-IPs (IP7/IP8) and the hierarchy of PP-IP importance in pathogenicity. Using gene deletion and inositol polyphosphate profiling, we identified Kcs1 as the major IP6 kinase (producing IP7) and Asp1 as an IP7 kinase (producing IP8). We show that Kcs1-derived IP7 is the most crucial PP-IP for cryptococcal drug susceptibility and the production of virulence determinants. In particular, Kcs1 kinase activity is essential for cryptococcal infection of mouse lungs, as reduced fungal burdens were observed in the absence of Kcs1 or when Kcs1 was catalytically inactive. Transcriptome and carbon source utilization analysis suggested that compromised growth of the KCS1 deletion strain (Δkcs1 mutant) in the low-glucose environment of the host lung is due to its inability to utilize alternative carbon sources. Despite this metabolic defect, the Δkcs1 mutant established persistent, low-level asymptomatic pulmonary infection but failed to elicit a strong immune response in vivo and in vitro and was not readily phagocytosed by primary or immortalized monocytes. Reduced recognition of the Δkcs1 cells by monocytes correlated with reduced exposure of mannoproteins on the Δkcs1 mutant cell surface. We conclude that IP7 is essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity. IMPORTANCE:Cryptococcus neoformans is responsible for 1 million cases of AIDS-associated meningitis and ~600,000 deaths annually. Understanding cellular pathways responsible for pathogenicity might have an impact on new drug development. We characterized the inositol polyphosphate kinases Kcs1 and Asp1, which are predicted to catalyze the production of inositol pyrophosphates containing one or two diphosphate moieties (PP-IPs). Using gene deletion analysis and inositol polyphosphate profiling, we confirmed that Kcs1 and Asp1 are major IP6 and IP7 kinases, respectively. Kcs1-derived IP7, but not Asp1-derived IP8, is crucial for pathogenicity. Global expression profiling and carbon source utilization testing suggest that IP7-deficient cryptococci cannot adapt their metabolism to allow growth in the glucose-poor environment of the host lung, and consequently, fungal burdens are significantly reduced. Persistent asymptomatic Δkcs1 mutant infection correlated with decreased mannoprotein exposure on the Δkcs1 mutant surface and reduced phagocytosis. We conclude that IP7 is crucial for the metabolic adaptation of C. neoformans to the host environment and for pathogenicity.
Project description:Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1?), and in combination with KCS1 (ipk1?kcs1?), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1?kcs1? and kcs1? lung burdens were also lower than those of ipk1?. Unlike ipk1?, ipk1?kcs1? and kcs1? failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1? produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1?kcs1?, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. Kcs1 is therefore a dual specificity (IP5 and IP6) kinase producing PP-IP4 and PP-IP5/IP7. All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced significantly in all mutants except ipk1?, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1? grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence.
Project description:We previously identified a series of inositol polyphosphate kinases (IPKs), Arg1, Ipk1, Kcs1 and Asp1, in the opportunistic fungal pathogen Cryptococcus neoformans. Using gene deletion analysis, we characterized Arg1, Ipk1 and Kcs1 and showed that they act sequentially to convert IP3 to PP-IP5 (IP7), a key metabolite promoting stress tolerance, metabolic adaptation and fungal dissemination to the brain. We have now directly characterized the enzymatic activity of Arg1, demonstrating that it is a dual specificity (IP3/IP4) kinase producing IP5. We showed previously that IP5 is further phosphorylated by Ipk1 to produce IP6, which is a substrate for the synthesis of PP-IP5 by Kcs1. Phenotypic comparison of the arg1Δ and kcs1Δ deletion mutants (both PP-IP5-deficient) reveals that arg1Δ has the most deleterious phenotype: while PP-IP5 is essential for metabolic and stress adaptation in both mutant strains, PP-IP5 is dispensable for virulence-associated functions such as capsule production, cell wall organization, and normal N-linked mannosylation of the virulence factor, phospholipase B1, as these phenotypes were defective only in arg1Δ. The more deleterious arg1Δ phenotype correlated with a higher rate of arg1Δ phagocytosis by human peripheral blood monocytes and rapid arg1Δ clearance from lung in a mouse model. This observation is in contrast to kcs1Δ, which we previously reported establishes a chronic, confined lung infection. In summary, we show that Arg1 is the most crucial IPK for cryptococcal virulence, conveying PP-IP5-dependent and novel PP-IP5-independent functions.
Project description:In the human-pathogenic fungus Cryptococcus neoformans, the inositol polyphosphate signaling pathway is critical for virulence. We recently demonstrated the key role of the inositol pyrophosphate IP7 (isomer 5-PP-IP5) in driving fungal virulence; however, the mechanism of action remains elusive. Using genetic and biochemical approaches, and mouse infection models, we show that IP7 synthesized by Kcs1 regulates fungal virulence by binding to a conserved lysine surface cluster in the SPX domain of Pho81. Pho81 is the cyclin-dependent kinase (CDK) inhibitor of the phosphate signaling (PHO) pathway. We also provide novel mechanistic insight into the role of IP7 in PHO pathway regulation by demonstrating that IP7 functions as an intermolecular "glue" to stabilize Pho81 association with Pho85/Pho80 and, hence, promote PHO pathway activation and phosphate acquisition. Blocking IP7-Pho81 interaction using site-directed mutagenesis led to a dramatic loss of fungal virulence in a mouse infection model, and the effect was similar to that observed following PHO81 gene deletion, highlighting the key importance of Pho81 in fungal virulence. Furthermore, our findings provide additional evidence of evolutionary divergence in PHO pathway regulation in fungi by demonstrating that IP7 isomers have evolved different roles in PHO pathway control in C. neoformans and nonpathogenic yeast.IMPORTANCE Invasive fungal diseases pose a serious threat to human health globally with >1.5 million deaths occurring annually, 180,000 of which are attributable to the AIDS-related pathogen, Cryptococcus neoformans Here, we demonstrate that interaction of the inositol pyrophosphate, IP7, with the CDK inhibitor protein, Pho81, is instrumental in promoting fungal virulence. IP7-Pho81 interaction stabilizes Pho81 association with other CDK complex components to promote PHO pathway activation and phosphate acquisition. Our data demonstrating that blocking IP7-Pho81 interaction or preventing Pho81 production leads to a dramatic loss in fungal virulence, coupled with Pho81 having no homologue in humans, highlights Pho81 function as a potential target for the development of urgently needed antifungal drugs.
Project description:In eukaryotes, inositol polyphosphates perform essential metabolic and signaling functions. Using human fungal pathogen Cryptococcus neoformans as a model, we created mutants in three inositol polyphosphates kinases: Arg1, Ipk1 and Kcs1. Each of the mutants produces a unique repertoire of inositol polyphosphates, different from the wild type strain. Comparative phenotypic and transcriptome analyses of wild type and mutant strains indicates that inositol polyphosphate PP-IP5 (IP7) is the key regulator of gene expression, fitness and virulence in C. neoformans. Comparison of WT and mutants (Darg1, Dkcs1 and Dipk1) grown in broth culture in the absence of stress.
Project description:Hyperphosphorylation of protein tau is a hallmark of Alzheimer's disease (AD). Changes in energy and lipid metabolism have been correlated with the late onset of this neurological disorder. However, it is uncertain if metabolic dysregulation is a consequence of AD or one of the initiating factors of AD pathophysiology. Also, it is unclear whether variations in lipid metabolism regulate the phosphorylation state of tau. Here, we show that in humanized yeast, tau hyperphosphorylation is stimulated by glucose starvation in coincidence with the downregulation of Pho85, the yeast ortholog of CDK5. Changes in inositol phosphate (IP) signaling, which has a central role in energy metabolism, altered tau phosphorylation. Lack of inositol hexakisphosphate kinases Kcs1 and Vip1 (IP<sub>6</sub> and IP<sub>7</sub> kinases in mammals) increased tau hyperphosphorylation. Similar effects were found by mutation of <i>IPK2</i> (inositol polyphosphate multikinase), or <i>PLC1</i>, the yeast phospholipase C gene. These effects may be explained by IP-mediated regulation of Pho85. Indeed, this appeared to be the case for <i>plc1</i>, <i>ipk2</i>, and <i>kcs1</i>. However, the effects of Vip1 on tau phosphorylation were independent of the presence of Pho85, suggesting additional mechanisms. Interestingly, <i>kcs1</i> and <i>vip1</i> strains, like <i>pho85</i>, displayed dysregulated sphingolipid (SL) metabolism. Moreover, genetic and pharmacological inhibition of SL biosynthesis stimulated the appearance of hyperphosphorylated forms of tau, while increased flux through the pathway reduced its abundance. Finally, we demonstrated that Sit4, the yeast ortholog of human PP2A protein phosphatase, is a downstream effector of SL signaling in mediating the tau phosphorylation state. Altogether, our results add new knowledge on the molecular effectors involved in tauopathies and identify new targets for pharmacological intervention.
Project description:Cryptococcus neoformans is an AIDS-associated human fungal pathogen and the most common cause of fungal meningitis, with a mortality rate over 40% in AIDS patients. Significant advances have been achieved in understanding its disease mechanisms. Yet the underlying mechanism of a high frequency of cryptococcal meningitis remains unclear. The existence of high inositol concentrations in brain and our earlier discovery of a large inositol transporter (ITR) gene family in C. neoformans led us to investigate the potential role of inositol in Cryptococcus-host interactions. In this study, we focus on functional analyses of two major ITR genes to understand their role in virulence of C. neoformans. Our results show that ITR1A and ITR3C are the only two ITR genes among 10 candidates that can complement the growth defect of a Saccharomyces cerevisiae strain lacking inositol transporters. Both S. cerevisiae strains heterologously expressing ITR1A or ITR3C showed high inositol uptake activity, an indication that they are major inositol transporters. Significantly, itr1a itr3c double mutants showed significant virulence attenuation in murine infection models. Mutating both ITR1A and ITR3C in an ino1 mutant background activates the expression of several remaining ITR candidates and does not show more severe virulence attenuation, suggesting that both inositol uptake and biosynthetic pathways are important for inositol acquisition. Overall, our study provides evidence that host inositol and fungal inositol transporters are important for Cryptococcus pathogenicity.
Project description:In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.
Project description:In recent years, sphingolipids have emerged as critical molecules in the regulation of microbial pathogenesis. In fungi, the synthesis of complex sphingolipids is important for the regulation of pathogenicity, but the role of sphingolipid degradation in fungal virulence is not known. Here, we isolated and characterized the inositol phosphosphingolipid-phospholipase C1 (ISC1) gene from the fungal pathogen Cryptococcus neoformans and showed that it encodes an enzyme that metabolizes fungal inositol sphingolipids. Isc1 protects C. neoformans from acidic, oxidative, and nitrosative stresses, which are encountered by the fungus in the phagolysosomes of activated macrophages, through a Pma1-dependent mechanism(s). In an immunocompetent mouse model, the C. neoformans Deltaisc1 mutant strain is almost exclusively found extracellularly and in a hyperencapsulated form, and its dissemination to the brain is remarkably reduced compared to that of control strains. Interestingly, the dissemination of the C. neoformans Deltaisc1 strain to the brain is promptly restored in these mice when alveolar macrophages are pharmacologically depleted or when infecting an immunodeficient mouse in which macrophages are not efficiently activated. These studies suggest that Isc1 plays a key role in protecting C. neoformans from the intracellular environment of macrophages, whose activation is important for preventing fungal dissemination of the Deltaisc1 strain to the central nervous system and the development of meningoencephalitis.