Search

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.IMPORTANCEThe 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., Competing Interests: The authors declare no conflict of interest.

Fungal pathogens uniquely regulate phosphate homeostasis via the cyclin-dependent kinase (CDK) signaling machinery of the phosphate acquisition (PHO) pathway (Pho85 kinase-Pho80 cyclin-CDK inhibitor Pho81), providing drug-targeting opportunities. Here, we investigate the impact of a PHO pathway activation-defective Cryptococcus neoformans mutant ( pho81 Δ) and a constitutively activated PHO pathway mutant ( pho80 Δ) on fungal virulence. Irrespective of phosphate availability, the PHO pathway was derepressed in pho80 Δ with all phosphate acquisition pathways upregulated and much of the excess phosphate stored as polyphosphate (polyP). Elevated phosphate in pho80 Δ coincided with elevated metal ions, metal stress sensitivity, and a muted calcineurin response, all of which were ameliorated by phosphate depletion. In contrast, metal ion homeostasis was largely unaffected in the pho81 Δ mutant, and P i , polyP, ATP, and energy metabolism were reduced, even under phosphate-replete conditions. A similar decline in polyP and ATP suggests that polyP supplies phosphate for energy production even when phosphate is available. Using calcineurin reporter strains in the wild-type, pho80 Δ, and pho81 Δ background, we also demonstrate that phosphate deprivation stimulates calcineurin activation, most likely by increasing the bioavailability of calcium. Finally, we show that blocking, as opposed to permanently activating, the PHO pathway reduced fungal virulence in mouse infection models to a greater extent and that this is most likely attributable to depleted phosphate stores and ATP, and compromised cellular bioenergetics, irrespective of phosphate availability. IMPORTANCE 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. In contrast to humans, fungal cells maintain phosphate homeostasis via a CDK complex, providing drug-targeting opportunities. To investigate which CDK components are the best targets for potential antifungal therapy, we used strains with a constitutively active ( pho80 Δ) and an activation-defective ( pho81 Δ) PHO pathway, to investigate the impact of dysregulated phosphate homeostasis on cellular function and virulence. Our studies suggest that inhibiting the function of Pho81, which has no human homologue, would have the most detrimental impact on fungal growth in the host due to depletion of phosphate stores and ATP, irrespective of phosphate availability in the host.

Cryptococcus neoformans ( Cn ) is a pathogenic yeast that is the leading cause of fungal meningitis in immunocompromised patients. Various Cn virulence factors, such as the enzyme laccase and its product melanin, phospholipase, and capsular polysaccharide have been identified. During a screen of knockout mutants, the gene resistance to aminocholesterol 1 ( RTA1 ) was identified, the function of which is currently unknown in Cn . Rta1 homologs in S. cerevisiae belong to a lipid-translocating exporter family of fungal proteins with transmembrane regions and confer resistance to the antimicrobial agent 7-aminocholesterol when overexpressed. To determine the role of RTA1 in Cn , the knock-out ( rta1Δ ) and reconstituted ( rta1Δ+RTA1 ) strains were created and phenotypically tested. RTA1 was involved in resistance to 7-aminocholesterol, and also in exocyst complex component 3 (Sec6)-mediated secretion of urease, laccase, and the major capsule component, glucuronoxylomannan (GXM), which coincided with significantly smaller capsules in the rta1Δ and rta1Δ+RTA1 strains compared to the wild-type H99 strain. Furthermore, RTA1 expression was reduced in a secretory 14 mutant ( sec14Δ ) and increased in an RNAi Sec6 mutant. Transmission electron microscopy demonstrated vesicle accumulation inside the rta1Δ strain, predominantly near the cell membrane. Given that Rta1 is likely to be a transmembrane protein located at the plasma membrane, these data suggest that Rta1 may be involved in both secretion of various fungal virulence factors and resistance to 7-aminocholesterol in Cn .

New antifungals with unique modes of action are urgently needed to treat the increasing global burden of invasive fungal infections. The fungal inositol polyphosphate kinase (IPK) pathway, comprised of IPKs that convert IP 3 to IP 8 , provides a promising new target due to its impact on multiple, critical cellular functions and, unlike in mammalian cells, its lack of redundancy. Nearly all IPKs in the fungal pathway are essential for virulence, with IP 3-4 kinase (IP 3-4 K) the most critical. The dibenzylaminopurine compound, N 2-( m -trifluorobenzylamino)- N 6-( p -nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP 3-4 K has not been investigated. We purified IP 3-4 K from the human pathogens, Cryptococcus neoformans and Candida albicans , and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC 50 ) and binding affinity (K D ), respectively, of TNP and 38 analogues. A novel chemical route was developed to efficiently prepare TNP analogues. TNP and its analogues demonstrated inhibition of recombinant IP 3-4 K from C. neoformans ( Cn Arg1) at low µM IC 50 s, but not IP 3-4 K from C. albicans ( Ca Ipk2) and many analogues exhibited selectivity for Cn Arg1 over the human equivalent, Hs IPMK. Our results provide a foundation for improving potency and selectivity of the TNP series for fungal IP 3-4 K.

Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular processes in eukaryotic cells. IPs and PP-IPs are highly negatively charged and exert their biological effects by interacting with specific protein targets. Studies performed predominantly in mammalian cells and model yeasts have shown that IPs and PP-IPs modulate target function through allosteric regulation, by promoting intra- and intermolecular stabilization and, in the case of PP-IPs, by donating a phosphate from their pyrophosphate (PP) group to the target protein. Technological advances in genetics have extended studies of IP function to microbial pathogens and demonstrated that disrupting PP-IP biosynthesis and PP-IP-protein interaction has a profound impact on pathogenicity. This review summarises the complexity of IP-mediated regulation in eukaryotes, including microbial pathogens. It also highlights examples of poor conservation of IP-protein interaction outcome despite the presence of conserved IP-binding domains in eukaryotic proteomes., (© 2021 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Devastating fires in Australia over 2019-20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p<0.0001). Despite evidence of cathelicidin expression within tissues known to be infected by Chlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species., Competing Interests: M.K is a Principal Scientist at Zoetis Veterinary Medicines Research and Development facility, Kalamazoo MI, USA. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The remaining authors declare no conflicts of interest.

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 IP 7 (isomer 5-PP-IP 5 ) in driving fungal virulence; however, the mechanism of action remains elusive. Using genetic and biochemical approaches, and mouse infection models, we show that IP 7 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 IP 7 in PHO pathway regulation by demonstrating that IP 7 functions as an intermolecular "glue" to stabilize Pho81 association with Pho85/Pho80 and, hence, promote PHO pathway activation and phosphate acquisition. Blocking IP 7 -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 IP 7 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, IP 7 , with the CDK inhibitor protein, Pho81, is instrumental in promoting fungal virulence. IP 7 -Pho81 interaction stabilizes Pho81 association with other CDK complex components to promote PHO pathway activation and phosphate acquisition. Our data demonstrating that blocking IP 7 -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., (Copyright © 2020 Desmarini et al.)

Intracellular calcium (Ca 2+ ) is crucial for signal transduction in Cryptococcus neoformans , the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca 2+ -requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca 2+ binding mediated by the EF-hand domains of the Ca 2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca 2+ homeostasis under stress and nonstress conditions, as the ncs1Δ mutant is sensitive to a high Ca 2+ concentration and has an elevated basal Ca 2+ level. Furthermore, NCS1 expression is induced by Ca 2+ , with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to the case with Saccharomyces cerevisiae , NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca 2+ sensitivity phenotype with the calcineurin and calmodulin mutants ( cna1 Δ and cam1Δ ), and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1 deficiency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data show that Ncs1 has a significant role as a Ca 2+ sensor in C. neoformans , working with calcineurin to regulate Ca 2+ homeostasis and, consequently, promote fungal growth and virulence. IMPORTANCE Cryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca 2+ signaling and homeostasis to the virulence of C. neoformans Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca 2+ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca 2+ homeostasis and cell cycle progression in C. neoformans ., (Copyright © 2020 Squizani et al.)

Cryptococcus neoformans is a human fungal pathogen that adapts its metabolism to cope with limited oxygen availability, nutrient deprivation and host phagocytes. To gain insight into cryptococcal metabolism, we optimized a protocol for the Seahorse Analyzer, which measures extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indications of glycolytic and respiratory activities. In doing so we achieved effective immobilization of encapsulated cryptococci, established Rotenone/Antimycin A and 2-deoxyglucose as effective inhibitors of mitochondrial respiration and glycolysis, respectively, and optimized a microscopy-based method of data normalization. We applied the protocol to monitor metabolic changes in the pathogen alone and in co-culture with human blood-derived monocytes. We also compared metabolic flux in wild-type C. neoformans, its isogenic 5-PP-IP 5 /IP 7 -deficient metabolic mutant kcs1 ∆, the sister species of C. neoformans, Cryptococcus deuterogattii/VGII, and two other yeasts, Saccharomyces cerevisiae and Candida albicans. Our findings show that in contrast to monocytes and C. albicans , glycolysis and respiration are tightly coupled in C. neoformans and C. deuterogattii, as no compensatory increase in glycolysis occurred following inhibition of respiration. We also demonstrate that kcs1 ∆ has reduced metabolic activity that correlates with reduced mitochondrial function. Metabolic inflexibility in C. neoformans is therefore consistent with its obligate aerobe status and coincides with phagocyte tolerance of ingested cryptococcal cells.

Invasive fungal pathogens cause more than 300 million serious human infections and 1.6 million deaths per year. A clearer understanding of the mechanisms by which these fungi cause disease is needed to identify novel targets for urgently needed therapies. Kinases are key components of the signaling and metabolic circuitry of eukaryotic cells, which include fungi, and kinase inhibition is currently being exploited for the treatment of human diseases. Inhibiting evolutionarily divergent kinases in fungal pathogens is a promising avenue for antifungal drug development. One such group of kinases is the phospholipase C1-dependent inositol polyphosphate kinases (IPKs), which act sequentially to transfer a phosphoryl group to a pre-phosphorylated inositol sugar (IP). This review focuses on the roles of fungal IPKs and their IP products in fungal pathogenicity, as determined predominantly from studies performed in the model fungal pathogen Cryptococcus neoformans , and compares them to what is known in non-pathogenic model fungi and mammalian cells to highlight potential drug targeting opportunities.

The phosphate sensing and acquisition (PHO) pathway of Cryptococcus neoformans is essential for growth in phosphate-limiting conditions and for dissemination of infection in a mouse model. Its key transcription factor, Pho4, regulates expression of genes controlling the acquisition of phosphate from both external and cellular sources. One such gene, BTA1, is highly up-regulated during phosphate starvation. Given that a significant proportion of cellular phosphate is incorporated into phospholipids, and that the Pho4-dependent BTA1 gene encodes an enzyme predicted to catalyse production of a phosphorus-free betaine lipid, we investigated whether phospholipids provide an accessible reservoir of phosphate during phosphate deficiency. By comparing lipid profiles of phosphate-starved WT C. neoformans, PHO4 (pho4Δ) and BTA1 (bta1Δ) deletion mutants using thin layer chromatography and liquid chromatography mass spectrometry, we showed that phosphatidylcholine (PC) is substituted by the phosphorus-free betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylgyceryl hydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA) in a Pho4- and Bta1-dependent manner, and that BTA1 encodes a functional DGTS synthase. Synthesis of DGTA tightly correlated with that of DGTS, consistent with DGTS being the precursor of DGTA. Similar to pho4Δ, bta1Δ grew more slowly than WT in cell culture medium (RPMI) and was hypovirulent in a murine model of cryptococcosis. In contrast to pho4Δ, bta1Δ tolerated alkaline pH and disseminated to the brain. Our results demonstrate that Bta1-dependent substitution of PC by betaine lipids is tightly regulated in C. neoformans by the PHO pathway, to conserve phosphate and preserve membrane integrity and function. This phospholipid remodeling strategy may also contribute to cryptococcal virulence during host infection., Competing Interests: The authors have declared that no competing interests exist.

Hmt1p is the predominant arginine methyltransferase in Saccharomyces cerevisiae Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes in hmt1 Δ, including acid phosphatases PHO5 , PHO11 , and PHO12 , phosphate transporters PHO84 and PHO89 and the vacuolar transporter chaperone VTC3 Analysis of the hmt1 Δ proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated in hmt1 Δ cells, showing decreased extracellular phosphatase levels and decreased total P i in phosphate-depleted medium. In vitro , we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation in hmt1 Δ if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated in vivo and the localization of a Pho4p-GFP fusion in hmt1 Δ was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate., (© 2018 Chia et al.)

A series of bis(4-pentylpyridinium) compounds with a variety of spacers between the pyridinium headgroups was synthesised, and the antifungal activity of these compounds was investigated. Lengthening the alkyl spacer between the pentylpyridinium headgroups from 12 to 16 methylene units resulted in increased antifungal activity against C. neoformans and C. albicans, but also resulted in increased hemolytic activity and cytotoxicity against mammalian cells. However, inclusion of an ortho-substituted benzene ring in the centre of the alkyl spacer resulted in decreased cytotoxicity and hemolytic activity, while maintaining antifungal potency. Replacement of the alkyl and aromatic-containing spacers by more hydrophilic ethylene glycol groups resulted in a loss of antifungal activity. Some of the compounds inhibited fungal PLB1 activity, but the low correlation of this inhibition with antifungal potency indicates PLB1 inhibition is unlikely to be the predominant mode of antifungal action of this class of compounds, with preliminary studies suggesting they may act via disruption of fungal mitochondrial function., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The innate immune system is the primary defense against cryptococcal infection, but paradoxically it promotes infection of the central nervous system. We performed a detailed longitudinal study of neurocryptococcosis in normal, chimeric, green fluorescent protein phagocyte-positive mice and phagocyte-depleted mice and interrogated the central nervous system innate immune response to Cryptococcus neoformans H99 using confocal microscopy, histology, flow cytometry, and quantification of brain cytokine/chemokines and fungal burdens. C. neoformans was present in the perivascular space (PVS) of post-capillary venules. This was associated with a massive influx of blood-derived monocytes, neutrophils, and T lymphocytes into the PVS and a predominantly proinflammatory cytokine/chemokine response. Phagocytes containing cryptococci were present only in the lumen and corresponding PVS of post-capillary venules. Free cryptococci were observed breaching the glia limitans, the protective barrier between the PVS and the cerebral parenchyma. Parenchymal cryptococcomas were typically in direct contact with post-capillary venules and lacked surrounding immune cell infiltrates. Phagocyte depletion abrogated cryptococcoma formation and PVS infiltrates. Together, these observations suggest that cryptococcomas can originate via phagocyte-dependent transport across post-capillary venular endothelium into the PVS and thence via passage of free cryptococci into the brain. In conclusion, we demonstrate for the first time that the PVS of cortical post-capillary venules is the major site of the early innate immune response to, and phagocyte-dependent entry of, C. neoformans., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Competing Interests: The authors have declared that no competing interests exist.

Invasive fungal diseases pose a serious threat, and new drugs are urgently needed. In this issue of Cell Chemical Biology, Pries et al. (2018) identified benzamide- and picolinamide-based small-molecule inhibitors with antifungal properties, including some active against pathogenic Candida species. These compounds target an essential component of the fungal secretion machinery, suggesting a new approach to antifungal development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cryptococcus neoformans is a basidiomycetous yeast and the cause of cryptococcosis in immunocompromised individuals. The most severe form of the disease is meningoencephalitis, which is one of the leading causes of death in HIV/AIDS patients. In order to access the central nervous system, C. neoformans relies on the activity of certain virulence factors such as urease, which allows transmigration through the blood-brain barrier. In this study, we demonstrate that the calcium transporter Pmc1 enables C. neoformans to penetrate the central nervous system, because the pmc1 null mutant failed to infect and to survive within the brain parenchyma in a murine systemic infection model. To investigate potential alterations in transmigration pathways in these mutants, global expression profiling of the pmc1 mutant strain was undertaken, and genes associated with urease, the Ca 2+ -calcineurin pathway, and capsule assembly were identified as being differentially expressed. Also, a decrease in urease activity was observed in the calcium transporter null mutants. Finally, we demonstrate that the transcription factor Crz1 regulates urease activity and that the Ca 2+ -calcineurin signalling pathway positively controls the transcription of calcium transporter genes and factors related to transmigration., (© 2017 John Wiley & Sons Ltd.)

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 IP 3 to PP-IP 5 (IP 7 ), 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 (IP 3 /IP 4 ) kinase producing IP 5 . We showed previously that IP 5 is further phosphorylated by Ipk1 to produce IP 6 , which is a substrate for the synthesis of PP-IP 5 by Kcs1. Phenotypic comparison of the arg1Δ and kcs1Δ deletion mutants (both PP-IP 5 -deficient) reveals that arg1Δ has the most deleterious phenotype: while PP-IP 5 is essential for metabolic and stress adaptation in both mutant strains, PP-IP 5 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-IP 5 -dependent and novel PP-IP 5 -independent functions.

With the growing demand for new antibiotics to combat increasing multi-drug resistance, a family of antimicrobial peptides known as cathelicidins has emerged as potential candidates. Expansions in cathelicidin-encoding genes in marsupials and monotremes are of specific interest as the peptides they encode have evolved to protect immunologically naive young in the harsh conditions of the pouch and burrow. Our previous work demonstrated that some marsupial and monotreme cathelicidins have broad-spectrum antibacterial activity and kill resistant bacteria, but the activity of many cathelicidins is unknown. To investigate associations between peptide antimicrobial activity and physiochemical properties, we tested 15 cathelicidin mature peptides from tammar wallaby, grey short-tailed opossum, platypus and echidna for antimicrobial activity against a range of bacterial and fungal clinical isolates. One opossum cathelicidin ModoCath4, tammar wallaby MaeuCath7 and echidna Taac-CATH1 had broad-spectrum antibacterial activity and killed methicillin-resistant Staphylococcus aureus. However, antimicrobial activity was reduced in the presence of serum or whole blood, and non-specific toxicity was observed at high concentrations. The active peptides were highly charged, potentially increasing binding to microbial surfaces, and contained amphipathic helical structures, which may facilitate membrane permeabilisation. Peptide sequence homology, net charge, amphipathicity and alpha helical content did not correlate with antimicrobial activity. However active peptides contained a significantly higher percentage of cationic residues than inactive ones, which may be used to predict active peptides in future work. Along with previous studies, our results indicate that marsupial and monotreme cathelicidins show potential for development as novel therapeutics to combat increasing antimicrobial resistance.

Influenza A virus (IAV) infects millions of people annually and predisposes to secondary bacterial infections. Inhalation of fungi within the Cryptococcus complex causes pulmonary disease with secondary meningo-encephalitis. Underlying pulmonary disease is a strong risk factor for development of C. gattii cryptococcosis though the effect of concurrent infection with IAV has not been studied. We developed an in vivo model of Influenza A H1N1 and C. gattii co-infection. Co-infection resulted in a major increase in morbidity and mortality, with severe lung damage and a high brain fungal burden when mice were infected in the acute phase of influenza multiplication. Furthermore, IAV alters the host response to C. gattii , leading to recruitment of significantly more neutrophils and macrophages into the lungs. Moreover, IAV induced the production of type 1 interferons (IFN-α4/β) and the levels of IFN-γ were significantly reduced, which can be associated with impairment of the immune response to Cryptococcus during co-infection. Phagocytosis, killing of cryptococci and production of reactive oxygen species (ROS) by IAV-infected macrophages were reduced, independent of previous IFN-γ stimulation, leading to increased proliferation of the fungus within macrophages. In conclusion, IAV infection is a predisposing factor for severe disease and adverse outcomes in mice co-infected with C. gattii .

Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signaling pathway. Cryptococcus neoformans disseminates from the lung to the brain and is the commonest cause of fungal meningitis worldwide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we characterized a transcription factor knockout strain ( hlh3Δ/pho4Δ ) defective in phosphate acquisition. Despite little similarity with other fungal Pho4 proteins, Hlh3/Pho4 functioned like a typical phosphate-responsive transcription factor in phosphate-deprived cryptococci, accumulating in nuclei and triggering expression of genes involved in phosphate acquisition. The pho4 Δ mutant strain was susceptible to a number of stresses, the effect of which, except for alkaline pH, was alleviated by phosphate supplementation. Even in the presence of phosphate, the PHO pathway was activated in wild-type cryptococci at or above physiological pH, and under these conditions, the pho4 Δ mutant had a growth defect and compromised phosphate uptake. The pho4Δ mutant was hypovirulent in a mouse inhalation model, where dissemination to the brain was reduced dramatically, and markedly hypovirulent in an intravenous dissemination model. The pho4Δ mutant was not detected in blood, nor did it proliferate significantly when cultured with peripheral blood monocytes. In conclusion, dissemination of infection and the pathogenesis of meningitis are dependent on cryptococcal phosphate uptake and stress tolerance at alkaline pH, both of which are Pho4 dependent. IMPORTANCE Cryptococcal meningitis is fatal without treatment and responsible for more than 500,000 deaths annually. To be a successful pathogen, C. neoformans must obtain an adequate supply of essential nutrients, including phosphate, from various host niches. Phosphate acquisition in fungi is regulated by the PHO signaling cascade, which is activated when intracellular phosphate decreases below a critical level. Induction of phosphate acquisition genes leads to the uptake of free phosphate via transporters. By blocking the PHO pathway using a Pho4 transcription factor mutant ( pho4Δ mutant), we demonstrate the importance of the pathway for cryptococcal dissemination and the establishment of brain infection in murine models. Specifically, we show that reduced dissemination of the pho4Δ mutant to the brain is due to an alkaline pH tolerance defect, as alkaline pH mimics the conditions of phosphate deprivation. The end result is inhibited proliferation in host tissues, particularly in blood.

Tasmanian devil joeys, like other marsupials, are born at a very early stage of development, prior to the development of their adaptive immune system, yet survive in a pathogen-laden pouch and burrow. Antimicrobial peptides, called cathelicidins, which provide innate immune protection during early life, are expressed in the pouch lining, skin and milk of devil dams. These peptides are active against pathogens identified in the pouch microbiome. Of the six characterised cathelicidins, Saha-CATH5 and 6 have broad-spectrum antibacterial activity and are capable of killing problematic human pathogens including methicillin-resistant S. aureus and vancomycin-resistant E. faecalis, while Saha-CATH3 is active against fungi. Saha-CATH5 and 6 were toxic to human A549 cells at 500 μg/mL, which is over seven times the concentration required to kill pathogens. The remaining devil cathelicidins were not active against tested bacterial or fungal strains, but are widely expressed throughout the body, such as in immune tissues, in digestive, respiratory and reproductive tracts, and in the milk and pouch, which indicates that they are likely also important components of the devil immune system. Our results suggest cathelicidins play a role in protecting naive young during pouch life by passive immune transfer in the milk and may modulate pouch microbe populations to reduce potential pathogens.

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.

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 (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1Δkcs1Δ and kcs1Δ lung burdens were also lower than those of ipk1Δ. Unlike ipk1Δ, ipk1Δkcs1Δ and kcs1Δ failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, 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 ipk1Δ, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1Δ grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence.

Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg) cause neurological disease and cross the BBB as free cells or in mononuclear phagocytes via the Trojan horse mechanism, although evidence for the latter is indirect. There is emerging evidence that Cn and the North American outbreak Cg strain (R265) more commonly cause neurological and lung disease, respectively. We have employed a widely validated in vitro model of the BBB, which utilizes the hCMEC/D3 cell line derived from human brain endothelial cells (HBEC) and the human macrophage-like cell line, THP-1, to investigate whether transport of dual fluorescence-labelled Cn and Cg across the BBB occurs within macrophages. We showed that phagocytosis of Cn by non-interferon (IFN)-γ stimulated THP-1 cells was higher than that of Cg. Although Cn and Cg-loaded THP-1 bound similarly to TNF-activated HBECs under shear stress, more Cn-loaded macrophages were transported across an intact HBEC monolayer, consistent with the predilection of Cn for CNS infection. Furthermore, Cn exhibited a higher rate of expulsion from transmigrated THP-1 compared with Cg. Our results therefore provide further evidence for transmigration of both Cn and Cg via the Trojan horse mechanism and a potential explanation for the predilection of Cn to cause CNS infection., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2015 Lev et al.)

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of fungal-infection-related fatalities, especially in immunocompromised hosts. Several virulence factors are known to play a major role in the pathogenesis of cryptococcal infections, including the enzyme phospholipase B1 (Plb1). Compared to other well-studied Cryptococcus neoformans virulence factors such as the polysaccharide capsule and melanin production, very little is known about the contribution of Plb1 to cryptococcal virulence. Phospholipase B1 is a phospholipid-modifying enzyme that has been implicated in multiple stages of cryptococcal pathogenesis, including initiation and persistence of pulmonary infection and dissemination to the central nervous system, but the underlying reason for these phenotypes remains unknown. Here we demonstrate that a Δplb1 knockout strain of C. neoformans has a profound defect in intracellular growth within host macrophages. This defect is due to a combination of a 50% decrease in proliferation and a 2-fold increase in cryptococcal killing within the phagosome. In addition, we show for the first time that the Δplb1 strain undergoes a morphological change during in vitro and in vivo intracellular infection, resulting in a subpopulation of very large titan cells, which may arise as a result of the attenuated mutant's inability to cope within the macrophage., (Copyright © 2015 Evans et al.)

Background: Cryptococcus neoformans is the commonest cause of fungal meningitis, with a substantial mortality despite appropriate therapy. Quantitative culture of cryptococci in cerebrospinal fluid (CSF) during antifungal therapy is of prognostic value and has therapeutic implications, but is slow and not practicable in many resource-poor countries., Methods: We piloted two rapid techniques for quantifying viable cryptococci using mixtures of live and heat-killed cryptococci cultured in vitro: (i) quantitative microscopy with exclusion staining using trypan blue dye, and (ii) flow cytometry, using the fluorescent dye 2'-7'-Bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM). Results were compared with standard quantitative cryptococcal cultures. Quantitative microscopy was also performed on cerebrospinal fluid (CSF) samples., Results: Both microscopy and flow cytometry distinguished between viable and non-viable cryptococci. Cell counting (on log scale) by microscopy and by quantitative culture were significantly linearly associated (p<0.0001) and Bland-Altman analysis showed a high level of agreement. Proportions of viable cells (on logit scale), as detected by flow cytometry were significantly linearly associated with proportions detected by microscopy (p<0.0001) and Bland-Altman analysis showed a high level of agreement., Conclusions: Direct microscopic examination of trypan blue-stained cryptococci and flow-cytometric assessment of BCECF-AM-stained cryptococci were in good agreement with quantitative cultures. These are promising strategies for rapid determination of the viability of cryptococci, and should be investigated in clinical practice.

Unlabelled: 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&#95;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., Importance: Infections with the AIDS-related fungal pathogen Cryptococcus neoformans cause more than 600,000 deaths per year worldwide. Strains of Cryptococcus neoformans isolated from patients with AIDS secrete acid phosphatase; however, the identity and role of the enzyme(s) are unknown. We have analyzed the secretome of the highly virulent serotype A strain H99 and identified Aph1, a canonically secreted acid phosphatase. By creating an APH1 deletion mutant and an Aph1-DsRed-expressing strain, we demonstrate that Aph1 is the major extracellular and vacuolar acid phosphatase in C. neoformans and that it is phosphate repressible. Furthermore, we show that Aph1 is produced in cryptococci during coculture with THP-1 monocytes and contributes to fungal virulence in Galleria mellonella and mouse models of cryptococcosis. Our findings suggest that Aph1 is secreted to the environment to scavenge phosphate from a wide range of physiological substrates and is targeted to vacuoles to recycle phosphate from the expendable macromolecules., (Copyright © 2014 Lev et al.)

Miltefosine (MI) is a novel, potential antifungal agent with activity against some yeast and filamentous fungal pathogens. We previously demonstrated in the model yeast, Saccharomyces cerevisiae, that MI causes disruption of mitochondrial membrane potential and apoptosis-like cell death via interaction with the Cox9p sub-unit of cytochrome c oxidase (COX). To identify additional mechanisms of antifungal action, MI resistance was induced in S. cerevisiae by exposure to the mutagen, ethyl methanesulfonate, and gene mutation(s) responsible for resistance were investigated. An MI-resistant haploid strain (H-C101) was created. Resistance was retained in the diploid strain (D-C101) following mating, confirming dominant inheritance. Phenotypic assessment of individual D-C101 tetrads revealed that only one mutant gene contributed to the MI-resistance phenotype. To identify this gene, the genome of H-C101 was sequenced and 17 mutated genes, including metacaspase-encoding MCA1, were identified. The MCA1 mutation resulted in substitution of asparagine (N) with aspartic acid (D) at position 164 (MCA1(N164D)). MI resistance was found to be primarily due to MCA1(N164D), as single-copy episomal expression of MCA1(N164D), but not two other mutated genes (FAS1(T1417I) and BCK2(T104A)), resulted in MI resistance in the wild-type strain. Furthermore, an MCA1 deletion mutant (mca1Δ) was MI-resistant. MI treatment led to accumulation of reactive oxygen species (ROS) in MI-resistant (MCA1(N164D)-expressing and mca1Δ) strains and MI-susceptible (MCA1-expressing) strains, but failed to activate Mca1 in the MI-resistant strains, demonstrating that ROS accumulation does not contribute to the fungicidal effect of MI. In conclusion, functional disruption of Mca1, leads to MI resistance and inability to mediate MI-induced apoptotic effects. Mca1-mediated apoptosis is therefore a major mechanism of MI-induced antifungal action., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Miltefosine (MI) has in vitro fungicidal activity against pathogenic fungi. However, mechanisms of resistance to MI have not been studied. By screening a genomic library of the model yeast, Saccharomyces cerevisiae, we identified HXT13 as a candidate genetic determinant of MI resistance. HXT13 belongs to the yeast hexose transporter family, which mediates hexose sugar uptake and is included in the major facilitator superfamily (MFS). We now report that overexpression of HXT13, but not of the closely-related genes, HXT15 and HXT17, and the more distantly related HXT14, resulted in a stable MI-resistant phenotype in S. cerevisiae. Resistance of the HXT13 overexpressing strain to MI correlated with higher cell viability following MI exposure as assessed by SYTOX® green staining compared with the control and overexpressing HXT14 strains. The mechanism of resistance in the HXT13 overexpressing strain was due to increased ATP-independent MI efflux. However, resistance to MI of the HXT13-overexpressing strain did not extend to other drugs including the echinocandins, amphotericin B, azoles, cycloheximide and sulfometuron methyl, ruling out the involvement of HXT13 in multidrug resistance. In summary, we have identified a new function of the hexose sugar transporter gene HXT13 when overexpressed in S. cerevisiae, namely, in efflux of MI and in mediating MI resistance., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: Antifungal treatment of uncommon filamentous fungal infections is problematic. This study determined the in vitro susceptibility of miltefosine, as a single agent and in combination with posaconazole or voriconazole, against these pathogens., Methods: Susceptibility to miltefosine of 34 uncommon filamentous fungi was tested using CLSI broth microdilution M38-A2 methodology. Twenty isolates were studied for potential synergy using miltefosine/posaconazole and miltefosine/voriconazole combinations and the chequerboard microdilution assay., Results: MICs of miltefosine were high (in general, >8 mg/L) for most isolates compared with amphotericin B, echinocandins and the azoles. Miltefosine had greatest activity against Scedosporium spp., Lichtheimia corymbifera and Rhizomucor sp. (MICs ≤ 4 mg/L). Miltefosine in combination either with posaconazole or voriconazole demonstrated synergy [fractional inhibitory concentration index (FICI) ≤ 0.5] in 12 instances (11 isolates): miltefosine/posaconazole combinations were synergistic against 3 of 4 Fusarium oxysporum strains (FICI range 0.37-0.5) and 5 of 10 mucormycete strains (FICI range 0.06-0.5). The combination of voriconazole with miltefosine showed synergy against one Scedosporium prolificans isolate and three mucormycetes-a single strain each of L. corymbifera, Rhizopus oryzae and Rhizomucor sp. No antagonism was observed., Conclusions: Miltefosine demonstrated synergy in 8/20 (40%) and 4/20 (20%) instances when combined with posaconazole and voriconazole, respectively. Synergy was most often observed against F. oxysporum and the mucormycetes. Study of miltefosine/azole combinations as a novel antifungal approach is indicated.

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 CnΔplc1 mutant and markedly increased in the CnΔarg1 mutant, while PIP(2) was increased in both mutants. The CnΔplc1 and CnΔarg1 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 (ScΔarg82) strain, the CnΔarg1 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.

The subtelomeric regions of organisms ranging from protists to fungi undergo a much higher rate of rearrangement than is observed in the rest of the genome. While characterizing these ~40-kb regions of the human fungal pathogen Cryptococcus neoformans, we have identified a recent gene amplification event near the right telomere of chromosome 3 that involves a gene encoding an arsenite efflux transporter (ARR3). The 3,177-bp amplicon exists in a tandem array of 2-15 copies and is present exclusively in strains with the C. neoformans var. grubii subclade VNI A5 MLST profile. Strains bearing the amplification display dramatically enhanced resistance to arsenite that correlates with the copy number of the repeat; the origin of increased resistance was verified as transport-related by functional complementation of an arsenite transporter mutant of Saccharomyces cerevisiae. Subsequent experimental evolution in the presence of increasing concentrations of arsenite yielded highly resistant strains with the ARR3 amplicon further amplified to over 50 copies, accounting for up to ~1% of the whole genome and making the copy number of this repeat as high as that seen for the ribosomal DNA. The example described here therefore represents a rare evolutionary intermediate-an array that is currently in a state of dynamic flux, in dramatic contrast to relatively common, static relics of past tandem duplications that are unable to further amplify due to nucleotide divergence. Beyond identifying and engineering fungal isolates that are highly resistant to arsenite and describing the first reported instance of microevolution via massive gene amplification in C. neoformans, these results suggest that adaptation through gene amplification may be an important mechanism that C. neoformans employs in response to environmental stresses, perhaps including those encountered during infection. More importantly, the ARR3 array will serve as an ideal model for further molecular genetic analyses of how tandem gene duplications arise and expand.

Secretion pathways in fungi are essential for the maintenance of cell wall architecture and for the export of a number of virulence factors. In the fungal pathogen, Cryptococcus neoformans, much evidence supports the existence of more than one route taken by secreted molecules to reach the cell periphery and extracellular space, and a significant degree of crosstalk between conventional and non-conventional secretion routes. The need for such complexity may be due to differences in the nature of the exported cargo, the spatial and temporal requirements for constitutive and non-constitutive protein secretion, and/or as a means of compensating for the extra burden on the secretion machinery imposed by the elaboration of the polysaccharide capsule. This review focuses on the role of specific components of the C. neoformans secretion machinery in protein and/or polysaccharide export, including Sec4, Sec6, Sec14, Golgi reassembly and stacking protein and extracellular exosome-like vesicles. We also address what is known about traffic of the lipid, glucosylceramide, a target of therapeutic antibodies and an important regulator of C. neoformans pathogenicity, and the role of signalling pathways in the regulation of secretion.

Recent studies indicate that mitochondrial functions impinge on cell wall integrity, drug tolerance, and virulence of human fungal pathogens. However, the mechanistic aspects of these processes are poorly understood. We focused on the mitochondrial outer membrane SAM (Sorting and Assembly Machinery) complex subunit Sam37 in Candida albicans. Inactivation of SAM37 in C. albicans leads to a large reduction in fitness, a phenotype not conserved with the model yeast Saccharomyces cerevisiae. Our data indicate that slow growth of the sam37ΔΔ mutant results from mitochondrial DNA loss, a new function for Sam37 in C. albicans, and from reduced activity of the essential SAM complex subunit Sam35. The sam37ΔΔ mutant was hypersensitive to drugs that target the cell wall and displayed altered cell wall structure, supporting a role for Sam37 in cell wall integrity in C. albicans. The sensitivity of the mutant to membrane-targeting antifungals was not significantly altered. The sam37ΔΔ mutant was avirulent in the mouse model, and bioinformatics showed that the fungal Sam37 proteins are distant from their animal counterparts and could thus represent potential drug targets. Our study provides the first direct evidence for a link between mitochondrial function and cell wall integrity in C. albicans and is further relevant for understanding mitochondrial function in fitness, antifungal drug tolerance, and virulence of this major pathogen. Beyond the relevance to fungal pathogenesis, this work also provides new insight into the mitochondrial and cellular roles of the SAM complex in fungi.

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a bona fide target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δcna1, and a CRZ1/SP1 mutant (Δcrz1) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, CHS6, was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT C. neoformans and Δcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30-37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of C. neoformans Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.

We have investigated the potential of the GTP synthesis pathways as chemotherapeutic targets in the human pathogen Cryptococcus neoformans, a common cause of fatal fungal meningoencephalitis. We find that de novo GTP biosynthesis, but not the alternate salvage pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway results in slow growth and virulence factor defects, while loss of the cognate phosphoribosyltransferase in the salvage pathway yielded no phenotypes. Further, the Cryptococcus species complex displays variable sensitivity to the IMPDH inhibitor mycophenolic acid, and we uncover a rare drug-resistant subtype of C. gattii that suggests an adaptive response to microbial IMPDH inhibitors in its environmental niche. We report the structural and functional characterization of IMPDH from Cryptococcus, revealing insights into the basis for drug resistance and suggesting strategies for the development of fungal-specific inhibitors. The crystal structure reveals the position of the IMPDH moveable flap and catalytic arginine in the open conformation for the first time, plus unique, exploitable differences in the highly conserved active site. Treatment with mycophenolic acid led to significantly increased survival times in a nematode model, validating de novo GTP biosynthesis as an antifungal target in Cryptococcus.

Miltefosine has antifungal properties and potential for development as a therapeutic for invasive fungal infections. However, its mode of action in fungi is poorly understood. We demonstrate that miltefosine is rapidly incorporated into yeast, where it penetrates the mitochondrial inner membrane, disrupting mitochondrial membrane potential and leading to an apoptosis-like cell death. COX9, which encodes subunit VIIa of the cytochrome c oxidase (COX) complex in the electron transport chain of the mitochondrial membrane, was identified as a potential target of miltefosine from a genomic library screen of the model yeast Saccharomyces cerevisiae. When overexpressed in S. cerevisiae, COX9, but not COX7 or COX8, led to a miltefosine-resistant phenotype. The effect of miltefosine on COX activity was assessed in cells expressing different levels of COX9. Miltefosine inhibited COX activity in a dose-dependent manner in Cox9p-positive cells. This inhibition most likely contributed to the miltefosine-induced apoptosis-like cell death.

Adaptation to host temperature is a prerequisite for any pathogen capable of causing deep infection in humans. Our previous studies demonstrated that a Cryptococcus neoformans ccr4Δ mutant lacking the major deadenylase involved in regulated mRNA decay was defective in host temperature adaptation and therefore virulence. In this study, the ccr4Δ mutant was found to exhibit characteristics of chronic unfolded-protein response (UPR) engagement in both the gene expression profile and phenotype. We demonstrate that host temperature adaptation in C. neoformans is accompanied by transient induction of the endoplasmic reticulum (ER) stress response and that Ccr4-dependent posttranscriptional gene regulation contributes to resolution of ER stress during host temperature adaptation.

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine-three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°-40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection.

Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (CnΔsec14-1 and CnΔsec14-1CnΔsfh5). Similar to the CnPLB1 deletion mutant (CnΔplb1), CnΔsec14-1 was hypovirulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live CnΔsec14-1 and CnΔplb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely related CnSEC14-1 homologue, and CnSFH5, a distantly related SEC fourteen like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of CnΔsec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14-dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis., (© Published 2011 This article is a US Government work and is in the public domain in the USA.)

The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs., (© 2010 Blackwell Publishing Ltd.)

Many pathogenic microbes, including many fungi, produce phospholipases which facilitate survival of the pathogen in vivo, invasion and dissemination throughout the host, expression of virulence traits and evasion of host immune defense mechanisms. These phospholipases are either secreted or produced intracellularly and act by physically disrupting host membranes, and/or by affecting fungal cell signaling and production of immunomodulatory effectors. Many of the secreted phospholipases acquire a glycosylphosphatidylinositol sorting motif to facilitate membrane and/or cell wall association and secretion. This review focuses primarily on the role of two members of the phospholipase enzyme family, phospholipase B (Plb) and phosphatidylinositol (PI)-specific phospholipase C (PI-C/Plc), in fungal pathogenesis and in particular, what has been learnt about their function from studies performed in the model pathogenic yeast, Cryptococcus neoformans. These studies have revealed how Plb has adapted to become an important part of the virulence repertoire of pathogenic fungi and how its secretion is regulated. They have also provided valuable insight into how the intracellular enzyme, Plc1, contributes to fungal fitness and pathogenicity - via a putative role in signal transduction pathways that regulate the production of stress-protecting pigments, polysaccharide capsule, cell wall integrity, and adaptation to growth at host temperature. Finally, this review will address the role fungal phospholipases have played in the development of a new class of antifungal drugs, which mimic their phospholipid substrates.