Sophie Lev - Academia.edu (original) (raw)

Papers by Sophie Lev

Humana Press eBooks, 2010

In host-pathogen interactions, identification of pathogen genes expressed during plant infection ... more In host-pathogen interactions, identification of pathogen genes expressed during plant infection poses a challenge, even though these genes may be strongly induced by signals from the host. Here, we describe the application of a PCR-based differential screening method to plant-fungal interactions. Suppression subtraction hybridization (SSH) provides a sensitive method to isolate fungal genes expressed in planta. Total RNA is isolated from infected plants for comparison with the pathogen in axenic culture, or, in the application described here, plants infected with a wild type isolate are compared with plants infected with a mutant. Following library construction, clones are sequenced and screened for differential expression in the two starting populations.

Molecular Plant-microbe Interactions, Jun 1, 2008

Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essentia... more Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways.

The Plant Cell, Mar 21, 2003

Conserved eukaryotic signaling elements play an important role in the development of fungal patho... more Conserved eukaryotic signaling elements play an important role in the development of fungal pathogens on their hosts. Chk1, a mitogen-activated protein kinase (MAPK), functions in virulence, mating, and sporulation of the maize leaf pathogen Cochliobolus heterostrophus. Suppression subtractive hybridization was used to identify fungal genes whose expression on the host plant is affected in chk1 deletion mutants. Two of the genes isolated in this screen were predicted to encode cellulolytic enzymes: a cellobiohydrolase, CBH7, and an endoglucanase, EG6. Expression of EG6 and CBH7 was followed by the fusion of their upstream regulatory regions to the coding sequence of the green fluorescent protein. Induction of both genes began at the onset of invasive growth and reached its maximal extent during leaf necrosis. Furthermore, EG6 was induced preferentially within necrotic lesions. Disruption of MAPK CHK1 resulted in a delay in the penetration of hyphae into the leaf and a concomitant delay in the induction of expression of both cellulase genes. In saprophytic culture, the absence of Chk1 resulted in a marked delay in the induction of CBH7 expression by crystalline cellulose. EG6 was expressed at a basal level in culture, and this expression was found to depend strictly on Chk1. Thus, the Chk1 MAPK signaling pathway is involved in the regulation of two cellulase-encoding genes and is necessary for their timely induction by environmental signals.

mBio

Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 o... more 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.

Biomolecules

New antifungals with unique modes of action are urgently needed to treat the increasing global bu... more 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 IP3 to IP8, 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 IP3-4 kinase (IP3-4K) the most critical. The dibenzylaminopurine compound, N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP3-4K has not been investigated. We purified IP3-4K from the human pathogens, Cryptococcus neoformans and Candida albicans, and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC50) and binding affinity (KD), respectively, of TNP...

Cellular Microbiology, 2021

Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular proc... more 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. K E Y W O R D S fungal pathogens, inositol polyphosphate kinases, inositol polyphosphates, inositol pyrophosphates, microbial pathogenesis, protein modification 1 | INTRODUCTION IPs and PP-IPs are produced by a series of sequentially acting IP kinases (IPKs). Using genetic and pharmacological approaches to modulate IP kinase (IPK) activity in conjunction with gel-, HPLC-and, more recently, mass spectrometry-based metabolic profiling strategies, the identification and role of IPs and PP-IPs were initially elucidated in mammalian cells and the model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. IPK enzymatic function has also been confirmed in vitro using chemically synthesized substrates. These studies revealed that IPs and PP-IPs function in a diverse range of cellular processes including glucose homeostasis, insulin sensitivity and secretion, fat metabolism and cellular energy dynamics, growth factor signalling, phosphate homeostasis, vesicular trafficking, DNA damage and repair, chromatin remodelling, spermatogenesis, neuronal migration, neutrophil activity, aging, apoptosis and platelet function (Lee, Kim, Ahn, & Kim, 2020). Hence, it is not surprising that dysregulated IP and PP-IP biosynthesis in human cells is associated with numerous diseases including Huntington's disease (Ahmed et al., 2015), diabetes and obesity (Chakraborty et al., 2010) and cancer (Rao et al., 2015). Advances in genome sequencing and genome manipulation technology have subsequently allowed investigation into the roles of IPs and PP-IPs in microbial pathogenicity. The most significant progress has been in: Cryptococcus neoformans, an AIDS-related fungal pathogen and the most common cause of fungal meningitis worldwide (Rajasingham et al., 2017); Candida albicans, the commensal and opportunistic nosocomial pathogen and most prevalent cause of fungal infections worldwide (Bongomin, Gago, Oladele, &

This article cites 46 articles, 23 of which can be accessed free

The American Journal of Pathology, 2018

The innate immune system is the primary defense against cryptococcal infection, but paradoxically... more 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 phagocyteepositive 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.

Cell Chemical Biology, 2018

Identification of new activators of mitochondrial fusion reveals a link between mitochondrial mor... more Identification of new activators of mitochondrial fusion reveals a link between mitochondrial morphology and pyrimidine metabolism. Cell Chem. Biol. 25, this issue, 268-278.

Molecular & Cellular Proteomics, 2018

Arginine methylation of proteins in the eukaryotic cell is predominantly catalyzed by one conserv... more Arginine methylation of proteins in the eukaryotic cell is predominantly catalyzed by one conserved enzyme; PRMT1 in mammals or Hmt1p in yeast. Knockout in mammals is embryonic lethal; however, Hmt1p in yeast is non-essential. The systems-level effects of hmt1 knockout in yeast were investigated. Unexpected but significant dysregulation in phosphate homeostasis was seen upon hmt1 knockout. Transcription factor-driven processes may explain these observations, or regulatory processes may link the sensing of S-adenosylmethionine to intracellular phosphate or polyphosphate.

mSphere

Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signal... more 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 pho4Δ 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 pho4Δ ...

Journal of Fungi, 2016

Opportunistic fungi are a major cause of morbidity and mortality worldwide , particularly in immu... more Opportunistic fungi are a major cause of morbidity and mortality worldwide , 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-IP 5 (IP 7) 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.

Molecular Plant Microbe Interactions, 2009

Filamentous fungi produce hydrophobins, small proteins that are localized on the outer surface of... more Filamentous fungi produce hydrophobins, small proteins that are localized on the outer surface of their cell walls and are involved in growth and development. Nutrient availability and light regulate hydrophobin gene expression. Recently it was demonstrated that signal transduction components such as a MAPK in Magnaporthe grisea and a G-protein alpha subunit in Cryphonectria parasitica are also involved. Here we conducted a comparative study on the effect of loss of function mutations in different signaling components on hydrophobin gene expression in Cochliobolus heterostrophus. Furthermore, a mutant deficient in both G-protein alpha and beta subunits (Cgab1) was constructed. This mutant has an albino phenotype, lacked conidia but still formed abortive pseudothecia in a backcross, and was unable to infect maize. Loss of the G-alpha subunit led, in some conditions, to increased hydrophobin expression, which may be involved in the spore aggregation phenotype of this mutant. Mutations...

PLoS ONE, 2012

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium... more 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, Dcna1, and a CRZ1/SP1 mutant (Dcrz1) 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 Dcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30-37uC vs 25uC) 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.

The Plant Cell, 2003

Conserved eukaryotic signaling elements play an important role in the development of fungal patho... more Conserved eukaryotic signaling elements play an important role in the development of fungal pathogens on their hosts. Chk1, a mitogen-activated protein kinase (MAPK), functions in virulence, mating, and sporulation of the maize leaf pathogen Cochliobolus heterostrophus. Suppression subtractive hybridization was used to identify fungal genes whose expression on the host plant is affected in chk1 deletion mutants. Two of the genes isolated in this screen were predicted to encode cellulolytic enzymes: a cellobiohydrolase, CBH7 , and an endoglucanase, EG6. Expression of EG6 and CBH7 was followed by the fusion of their upstream regulatory regions to the coding sequence of the green fluorescent protein. Induction of both genes began at the onset of invasive growth and reached its maximal extent during leaf necrosis. Furthermore, EG6 was induced preferentially within necrotic lesions. Disruption of MAPK CHK1 resulted in a delay in the penetration of hyphae into the leaf and a concomitant delay in the induction of expression of both cellulase genes. In saprophytic culture, the absence of Chk1 resulted in a marked delay in the induction of CBH7 expression by crystalline cellulose. EG6 was expressed at a basal level in culture, and this expression was found to depend strictly on Chk1. Thus, the Chk1 MAPK signaling pathway is involved in the regulation of two cellulase-encoding genes and is necessary for their timely induction by environmental signals.

mBio, 2020

Invasive fungal diseases pose a serious threat to human health globally with >1.5 million deat... more 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.

Humana Press eBooks, 2010

In host-pathogen interactions, identification of pathogen genes expressed during plant infection ... more In host-pathogen interactions, identification of pathogen genes expressed during plant infection poses a challenge, even though these genes may be strongly induced by signals from the host. Here, we describe the application of a PCR-based differential screening method to plant-fungal interactions. Suppression subtraction hybridization (SSH) provides a sensitive method to isolate fungal genes expressed in planta. Total RNA is isolated from infected plants for comparison with the pathogen in axenic culture, or, in the application described here, plants infected with a wild type isolate are compared with plants infected with a mutant. Following library construction, clones are sequenced and screened for differential expression in the two starting populations.

Molecular Plant-microbe Interactions, Jun 1, 2008

Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essentia... more Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways.

The Plant Cell, Mar 21, 2003

Conserved eukaryotic signaling elements play an important role in the development of fungal patho... more Conserved eukaryotic signaling elements play an important role in the development of fungal pathogens on their hosts. Chk1, a mitogen-activated protein kinase (MAPK), functions in virulence, mating, and sporulation of the maize leaf pathogen Cochliobolus heterostrophus. Suppression subtractive hybridization was used to identify fungal genes whose expression on the host plant is affected in chk1 deletion mutants. Two of the genes isolated in this screen were predicted to encode cellulolytic enzymes: a cellobiohydrolase, CBH7, and an endoglucanase, EG6. Expression of EG6 and CBH7 was followed by the fusion of their upstream regulatory regions to the coding sequence of the green fluorescent protein. Induction of both genes began at the onset of invasive growth and reached its maximal extent during leaf necrosis. Furthermore, EG6 was induced preferentially within necrotic lesions. Disruption of MAPK CHK1 resulted in a delay in the penetration of hyphae into the leaf and a concomitant delay in the induction of expression of both cellulase genes. In saprophytic culture, the absence of Chk1 resulted in a marked delay in the induction of CBH7 expression by crystalline cellulose. EG6 was expressed at a basal level in culture, and this expression was found to depend strictly on Chk1. Thus, the Chk1 MAPK signaling pathway is involved in the regulation of two cellulase-encoding genes and is necessary for their timely induction by environmental signals.

mBio

Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 o... more 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.

Biomolecules

New antifungals with unique modes of action are urgently needed to treat the increasing global bu... more 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 IP3 to IP8, 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 IP3-4 kinase (IP3-4K) the most critical. The dibenzylaminopurine compound, N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP3-4K has not been investigated. We purified IP3-4K from the human pathogens, Cryptococcus neoformans and Candida albicans, and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC50) and binding affinity (KD), respectively, of TNP...

Cellular Microbiology, 2021

Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular proc... more 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. K E Y W O R D S fungal pathogens, inositol polyphosphate kinases, inositol polyphosphates, inositol pyrophosphates, microbial pathogenesis, protein modification 1 | INTRODUCTION IPs and PP-IPs are produced by a series of sequentially acting IP kinases (IPKs). Using genetic and pharmacological approaches to modulate IP kinase (IPK) activity in conjunction with gel-, HPLC-and, more recently, mass spectrometry-based metabolic profiling strategies, the identification and role of IPs and PP-IPs were initially elucidated in mammalian cells and the model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. IPK enzymatic function has also been confirmed in vitro using chemically synthesized substrates. These studies revealed that IPs and PP-IPs function in a diverse range of cellular processes including glucose homeostasis, insulin sensitivity and secretion, fat metabolism and cellular energy dynamics, growth factor signalling, phosphate homeostasis, vesicular trafficking, DNA damage and repair, chromatin remodelling, spermatogenesis, neuronal migration, neutrophil activity, aging, apoptosis and platelet function (Lee, Kim, Ahn, & Kim, 2020). Hence, it is not surprising that dysregulated IP and PP-IP biosynthesis in human cells is associated with numerous diseases including Huntington's disease (Ahmed et al., 2015), diabetes and obesity (Chakraborty et al., 2010) and cancer (Rao et al., 2015). Advances in genome sequencing and genome manipulation technology have subsequently allowed investigation into the roles of IPs and PP-IPs in microbial pathogenicity. The most significant progress has been in: Cryptococcus neoformans, an AIDS-related fungal pathogen and the most common cause of fungal meningitis worldwide (Rajasingham et al., 2017); Candida albicans, the commensal and opportunistic nosocomial pathogen and most prevalent cause of fungal infections worldwide (Bongomin, Gago, Oladele, &

This article cites 46 articles, 23 of which can be accessed free

The American Journal of Pathology, 2018

The innate immune system is the primary defense against cryptococcal infection, but paradoxically... more 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 phagocyteepositive 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.

Cell Chemical Biology, 2018

Identification of new activators of mitochondrial fusion reveals a link between mitochondrial mor... more Identification of new activators of mitochondrial fusion reveals a link between mitochondrial morphology and pyrimidine metabolism. Cell Chem. Biol. 25, this issue, 268-278.

Molecular & Cellular Proteomics, 2018

Arginine methylation of proteins in the eukaryotic cell is predominantly catalyzed by one conserv... more Arginine methylation of proteins in the eukaryotic cell is predominantly catalyzed by one conserved enzyme; PRMT1 in mammals or Hmt1p in yeast. Knockout in mammals is embryonic lethal; however, Hmt1p in yeast is non-essential. The systems-level effects of hmt1 knockout in yeast were investigated. Unexpected but significant dysregulation in phosphate homeostasis was seen upon hmt1 knockout. Transcription factor-driven processes may explain these observations, or regulatory processes may link the sensing of S-adenosylmethionine to intracellular phosphate or polyphosphate.

mSphere

Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signal... more 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 pho4Δ 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 pho4Δ ...

Journal of Fungi, 2016

Opportunistic fungi are a major cause of morbidity and mortality worldwide , particularly in immu... more Opportunistic fungi are a major cause of morbidity and mortality worldwide , 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-IP 5 (IP 7) 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.

Molecular Plant Microbe Interactions, 2009

Filamentous fungi produce hydrophobins, small proteins that are localized on the outer surface of... more Filamentous fungi produce hydrophobins, small proteins that are localized on the outer surface of their cell walls and are involved in growth and development. Nutrient availability and light regulate hydrophobin gene expression. Recently it was demonstrated that signal transduction components such as a MAPK in Magnaporthe grisea and a G-protein alpha subunit in Cryphonectria parasitica are also involved. Here we conducted a comparative study on the effect of loss of function mutations in different signaling components on hydrophobin gene expression in Cochliobolus heterostrophus. Furthermore, a mutant deficient in both G-protein alpha and beta subunits (Cgab1) was constructed. This mutant has an albino phenotype, lacked conidia but still formed abortive pseudothecia in a backcross, and was unable to infect maize. Loss of the G-alpha subunit led, in some conditions, to increased hydrophobin expression, which may be involved in the spore aggregation phenotype of this mutant. Mutations...

PLoS ONE, 2012

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium... more 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, Dcna1, and a CRZ1/SP1 mutant (Dcrz1) 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 Dcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30-37uC vs 25uC) 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.

The Plant Cell, 2003

Conserved eukaryotic signaling elements play an important role in the development of fungal patho... more Conserved eukaryotic signaling elements play an important role in the development of fungal pathogens on their hosts. Chk1, a mitogen-activated protein kinase (MAPK), functions in virulence, mating, and sporulation of the maize leaf pathogen Cochliobolus heterostrophus. Suppression subtractive hybridization was used to identify fungal genes whose expression on the host plant is affected in chk1 deletion mutants. Two of the genes isolated in this screen were predicted to encode cellulolytic enzymes: a cellobiohydrolase, CBH7 , and an endoglucanase, EG6. Expression of EG6 and CBH7 was followed by the fusion of their upstream regulatory regions to the coding sequence of the green fluorescent protein. Induction of both genes began at the onset of invasive growth and reached its maximal extent during leaf necrosis. Furthermore, EG6 was induced preferentially within necrotic lesions. Disruption of MAPK CHK1 resulted in a delay in the penetration of hyphae into the leaf and a concomitant delay in the induction of expression of both cellulase genes. In saprophytic culture, the absence of Chk1 resulted in a marked delay in the induction of CBH7 expression by crystalline cellulose. EG6 was expressed at a basal level in culture, and this expression was found to depend strictly on Chk1. Thus, the Chk1 MAPK signaling pathway is involved in the regulation of two cellulase-encoding genes and is necessary for their timely induction by environmental signals.

mBio, 2020

Invasive fungal diseases pose a serious threat to human health globally with >1.5 million deat... more 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.