scholarly journals Role of CTR4 in the Virulence of Cryptococcus neoformans

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Scott R. Waterman ◽  
Yoon-Dong Park ◽  
Meera Raja ◽  
Jin Qiu ◽  
Dima A. Hammoud ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Ex Vivo ◽  
Copper Homeostasis ◽  
Fluorescent Imaging ◽  
Microbial Pathogenesis ◽  
Copper Transporter ◽  
Content Type ◽  
Expression Levels ◽  
Patient Cohort

ABSTRACTWhile research has identified an important contribution for metals, such as iron, in microbial pathogenesis, the roles of other transition metals, such as copper, remain mostly unknown. Recent evidence points to a requirement for copper homeostasis in the virulence ofCryptococcus neoformansbased on a role for aCUF1copper regulatory factor in mouse models and in a human patient cohort.C. neoformansis an important fungal pathogen that results in an estimated 600,000 AIDS-related deaths yearly. In the present studies, we found that aC. neoformansmutant lacking theCUF1-dependent copper transporter,CTR4, grows normally in rich medium at 37°C but has reduced survival in macrophages and attenuated virulence in a mouse model. This reduced survival and virulence were traced to a growth defect under nutrient-restricted conditions. Expression studies using a full-lengthCTR4-fluorescent fusion reporter construct demonstrated robust expression in macrophages, brain, and lung, the latter shown byex vivofluorescent imaging. Inductively coupled mass spectroscopy (ICP-MS) was used to probe the copper quota of fungal cells grown in defined medium and recovered from brain, which suggested a role for a copper-protective function ofCTR4in combination with cell metallothioneins under copper-replete conditions. In summary, these data suggest a role forCTR4in copper-related homeostasis and subsequently in fungal virulence.IMPORTANCECrytococcus neoformansis a significant global fungal pathogen, and copper homeostasis is a relatively unexplored aspect of microbial pathogenesis that could lead to novel therapeutics. Previous studies correlated expression levels of a Ctr4 copper transporter to development of meningoencephalitis in a patient cohort of solid-organ transplants, but a direct role for Ctr4 in mammalian pathogenesis has not been demonstrated. The present studies utilize a Δctr4mutant strain which revealed an important role forCTR4inC. neoformansinfections in mice and relate the gene product to homeostatic control of copper and growth under nutrient-restricted conditions. Robust expression levels ofCTR4during fungal infection were exploited to demonstrate expression and lung cryptococcal disease usingex vivofluorescence imaging. In summary, these studies are the first to directly demonstrate a role for a copper transporter in fungal disease and provide anex vivoimaging tool for further study of cryptococcal gene expression and pathogenesis.

scholarly journals Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Lucy X. Li ◽  
Camaron R. Hole ◽  
Javier Rangel-Moreno ◽  
Shabaana A. Khader ◽  
Tamara L. Doering
Keyword(s):  
Cryptococcus Neoformans ◽  
Mutant Strain ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Helper Cell Type ◽  
Lymphoid Structures ◽  
Type Infection

ABSTRACT Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

scholarly journals Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Shannon K. Esher ◽  
Kyla S. Ost ◽  
Lukasz Kozubowski ◽  
Dong-Hoon Yang ◽  
Min Su Kim ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Posttranslational Modification ◽  
Fungal Pathogen ◽  
Content Type ◽  
Processing Enzymes ◽  
Human Fungal Pathogen ◽  
Modification Process ◽  
Substrate Proteins ◽  
Processing Steps

ABSTRACT Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus. Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.

scholarly journals Laccases Involved in 1,8-Dihydroxynaphthalene Melanin Biosynthesis in Aspergillus fumigatus Are Regulated by Developmental Factors and Copper Homeostasis

2013 ◽  
Vol 12 (12) ◽  
pp. 1641-1652 ◽  
Author(s):  
Srijana Upadhyay ◽  
Guadalupe Torres ◽  
Xiaorong Lin
Keyword(s):  
Immune Responses ◽  
Aspergillus Fumigatus ◽  
Gene Cluster ◽  
Gene Expression Pattern ◽  
Copper Homeostasis ◽  
Copper Level ◽  
Fungal Virulence ◽  
Copper Transporter ◽  
Melanin Biosynthesis ◽  
Content Type

ABSTRACTAspergillus fumigatusproduces heavily melanized infectious conidia. The conidial melanin is associated with fungal virulence and resistance to various environmental stresses. This 1,8-dihydroxynaphthalene (DHN) melanin is synthesized by enzymes encoded in a gene cluster inA. fumigatus, including two laccases, Abr1 and Abr2. Although this gene cluster is not conserved in all aspergilli, laccases are critical for melanization in all species examined. Here we show that the expression ofA. fumigatuslaccases Abr1/2 is upregulated upon hyphal competency and drastically increased during conidiation. The Abr1 protein is localized at the surface of stalks and conidiophores, but not in young hyphae, consistent with the gene expression pattern and its predicted role. The induction of Abr1/2 upon hyphal competency is controlled by BrlA, the master regulator of conidiophore development, and is responsive to the copper level in the medium. We identified a developmentally regulated putative copper transporter, CtpA, and found that CtpA is critical for conidial melanization under copper-limiting conditions. Accordingly, disruption of CtpA enhanced the induction ofabr1andabr2, a response similar to that induced by copper starvation. Furthermore, nonpigmentedctpAΔ conidia elicited much stronger immune responses from the infected invertebrate hostGalleria mellonellathan the pigmentedctpAΔ or wild-type conidia. Such enhancement in elicitingGalleriaimmune responses was independent of thectpAΔ conidial viability, as previously observed for the DHN melanin mutants. Taken together, our findings indicate that both copper homeostasis and developmental regulators control melanin biosynthesis, which affects conidial surface properties that shape the interaction between this pathogen and its host.

scholarly journals Cell Wall Chitosan Is Necessary for Virulence in the Opportunistic Pathogen Cryptococcus neoformans

2011 ◽  
Vol 10 (9) ◽  
pp. 1264-1268 ◽  
Author(s):  
Lorina G. Baker ◽  
Charles A. Specht ◽  
Jennifer K. Lodge
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Slow Growth ◽  
Opportunistic Pathogen ◽  
Mammalian Host ◽  
Cell Integrity ◽  
Content Type ◽  
Opportunistic Fungal Pathogen ◽  
Chitin And Chitosan

ABSTRACTCryptococcus neoformansis an opportunistic fungal pathogen that causes meningoencephalitis. Its cell wall is composed of glucans, proteins, chitin, and chitosan. Multiple genetic approaches have defined a chitosan-deficient syndrome that includes slow growth and decreased cell integrity. Here we demonstrate chitosan is necessary for virulence and persistence in the mammalian host.

scholarly journals Dynamic Virulence: Real-Time Assessment of Intracellular Pathogenesis Links Cryptococcus neoformans Phenotype with Clinical Outcome

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Michael K. Mansour ◽  
Jatin M. Vyas ◽  
Stuart M. Levitz
Keyword(s):  
Cryptococcus Neoformans ◽  
Patient Outcomes ◽  
Fungal Pathogen ◽  
Fungal Isolate ◽  
Host Factors ◽  
Intracellular Replication ◽  
Phenotypic Properties ◽  
Content Type ◽  
Opportunistic Fungal Pathogen ◽  
Time Assessment

ABSTRACT While a myriad of studies have examined host factors that predispose persons to infection with the opportunistic fungal pathogen Cryptococcus neoformans, comparatively little has been done to examine how virulence factor differences among cryptococcal isolates may impact outcome. In the recent report by Alanio et al. (A. Alanio, M. Desnos-Ollivier, and F. Dromer, mBio 2:e00158-11, 2011), novel flow cytometry-based techniques were employed to demonstrate an association between the phenotype of C. neoformans-macrophage interactions, as measured by phagocytosis and intracellular replication, and patient outcomes, as determined by positive cultures on therapy and survival. These experiments establish that the prognosis of patients with cryptococcosis is influenced by the phenotypic properties of the infecting fungal isolate.

scholarly journals Thioredoxin Reductase Is Essential for Viability in the Fungal Pathogen Cryptococcus neoformans

2005 ◽  
Vol 4 (2) ◽  
pp. 487-489 ◽  
Author(s):  
Tricia A. Missall ◽  
Jennifer K. Lodge
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Nitrosative Stress ◽  
Pathogenic Fungus ◽  
Thioredoxin Reductase ◽  
Mammalian Host ◽  
Low Molecular Weight ◽  
Oxidative And Nitrosative Stress ◽  
Copper Transporter ◽  
Resistance Pathway

ABSTRACT Thioredoxin reductase (TRR1) is an important component of the thioredoxin oxidative stress resistance pathway. Here we show that it is induced during oxidative and nitrosative stress and is preferentially localized to the mitochondria in Cryptococcus neoformans. The C. neoformans TRR1 gene encodes the low-molecular-weight isoform of the thioredoxin reductase enzyme, which shares little homology with that of its mammalian host. By replacing the endogenous TRR1 promoter with an inducible copper transporter promoter, we showed that Trr1 appears to be essential for viability of this pathogenic fungus, making it a potential antifungal target.

scholarly journals Dectin-2 Deficiency Promotes Th2 Response and Mucin Production in the Lungs after Pulmonary Infection with Cryptococcus neoformans

2014 ◽  
Vol 83 (2) ◽  
pp. 671-681 ◽  
Author(s):  
Yuri Nakamura ◽  
Ko Sato ◽  
Hideki Yamamoto ◽  
Kana Matsumura ◽  
Ikumi Matsumoto ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mhc Class Ii ◽  
Proinflammatory Cytokines ◽  
Fungal Pathogen ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Interleukin 4 ◽  
Th2 Response ◽  
Content Type ◽  
Mucin Production

Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides.Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense againstC. neoformansinfection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of β1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. Inin vitroexperiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates ofC. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection withC. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.

scholarly journals Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

2014 ◽  
Vol 13 (5) ◽  
pp. 657-663 ◽  
Author(s):  
Katie Glenn ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Allosteric Regulation ◽  
Valuable Insight ◽  
Kinetic Characterization ◽  
Content Type ◽  
Metabolic Role ◽  
Separate Site ◽  
Opportunistic Fungal Pathogen

ABSTRACTXylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogenCryptococcus neoformans, which has twoXFPgenes (designatedXFP1andXFP2). Our kinetic characterization ofC. neoformansXfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics.C. neoformansXfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC50) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogenCryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.

scholarly journals Troponoids Can Inhibit Growth of the Human Fungal Pathogen Cryptococcus neoformans

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Maureen J. Donlin ◽  
Anthony Zunica ◽  
Ashlyn Lipnicky ◽  
Aswin K. Garimallaprabhakaran ◽  
Alex J. Berkowitz ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Fungal Pathogen ◽  
Antimicrobial Activities ◽  
Fungal Species ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Red Cedar ◽  
Immunosuppressed Patients ◽  
Western Red Cedar

ABSTRACT Cryptococcus neoformans is a pathogen that is common in immunosuppressed patients. It can be treated with amphotericin B and fluconazole, but the mortality rate remains 15 to 30%. Thus, novel and more effective anticryptococcal therapies are needed. The troponoids are based on natural products isolated from western red cedar, and have a broad range of antimicrobial activities. Extracts of western red cedar inhibit the growth of several fungal species, but neither western red cedar extracts nor troponoid derivatives have been tested against C. neoformans. We screened 56 troponoids for their ability to inhibit C. neoformans growth and to assess whether they may be attractive candidates for development into anticryptococcal drugs. We determined MICs at which the compounds inhibited 80% of cryptococcal growth relative to vehicle-treated controls and identified 12 compounds with MICs ranging from 0.2 to 15 μM. We screened compounds with MICs of ≤20 μM for cytotoxicity in liver hepatoma cells. Fifty percent cytotoxicity values (CC50s) ranged from 4 to >100 μM. The therapeutic indexes (TI, CC50/MIC) for most of the troponoids were fairly low, with most being <8. However, two compounds had TI values that were >8, including a tropone with a TI of >300. These tropones are fungicidal and are not antagonistic when used in combination with fluconazole or amphotericin B. Inhibition by these two tropones remains unchanged under conditions favoring cryptococcal capsule formation. These data support the hypothesis that troponoids may be a productive scaffold for the development of novel anticryptococcal therapies.

scholarly journals Expression of Plasmodium vivaxcrt-oIs Related to Parasite Stage but NotEx VivoChloroquine Susceptibility

2015 ◽  
Vol 60 (1) ◽  
pp. 361-367 ◽  
Author(s):  
Zuleima Pava ◽  
Irene Handayuni ◽  
Grennady Wirjanata ◽  
Sheren To ◽  
Leily Trianty ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Drug Susceptibility ◽  
Fold Change ◽  
Low Grade ◽  
Content Type ◽  
Expression Levels ◽  
Stage Of Development ◽  
Parasite Stage

ABSTRACTChloroquine (CQ)-resistantPlasmodium vivaxis present in most countries whereP. vivaxinfection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression ofP. vivaxcrt-o(pvcrt-o) has been correlated within vivoCQ resistance in an area with low-grade resistance. We assessedpvcrt-oexpression in isolates from Papua (Indonesia), whereP. vivaxis highly CQ resistant.Ex vivodrug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels ofpvcrt-owere measured using a novel real-time quantitative reverse transcription-PCR method. Large variations inpvcrt-oexpression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with theP. vivaxβ-tubulin gene and from 0.01 to 24 relative to that seen with theP. vivaxaldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5;P< 0.001). Twenty-nine isolates fulfilled the criteria forex vivodrug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found thatpvcrt-oexpression levels did not correlate with theex vivoresponse to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuringpvcrt-oexpression and suggest thatpvcrt-otranscription is not a primary determinant ofex vivodrug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels andP. vivaxdrug resistance.

Sign in / Sign up

Export Citation Format

Share Document