scholarly journals Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

10.3791/52666 ◽  
2015 ◽  
Author(s):  
Tonya Taylor ◽  
Indrani Bose ◽  
Taylor Luckie ◽  
Kerry Smith
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Biolistic Transformation ◽  
Opportunistic Fungal Pathogen

scholarly journals Susceptibility of melanized and nonmelanized Cryptococcus neoformans to the melanin-binding compounds trifluoperazine and chloroquine.

1996 ◽  
Vol 40 (3) ◽  
pp. 541-545 ◽  
Author(s):  
Y Wang ◽  
A Casadevall
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Antipsychotic Agent ◽  
Measuring Cell ◽  
High Affinity ◽  
Propidium Iodide Staining ◽  
Opportunistic Fungal Pathogen ◽  
Melanin Binding

Cryptococcus neoformans is an opportunistic fungal pathogen which becomes heavily melanized in the presence of phenolic substrates such as L-dopa. Various drugs are known to bind to melanin with high affinity, including the antipsychotic agent trifluoperazine and the antimalarial agent chloroquine. We hypothesized that drugs which bind melanin may have different toxicities for melanized and nonmelanized C. neoformans cells. The effects of trifluoperazine and chloroquine or C. neoformans were determined by measuring cell viability after exposure to these drugs. Cell viability was measured by CFU determination and flow cytometry with propidium iodide staining. Melanized cells were more susceptible than nonmelanized cells to the fungicidal effects of trifluoperazine. Chloroquine had no fungicidal effect on either melanized or nonmelanized C. neoformans under the conditions studied. Flow cytometry of trifluoperazine-treated C. neoformans cells stained with the mitochondrial stain dihydrorhodamine 123 revealed fluorescence changes consistent with mitochondrial damage. Our results indicate that melanized and nonmelanized C. neoformans cells can differ in susceptibility to certain drugs and suggest that strategies which target melanin may be productive for antifungal-drug discovery.

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.

Identification of Virulence Mutants of the Fungal Pathogen Cryptococcus neoformans Using Signature-Tagged Mutagenesis

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 935-947 ◽  
Author(s):  
Rex T Nelson ◽  
Jun Hua ◽  
Bryant Pryor ◽  
Jennifer K Lodge
Keyword(s):  
Mouse Model ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Virulent Strain ◽  
Critical Parameters ◽  
Avirulent Strain ◽  
Multiple Copies ◽  
Actin Promoter ◽  
Opportunistic Fungal Pathogen ◽  
Different Strains

Abstract Cryptococcus neoformans var. neoformans is an important opportunistic fungal pathogen of patients whose immune system has been compromised due to viral infection, antineoplastic chemotherapy, or tissue transplantation. As many as 13% of all AIDS patients suffer a life-threatening cryptococcal infection at some time during the course of their HIV disease. To begin to understand the molecular basis for virulence in Cryptococcus neoformans var. neoformans serotype A, we have employed signature-tagged mutagenesis (STM) to identify mutants with altered virulence in a mouse model. The critical parameters of signature-tagged mutagenesis in C. neoformans are explored. Data are presented showing that at least 100 different strains can be mixed together in a single animal with each participating in the infection and that there is no apparent interaction between a virulent strain and an avirulent strain in our animal model. Using signature-tagged mutagenesis, we identified 39 mutants with significantly altered growth in a competitive assay. Molecular analyses of these mutants indicated that 19 (49%) contained an insertion in the actin promoter by homologous recombination from a single crossover event, creating a duplication of the actin promoter and the integration of single or multiple copies of the vector. Analysis of the chromosomal insertion sites of those mutants that did not have an integration event in the actin promoter revealed an approximately random distribution among the chromosomes. Individual challenge of the putative mutants in a mouse model revealed five hypovirulent mutants and one hypervirulent mutant.

scholarly journals Mating-Type-Specific and Nonspecific PAK Kinases Play Shared and Divergent Roles in Cryptococcus neoformans

2002 ◽  
Vol 1 (2) ◽  
pp. 257-272 ◽  
Author(s):  
Ping Wang ◽  
Connie B. Nichols ◽  
Klaus B. Lengeler ◽  
Maria E. Cardenas ◽  
Gary M. Cox ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mating Type ◽  
Fungal Pathogen ◽  
Sexual Cycle ◽  
Type Locus ◽  
Serotype A ◽  
A Cells ◽  
Genes Encoding ◽  
Opportunistic Fungal Pathogen ◽  
Synthetically Lethal

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid MATα and MATa cells. Virulence has been linked to the mating type, and MATα cells are more virulent than congenic MATa cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family: STE20α, STE20a, and PAK1. In contrast to the STE20 genes that were previously shown to be in the mating-type locus, the PAK1 gene is unlinked to the mating type. The STE20α, STE20a, and PAK1 genes were disrupted in serotype A and D strains of C. neoformans, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence. ste20α pak1 and ste20a pak1 double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the STE20α gene, the MATα locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence 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 The interplay of phenotype and genotype in Cryptococcus neoformans disease

2020 ◽  
Vol 40 (10) ◽  
Author(s):  
Sophie Altamirano ◽  
Katrina M. Jackson ◽  
Kirsten Nielsen
Keyword(s):  
Patient Outcome ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Proper Treatment ◽  
Rapid Adaptation ◽  
Complete Understanding ◽  
Phenotypic Changes ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen

Abstract Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis primarily in immunocompromised individuals. In order to survive and proliferate during infection, C. neoformans must adapt to a variety of stresses it encounters within the host. Patient outcome depends on the interaction between the pathogen and the host. Understanding the mechanisms that C. neoformans uses to facilitate adaptation to the host and promote pathogenesis is necessary to better predict disease severity and establish proper treatment. Several virulence phenotypes have been characterized in C. neoformans, but the field still lacks a complete understanding of how genotype and phenotype contribute to clinical outcome. Furthermore, while it is known that C. neoformans genotype impacts patient outcome, the mechanisms remain unknown. This lack of understanding may be due to the genetic heterogeneity of C. neoformans and the extensive phenotypic variation observed between and within isolates during infection. In this review, we summarize the current understanding of how the various genotypes and phenotypes observed in C. neoformans correlate with human disease progression in the context of patient outcome and recurrence. We also postulate the mechanisms underlying the genetic and phenotypic changes that occur in vivo to promote rapid adaptation in the host.

scholarly journals Adenylyl Cyclase Functions Downstream of the Gα Protein Gpa1 and Controls Mating and Pathogenicity of Cryptococcus neoformans

2002 ◽  
Vol 1 (1) ◽  
pp. 75-84 ◽  
Author(s):  
J. Andrew Alspaugh ◽  
Read Pukkila-Worley ◽  
Toshiaki Harashima ◽  
Lora M. Cavallo ◽  
Deanna Funnell ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Signal Transduction Pathway ◽  
Growth Defect ◽  
Wild Type ◽  
Camp Production ◽  
Mutant Strains ◽  
Opportunistic Fungal Pathogen ◽  
Mutant Phenotypes

ABSTRACT The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

scholarly journals Chitosan, the Deacetylated Form of Chitin, Is Necessary for Cell Wall Integrity in Cryptococcus neoformans

2007 ◽  
Vol 6 (5) ◽  
pp. 855-867 ◽  
Author(s):  
Lorina G. Baker ◽  
Charles A. Specht ◽  
Maureen J. Donlin ◽  
Jennifer K. Lodge
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Vegetative Growth ◽  
Fungal Pathogen ◽  
Cell Structure ◽  
Chitin Synthase ◽  
Immunocompromised Patients ◽  
Cell Integrity ◽  
Fungal Cell ◽  
Opportunistic Fungal Pathogen

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. The fungal cell wall is an excellent target for antifungal therapies as it is an essential organelle that provides cell structure and integrity, it is needed for the localization or attachment of known virulence factors, including the polysaccharide capsule, melanin, and phospholipase, and it is critical for host-pathogen interactions. In C. neoformans, chitosan produced by the enzymatic removal of acetyl groups from nascent chitin polymers has been implicated as an important component of the vegetative cell wall. In this study, we identify four putative chitin/polysaccharide deacetylases in C. neoformans. We have demonstrated that three of these deacetylases, Cda1, Cda2, and Cda3, can account for all of the chitosan produced during vegetative growth in culture, but the function for one, Fpd1, remains undetermined. The data suggest a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase Chs3 into chitosan. Utilizing a collection of chitin/polysaccharide deacetylase deletion strains, we determined that during vegetative growth, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width and the lack of chitosan results in a “leaky melanin” phenotype. Our analysis indicates that chitin deacetylases and the chitosan made by them may prove to be excellent antifungal targets.

scholarly journals Development of Positive Selectable Markers for the Fungal Pathogen Cryptococcus neoformans

2000 ◽  
Vol 7 (1) ◽  
pp. 125-128 ◽  
Author(s):  
Jun Hua ◽  
Justin D. Meyer ◽  
Jennifer K. Lodge
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Selectable Marker ◽  
Single Copy ◽  
Selectable Markers ◽  
Opportunistic Fungal Pathogen

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in ∼10% of patients with AIDS. New selectable markers which confer resistance to G418 or phleomycin when transformed into C. neoformans were made. A hygromycin-selectable marker was modified to allow selection with a single copy of the marker.

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