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 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 Chitinases Are Essential for Sexual Development but Not Vegetative Growth in Cryptococcus neoformans

2009 ◽  
Vol 8 (11) ◽  
pp. 1692-1705 ◽  
Author(s):  
Lorina G. Baker ◽  
Charles A. Specht ◽  
Jennifer K. Lodge
Keyword(s):  
Cell Wall ◽  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Vegetative Growth ◽  
Cell Walls ◽  
Cell Structure ◽  
Complex Structure ◽  
Hydrolytic Enzymes ◽  
Fungal Cell ◽  
Fungal Cell Walls

ABSTRACT Cryptococcus neoformans is an opportunistic pathogen that mainly infects immunocompromised individuals. The fungal cell wall of C. neoformans is an excellent target for antifungal therapies since it is an essential organelle that provides cell structure and integrity. Importantly, it is needed for localization or attachment of known virulence factors, including melanin, phospholipase, and the polysaccharide capsule. The polysaccharide fraction of the cryptococcal cell wall is a complex structure composed of chitin, chitosan, and glucans. Chitin is an indispensable component of many fungal cell walls that contributes significantly to cell wall strength and integrity. Fungal cell walls are very dynamic, constantly changing during cell division and morphogenesis. Hydrolytic enzymes, such as chitinases, have been implicated in the maintenance of cell wall plasticity and separation of the mother and daughter cells at the bud neck during vegetative growth in yeast. In C. neoformans we identified four predicted endochitinases, CHI2, CHI21, CHI22, and CHI4, and a predicted exochitinase, hexosaminidase, HEX1. Enzymatic analysis indicated that Chi2, Chi22, and Hex1 actively degraded chitinoligomeric substrates. Chi2 and Hex1 activity was associated mostly with the cellular fraction, and Chi22 activity was more prominent in the supernatant. The enzymatic activity of Hex1 increased when grown in media containing only N-acetylglucosamine as a carbon source, suggesting that its activity may be inducible by chitin degradation products. Using a quadruple endochitinase deletion strain, we determined that the endochitinases do not affect the growth or morphology of C. neoformans during asexual reproduction. However, mating assays indicated that Chi2, Chi21, and Chi4 are each involved in sexual reproduction. In summary, the endochitinases were found to be dispensable for routine vegetative growth but not sexual reproduction.

scholarly journals Cryptococcus neoformans Chitin Synthase 3 (Chs3) Plays a Critical Role in Dampening Host Inflammatory Responses

2019 ◽  
Author(s):  
Camaron R. Hole ◽  
Woei C. Lam ◽  
Rajendra Upadhya ◽  
Jennifer K. Lodge
Keyword(s):  
Immune Response ◽  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Inflammatory Responses ◽  
Critical Role ◽  
Pathogenic Fungi ◽  
Fungal Cell Wall ◽  
Chitin Synthase ◽  
Aids Patients ◽  
Fungal Cell

ABSTRACTCryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan deficient C. neoformans lacking three chitin deacetylases (cda1Δ2Δ3Δ) was found to confer protective immunity to a subsequent challenge with a virulent wild type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase mediated formation of chitosan. Mice inoculated with chs3Δ at a dose previously shown to induce protection with cda1Δ2Δ3Δ die within 36 hours after installation of the organism. Mortality was not dependent on viable fungi as mice inoculated with heat-killed preparation of chs3Δ died at the same rate as mice inoculated with live chs3Δ, suggesting the rapid onset of death was host mediated likely caused by an over exuberant immune response. Histology, cytokine profiling, and flow cytometry indicates a massive neutrophil influx in the mice inoculated with chs3Δ. Mice depleted of neutrophils survived chs3Δ inoculation indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcal induced host inflammatory responses.IMPORTANCECryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in ∼200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the host’s immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different than many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study we investigated the host responses to chs3Δ, a chitosan-deficient strain, and found mice inoculated with chs3Δ all died within 36 hours and death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

scholarly journals A Chitin Synthase and Its Regulator Protein Are Critical for Chitosan Production and Growth of the Fungal Pathogen Cryptococcus neoformans

2005 ◽  
Vol 4 (11) ◽  
pp. 1902-1912 ◽  
Author(s):  
Isaac R. Banks ◽  
Charles A. Specht ◽  
Maureen J. Donlin ◽  
Kimberly J. Gerik ◽  
Stuart M. Levitz ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Liquid Culture ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Chitin Synthesis ◽  
Chitin Synthases ◽  
Regulator Protein ◽  
Chitin And Chitosan

ABSTRACT Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30°C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37°C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Δ and the csr2Δ mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.

scholarly journals Cryptococcus neoformans Chitin Synthase 3 Plays a Critical Role in Dampening Host Inflammatory Responses

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Camaron R. Hole ◽  
Woei C. Lam ◽  
Rajendra Upadhya ◽  
Jennifer K. Lodge
Keyword(s):  
Immune Response ◽  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Inflammatory Responses ◽  
Pathogenic Fungi ◽  
Fungal Cell Wall ◽  
Chitin Synthase ◽  
Aids Patients ◽  
Fungal Cell ◽  
Content Type

ABSTRACT Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human-pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan-deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan-deficient C. neoformans strain lacking three chitin deacetylases (cda1Δcda2Δcda3Δ) was found to confer protective immunity to a subsequent challenge with a virulent wild-type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase-mediated formation of chitosan. Mice inoculated with the chs3Δ strain at a dose previously shown to induce protection with the cda1Δcda2Δcda3Δ strain die within 36 h after installation of the organism. Mortality was not dependent on viable fungi, as mice inoculated with a heat-killed preparation of the chs3Δ strain died at the same rate as mice inoculated with a live chs3Δ strain, suggesting that the rapid onset of death was host mediated, likely caused by an overexuberant immune response. Histology, cytokine profiling, and flow cytometry indicate a massive neutrophil influx in the mice inoculated with the chs3Δ strain. Mice depleted of neutrophils survived chs3Δ inoculation, indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcus-induced host inflammatory responses. IMPORTANCE Cryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in ∼200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the host’s immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different from the cell walls of many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study, we investigated the host responses to a chs3Δ strain, a chitosan-deficient strain, and found that mice inoculated with the chs3Δ strain all died within 36 h and that death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

scholarly journals Chitosan Biosynthesis and Virulence in the Human Fungal Pathogen Cryptococcus gattii

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Woei C. Lam ◽  
Rajendra Upadhya ◽  
Charles A. Specht ◽  
Abigail E. Ragsdale ◽  
Camaron R. Hole ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fungal Pathogen ◽  
Cryptococcus Gattii ◽  
Fungal Cell Wall ◽  
Fungal Pathogenesis ◽  
Protective Response ◽  
Fungal Cell ◽  
Fungal Virulence ◽  
Content Type ◽  
Mammalian Infection

ABSTRACT Cryptococcus gattii R265 is a hypervirulent fungal strain responsible for the recent outbreak of cryptococcosis in Vancouver Island of British Columbia in Canada. It differs significantly from Cryptococcus neoformans in its natural environment, its preferred site in the mammalian host, and its pathogenesis. Our previous studies of C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains a two- to threefold larger amount of chitosan than that of C. neoformans. The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDAs) have, however, been modified. To deduce their roles, single and double CDA deletion strains and a triple CDA deletion strain were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in strain R265, Cda3 is critical for virulence. Deletion of either CDA3 alone or in combination with another CDA (cda1Δ3Δ or cda2Δ3Δ) or both (cda1Δ2Δ3Δ) rendered the fungus avirulent and cleared from the infected host. Moreover, the cda1Δ2Δ3Δ strain of R265 induced a protective response to a subsequent infection with R265. These studies begin to illuminate the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence. IMPORTANCE The fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offer new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans.

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 Screening and Antifungal Activity of a β-Carboline Derivative against Cryptococcus neoformans and C. gattii

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Kátia Santana Cruz ◽  
Emerson Silva Lima ◽  
Marcia de Jesus Amazonas da Silva ◽  
Erica Simplício de Souza ◽  
Andreia Montoia ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Active Compound ◽  
Cell Walls ◽  
Human Fibroblasts ◽  
Lead Compound ◽  
Fungal Cell ◽  
Fungal Cell Walls

Background. Cryptococcosis is a fungal disease of bad prognosis due to its pathogenicity and the toxicity of the drugs used for its treatment. The aim of this study was to investigate the medicinal potential of carbazole and β-carboline alkaloids and derivatives against Cryptococcus neoformans and C. gattii. Methods. MICs were established in accordance with the recommendations of the Clinical and Laboratory Standards Institute for alkaloids and derivatives against C. neoformans and C. gattii genotypes VNI and VGI, respectively. A single active compound was further evaluated against C. neoformans genotypes VNII, VNIII, and VNIV, C. gattii genotypes VGI, VGIII, and VGIV, Candida albicans ATCC 36232, for cytotoxicity against the MRC-5 lineage of human fibroblasts and for effects on fungal cells (cell wall, ergosterol, and leakage of nucleic acids). Results. Screening of 11 compounds revealed 8-nitroharmane as a significant inhibitor (MIC 40 μg/mL) of several C. neoformans and C. gattii genotypes. It was not toxic to fibroblasts (IC50 > 50 µg/mL) nor did it alter fungal cell walls or the concentration of ergosterol in C. albicans or C. neoformans. It increased leakage of substances that absorb at 260 nm. Conclusions. The synthetic β-carboline 8-nitroharmane significantly inhibits pathogenic Cryptococcus species and is interesting as a lead compound towards new therapy for Cryptococcus infections.

scholarly journals Targeting a critical step in fungal hexosamine biosynthesis

2020 ◽  
Vol 295 (26) ◽  
pp. 8678-8691 ◽  
Author(s):  
Deborah E. A. Lockhart ◽  
Mathew Stanley ◽  
Olawale G. Raimi ◽  
David A. Robinson ◽  
Dominika Boldovjakova ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antifungal Drugs ◽  
Fungal Cell Wall ◽  
Fungal Cell ◽  
Hexosamine Biosynthetic Pathway ◽  
Hexosamine Biosynthesis ◽  
Key Enzyme ◽  
Opportunistic Fungal Pathogen ◽  
Extracellular Environment ◽  
Chemical Evidence

Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment including host defenses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. As this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective submicromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.

scholarly journals Laccase Expression in Murine Pulmonary Cryptococcus neoformans Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 3124-3127 ◽  
Author(s):  
Javier Garcia-Rivera ◽  
Stephanie C. Tucker ◽  
Marta Feldmesser ◽  
Peter R. Williamson ◽  
Arturo Casadevall
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Lung Tissue ◽  
Immunogold Electron Microscopy ◽  
Cell Cytoplasm ◽  
Fungal Cell ◽  
Time Of Infection

ABSTRACT Cryptococcus neoformans laccase expression during murine infection was investigated in lung tissue by immunohistochemistry and immunogold electron microscopy. Laccase was detected in the fungal cell cytoplasm, cell wall, and capsule in vivo. The amount of laccase found in different sites varied as a function of the time of infection.

Sign in / Sign up

Export Citation Format

Share Document