scholarly journals Cryptococcus neoformans Dual GDP-Mannose Transporters and Their Role in Biology and Virulence

2014 ◽  
Vol 13 (6) ◽  
pp. 832-842 ◽  
Author(s):  
Zhuo A. Wang ◽  
Cara L. Griffith ◽  
Michael L. Skowyra ◽  
Nichole Salinas ◽  
Matthew Williams ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Protein Glycosylation ◽  
Content Type ◽  
Phenotypic Differences ◽  
Major Virulence Factor ◽  
Transmembrane Transporters ◽  
Mutant Cells ◽  
Made In

ABSTRACTCryptococcus neoformansis an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed inSaccharomyces cerevisiaeshowed that both are functional, with similar kinetics and substrate specificitiesin vitro. We have now examined these proteinsin vivoand demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore,gmt1 gmt2double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

scholarly journals New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ren-Yi Lu ◽  
Ting-Jun-Hong Ni ◽  
Jing Wu ◽  
Lan Yan ◽  
Quan-Zhen Lv ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Pathogenic Fungi ◽  
Control Group ◽  
Survival Times ◽  
Content Type ◽  
Fungal Burden ◽  
Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

scholarly journals Physiological Differences in Cryptococcus neoformans Strains In Vitro versus In Vivo and Their Effects on Antifungal Susceptibility

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Nina T. Grossman ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Cryptococcal Meningitis ◽  
Antifungal Susceptibility ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Laboratory Conditions ◽  
Physiological Differences ◽  
Sub Saharan

ABSTRACT Cryptococcus neoformans is an environmentally ubiquitous fungal pathogen that primarily causes disease in people with compromised immune systems, particularly those with advanced AIDS. There are estimated to be almost 1 million cases per year of cryptococcal meningitis in patients infected with human immunodeficiency virus, leading to over 600,000 annual deaths, with a particular burden in sub-Saharan Africa. Amphotericin B (AMB) and fluconazole (FLC) are key components of cryptococcal meningitis treatment: AMB is used for induction, and FLC is for consolidation, maintenance and, for occasional individuals, prophylaxis. However, the results of standard antifungal susceptibility testing (AFST) for AMB and FLC do not correlate well with therapeutic outcomes and, consequently, no clinical breakpoints have been established. While a number of explanations for this absence of correlation have been proffered, one potential reason that has not been adequately explored is the possibility that the physiological differences between the in vivo infection environment and the in vitro AFST environment lead to disparate drug susceptibilities. These susceptibility-influencing factors include melanization, which does not occur during AFST, the size of the polysaccharide capsule, which is larger in infecting cells than in those grown under normal laboratory conditions, and the presence of large polyploid “titan cells,” which rarely occur under laboratory conditions. Understanding whether and how C. neoformans differentially expresses mechanisms of resistance to AMB and FLC in the AFST environment compared to the in vivo environment could enhance our ability to interpret AFST results and possibly lead to the development of more applicable testing methods.

scholarly journals Azole Heteroresistance in Cryptococcus neoformans: Emergence of Resistant Clones with Chromosomal Disomy in the Mouse Brain during Fluconazole Treatment

2013 ◽  
Vol 57 (10) ◽  
pp. 5127-5130 ◽  
Author(s):  
Edward Sionov ◽  
Yun C. Chang ◽  
Kyung J. Kwon-Chung
Keyword(s):  
Mouse Model ◽  
Cryptococcus Neoformans ◽  
Mouse Brain ◽  
Content Type ◽  
The Brain ◽  
Strain Dependent ◽  
Is Strain

ABSTRACTWe have previously reported thatCryptococcus neoformansstrains are innately heteroresistant to fluconazolein vitro, producing minor, highly resistant subpopulations due to adaptive formation of disomic chromosomes. Using a mouse model, we assessed the emergence of heteroresistant clones in the brain during fluconazole treatment and found that the occurrence of heteroresistant clonesin vivowith chromosomal disomy is strain dependent. Interestingly, emergence of heteroresistant clonesin vivowas unrelated to the strain's MIC to fluconazole.

scholarly journals Cryptococcal Phospholipase B1 Is Required for Intracellular Proliferation and Control of Titan Cell Morphology during Macrophage Infection

2015 ◽  
Vol 83 (4) ◽  
pp. 1296-1304 ◽  
Author(s):  
Robert J. Evans ◽  
Zhongming Li ◽  
William S. Hughes ◽  
Julianne T. Djordjevic ◽  
Kirsten Nielsen ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Virulence Factors ◽  
Fold Increase ◽  
Macrophage Infection ◽  
Content Type ◽  
The Central Nervous System ◽  
Opportunistic Fungal Pathogen ◽  
And Control

Cryptococcus neoformansis an opportunistic fungal pathogen and a leading cause of fungal-infection-related fatalities, especially in immunocompromised hosts. Several virulence factors are known to play a major role in the pathogenesis of cryptococcal infections, including the enzyme phospholipase B1 (Plb1). Compared to other well-studiedCryptococcus neoformansvirulence factors such as the polysaccharide capsule and melanin production, very little is known about the contribution of Plb1 to cryptococcal virulence. Phospholipase B1 is a phospholipid-modifying enzyme that has been implicated in multiple stages of cryptococcal pathogenesis, including initiation and persistence of pulmonary infection and dissemination to the central nervous system, but the underlying reason for these phenotypes remains unknown. Here we demonstrate that a Δplb1knockout strain ofC. neoformanshas a profound defect in intracellular growth within host macrophages. This defect is due to a combination of a 50% decrease in proliferation and a 2-fold increase in cryptococcal killing within the phagosome. In addition, we show for the first time that the Δplb1strain undergoes a morphological change duringin vitroandin vivointracellular infection, resulting in a subpopulation of very large titan cells, which may arise as a result of the attenuated mutant's inability to cope within the macrophage.

scholarly journals Cryptococcus neoformans Host Adaptation: Toward Biological Evidence of Dormancy

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Alexandre Alanio ◽  
Frédérique Vernel-Pauillac ◽  
Aude Sturny-Leclère ◽  
Françoise Dromer
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Cryptococcus Neoformans ◽  
Immune Cells ◽  
Gene Expression Analysis ◽  
Yeast Cells ◽  
Content Type ◽  
Dormant Cells

ABSTRACTCryptococcosis is an opportunistic infection due to the ubiquitous yeastCryptococcus neoformans. This yeast interacts closely with innate immune cells, leading to various fates, including fungal persistence within cells, making possible the dissemination of the yeast cells with monocytes via a Trojan horse strategy. In humans, the natural history of the infection begins with primoinfection during childhood, which is followed by dormancy and, in some individuals, reactivation upon immunosuppression. To address the question of dormancy, we studiedC. neoformansinfection at the macrophage level (in vitroH99-macrophage interaction) and at the organ level in a murine model of cryptococcosis. We analyzed the diversity of yeast adaptation to the host by characterizing severalC. neoformanspopulations with new assays based on flow cytometry (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), and gene expression analysis. On the basis of parameters of multiplication and stress response, various populations of yeast cells were observed over timein vivoandin vitro. Cell sorting allowed the identification of a subpopulation that was less prone to grow under standard conditions than the other populations, with growth enhanced by the addition of serum. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. Our data suggest that dormant yeast cells could existin vitroandin vivo.C. neoformansexhibits a huge plasticity and adaptation to hosts that deserves further study.In vitrogeneration of dormant cells is now the main challenge to overcome the limited number of yeast cells recovered in our models.IMPORTANCECryptococcus neoformansis a sugar-coated unicellular fungus that interacts closely with various cells and organisms, including amoebas, nematodes, and immune cells of mammals. This yeast is able to proliferate and survive in the intracellular environment.C. neoformanscauses cryptococcosis, and yeast dormancy in humans has been suggested on the basis of epidemiological evidence obtained years ago. By studying anin vitromodel of yeast-macrophage interaction and murine models of cryptococcosis, we observed that yeast cells evolve in heterogeneous populations during infection on the basis of global metabolic activity. We compared the growth ability and gene expression of yeast cells belonging to various populations in those two models. We eventually found a population of yeast cells with low metabolism that fit some of the criteria for dormant cells. This paves the way for further characterization of dormancy inC. neoformans.

scholarly journals Surfactant Protein D Facilitates Cryptococcus neoformans Infection

2012 ◽  
Vol 80 (7) ◽  
pp. 2444-2453 ◽  
Author(s):  
Scarlett Geunes-Boyer ◽  
Michael F. Beers ◽  
John R. Perfect ◽  
Joseph Heitman ◽  
Jo Rae Wright
Keyword(s):  
Fungal Infection ◽  
Cryptococcus Neoformans ◽  
Defense Mechanisms ◽  
Surfactant Protein ◽  
Host Susceptibility ◽  
Yeast Cells ◽  
Surfactant Protein D ◽  
Content Type

ABSTRACTConcurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Furthermore,Cryptococcus neoformanshas become a primary human pathogen, causing infection in seemingly healthy individuals. Although numerous studies have elucidated the virulence properties ofC. neoformans, less is understood regarding lung host immune factors during early stages of fungal infection. Based on our previous studies documenting that pulmonary surfactant protein D (SP-D) protectsC. neoformanscells against macrophage-mediated defense mechanismsin vitro(S. Geunes-Boyer et al., Infect. Immun. 77:2783–2794, 2009), we postulated that SP-D would facilitate fungal infectionin vivo. To test this hypothesis, we examined the role of SP-D in response toC. neoformansusing SP-D−/−mice. Here, we demonstrate that mice lacking SP-D were partially protected duringC. neoformansinfection; they displayed a longer mean time to death and decreased fungal burden at several time points postinfection than wild-type mice. This effect was reversed by the administration of exogenous SP-D. Furthermore, we show that SP-D bound to the surface of the yeast cells and protected the pathogenic microbes against macrophage-mediated defense mechanisms and hydrogen peroxide (H2O2)-induced oxidative stressin vitroandin vivo. These findings indicate thatC. neoformansis capable of coopting host SP-D to increase host susceptibility to the yeast. This study establishes a new paradigm for the role played by SP-D during host responses toC. neoformansand consequently imparts insight into potential future preventive and/or treatment strategies for cryptococcosis.

scholarly journals Hammondia hammondi Harbors Functional Orthologs of the Host-Modulating Effectors GRA15 and ROP16 but Is Distinguished from Toxoplasma gondii by a Unique Transcriptional Profile

2014 ◽  
Vol 13 (12) ◽  
pp. 1507-1518 ◽  
Author(s):  
Katelyn A. Walzer ◽  
Gregory M. Wier ◽  
Rachel A. Dam ◽  
Ananth R. Srinivasan ◽  
Adair L. Borges ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Transcript Abundance ◽  
Dense Granule ◽  
Transcriptional Analysis ◽  
Content Type ◽  
Phenotypic Differences ◽  
Functional Conservation ◽  
High Transcript Abundance

ABSTRACTToxoplasma gondiiand its nearest extant relative,Hammondia hammondi, are phenotypically distinct despite their remarkable similarity in gene content, synteny, and functionality. To begin to identify genetic differences that might drive distinct infection phenotypes ofT. gondiiandH. hammondi, in the present study we (i) determined whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16), were functionally conserved inH. hammondiand (ii) performed the first comparative transcriptional analysis ofH. hammondiandT. gondiisporulated oocysts. We found that GRA15 and ROP16 fromH. hammondi(HhGRA15 and HhROP16) modulate the host NF-κB and STAT6 pathways, respectively, when expressed heterologously inT. gondii. We also found the transcriptomes ofH. hammondiandT. gondiito be highly distinct. Consistent with the spontaneous conversion ofH. hammonditachyzoites into bradyzoites bothin vitroandin vivo,H. hammondihigh-abundance transcripts are enriched for genes that are of greater abundance inT. gondiibradyzoites. We also identified genes that are of high transcript abundance inH. hammondibut are poorly expressed in multipleT. gondiilife stages, suggesting that these genes are uniquely expressed inH. hammondi. Taken together, these data confirm the functional conservation of knownT. gondiivirulence effectors inH. hammondiand point to transcriptional differences as a potential source of the phenotypic differences between these species.

scholarly journals Nonlytic Exocytosis of Cryptococcus neoformans from Macrophages Occurs In Vivo and Is Influenced by Phagosomal pH

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
André Moraes Nicola ◽  
Emma J. Robertson ◽  
Patrícia Albuquerque ◽  
Lorena da Silveira Derengowski ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Ammonium Chloride ◽  
Vacuolar Atpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Major Question ◽  
Content Type ◽  
Weak Bases

ABSTRACTA unique aspect of the interaction of the fungusCryptococcus neoformanswith macrophages is the phenomenon of nonlytic exocytosis, also referred to as “vomocytosis” or phagosome extrusion/expulsion, which involves the escape of fungal cells from the phagocyte with the survival of both cell types. This phenomenon has been observed onlyin vitrousing subjective and time-consuming microscopic techniques. In spite of recent advances in our knowledge about its mechanisms, a major question still remaining is whether this phenomenon also occursin vivo. In this study, we describe a novel flow cytometric method that resulted in a substantial gain in throughput for studying phagocytosis and nonlytic exocytosisin vitroand used it to explore the occurrence of this phenomenon in a mouse model of infection. Furthermore, we tested the hypothesis that host cell phagosomal pH affected nonlytic exocytosis. The addition of the weak bases ammonium chloride and chloroquine resulted in a significant increase of nonlytic exocytosis events, whereas the vacuolar ATPase inhibitor bafilomycin A1 had the opposite effect. Although all three agents are known to neutralize phagosomal acidity, their disparate effects suggest that phagosomal pH is an important and complex variable in this process. Our experiments established that nonlytic exocytosis occurredin vivowith a frequency that is possibly much higher than that observedin vitro. These results in turn suggest that nonlytic exocytosis has a potential role in the pathogenesis of cryptococcosis.IMPORTANCECryptococcus neoformanscauses disease in people with immune deficiencies such as AIDS. Upon infection,C. neoformanscells are ingested by macrophage immune cells, which provide a niche for survival and replication. After ingestion, macrophages can expel the fungi without causing harm to either cell type, a process named nonlytic exocytosis. To dissect this phenomenon, we evaluated its dependence on the pH inside the macrophage and addressed its occurrence during infection of mice. We developed new techniques using flow cytometry to measureC. neoformansinternalization by and nonlytic exocytosis from macrophages. Neutralizing the phagosome acidity changed the rate of nonlytic exocytosis: activity increased with the weak bases chloroquine and ammonium chloride, whereas the vacuolar ATPase inhibitor bafilomycin A1 caused it to decrease. Experiments in mice suggested that nonlytic exocytosis occurred during infection withC. neoformans. These results shed new light on the interaction betweenC. neoformansand host macrophages.

scholarly journals Genomic and Phenotypic Diversity among Ten Laboratory Isolates ofPseudomonas aeruginosaPAO1

2018 ◽  
Vol 201 (5) ◽  
Author(s):  
Courtney E. Chandler ◽  
Alexander M. Horspool ◽  
Preston J. Hill ◽  
Daniel J. Wozniak ◽  
Jeffrey W. Schertzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phenotypic Diversity ◽  
Phenotypic Variability ◽  
Membrane Vesicle ◽  
Genomic Analysis ◽  
Extracellular Dna ◽  
Content Type ◽  
Phenotypic Differences

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen found ubiquitously in the environment and commonly associated with airway infection in patients with cystic fibrosis.P. aeruginosastrain PAO1 is one of the most commonly used laboratory-adapted research strains and is a standard laboratory-adapted strain in multiple laboratories and strain banks worldwide. Due to potential isolate-to-isolate variability, we investigated the genomic and phenotypic diversity among 10 PAO1 strains (henceforth called sublines) obtained from multiple research laboratories and commercial sources. Genomic analysis predicted a total of 5,682 genes, with 5,434 (95.63%) being identical across all 10 strains. Phenotypic analyses revealed comparable growth phenotypes in rich media and biofilm formation profiles. Limited differences were observed in antibiotic susceptibility profiles and immunostimulatory potential, measured using heat-killed whole-cell preparations in four immortalized cell lines followed by quantification of interleukin-6 (IL-6) and IL-1β secretion. However, variability was observed in the profiles of secreted molecular products, most notably, in rhamnolipid, pyoverdine, pyocyanin,Pseudomonasquinolone signal (PQS), extracellular DNA, exopolysaccharide, and outer membrane vesicle production. Many of the observed phenotypic differences did not correlate with subline-specific genetic changes, suggesting alterations in transcriptional and translational regulation. Taken together, these results suggest that individually maintained sublines of PAO1, even when acquired from the same parent subline, are continuously undergoing microevolution during culture and storage that results in alterations in phenotype, potentially affecting the outcomes ofin vitrophenotypic analyses andin vivopathogenesis studies.IMPORTANCELaboratory-adapted strains of bacteria are used throughout the world for microbiology research. These prototype strains help keep research data consistent and comparable between laboratories. However, we have observed phenotypic variability when using different strains ofPseudomonas aeruginosaPAO1, one of the major laboratory-adopted research strains. Here, we describe the genomic and phenotypic differences among 10 PAO1 strains acquired from independent sources over 15 years to understand how individual maintenance affects strain characteristics. We observed limited genomic changes but variable phenotypic changes, which may have consequences for cross-comparison of data generated using different PAO1 strains. Our research highlights the importance of limiting practices that may promote the microevolution of model strains and calls for researchers to specify the strain origin to ensure reproducibility.

scholarly journals Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

NAR Cancer ◽  
2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Amrita Sule ◽  
Jinny Van Doorn ◽  
Ranjini K Sundaram ◽  
Sachita Ganesa ◽  
Juan C Vasquez ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Parp Inhibitor ◽  
Parp Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Isocitrate Dehydrogenase 1 ◽  
Normal Product ◽  
Synthetic Lethal Interactions ◽  
Mutant Cells

Abstract Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as ‘BRCAness’, which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.

Sign in / Sign up

Export Citation Format

Share Document