Browsing by Subject "Candida albicans"
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Item Open Access Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity.(Nat Commun, 2015-01-22) Kanayama, M; Inoue, M; Danzaki, K; Hammer, G; He, Y; Shinohara, MLImmune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert antimicrobial responses. Here we report a role of autophagy in an early host antifungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80(hi) macrophages in the spleen, peritoneum and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80(hi) macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.Item Open Access Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.(Microbiology, 2010-03) Kingsbury, Joanne M; McCusker, John HThe isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.Item Open Access Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood.(Eur J Clin Microbiol Infect Dis, 2012-09) Schell, WA; Benton, JL; Smith, PB; Poore, M; Rouse, JL; Boles, DJ; Johnson, MD; Alexander, BD; Pamula, VK; Eckhardt, AE; Pollack, MG; Benjamin, DK; Perfect, JR; Mitchell, TGSpecies of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.Item Open Access Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.(Nature communications, 2019-09) Juvvadi, Praveen R; Fox, David; Bobay, Benjamin G; Hoy, Michael J; Gobeil, Sophie MC; Venters, Ronald A; Chang, Zanetta; Lin, Jackie J; Averette, Anna Floyd; Cole, D Christopher; Barrington, Blake C; Wheaton, Joshua D; Ciofani, Maria; Trzoss, Michael; Li, Xiaoming; Lee, Soo Chan; Chen, Ying-Lien; Mutz, Mitchell; Spicer, Leonard D; Schumacher, Maria A; Heitman, Joseph; Steinbach, William JCalcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.Item Open Access Insight into the Role of Metal Homeostasis in Fungal Adaptation to Drug Stress(2019) Hunsaker, ElizabethMaintenance of metal homeostasis is critical to cell survival due to the multitude of cellular processes that depend on one or more metal cofactors. We show that the opportunistic fungal pathogen Candida albicans becomes sensitized to both Cu limitation and Cu elevation during exposure in liquid culture to the antifungal drug fluconazole, a widely prescribed antifungal agent. Cu supplementation reduces tolerance of C. albicans to fluconazole in a way that does not require formation of a Cu–fluconazole complex. Rather, our data point to a less obvious relationship between drug stress and Cu availability that gives rise to metal-mediated outcomes of drug treatment. qRT-PCR, EPR, fluorescence, and ICP-MS studies demonstrate that C. albicans extensively remodels its metal homeostasis networks to respond to treatment with fluconazole. These adaptation strategies include increased Cu import and storage, increased retention of Fe, Mn, and Zn, altered utilization of Cu- and Mn-dependent enzymes, mobilization of intracellular Fe stores, and increased production of the heme prosthetic group utilized by the enzyme target of fluconazole. Furthermore, RNA-seq analysis reveals that co-treatment with fluconazole and Cu gives rise to unique patterns of gene expression that illustrate the profound impacts of small fluctuations in Cu availability on the transcriptomic response to fluconazole stress. Finally, we show that fluconazole causes substantial changes to the metalloproteome of C. albicans, most notably to the levels of Cu proteins. The findings offer a new perspective for thinking about fungal response to drug stress that pushes cells out of their metal homeostatic zones, leading them to enact metal-associated adaptation mechanisms to restore homeostasis to survive.
Item Embargo Insights into the Role of Copper and Zinc on the Uptake and Antifungal Activity of the Salivary Peptide Histatin-5(2023) Campbell, Joanna XianzhenHistatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 can bind metals in vitro, and metals have been shown to alter the fungicidal activity of the peptide. The goal of this work is to gain insight into the role of metals in the biological activity Hist-5. Toward this goal, we developed and characterized a novel fluorescently labeled Hist-5 peptide (Hist-5*) to visualize changes in internalization and localization of the peptide in fungal cells as a function of metal availability in the growth medium. Here, we provide evidence for Zn-modulated antifungal activity of Hist-5 in which the availability of Zn2+ in the surrounding environment inhibits Hist-5 cellular uptake and cidality. Cellular growth assays revealed a concentration-dependent inhibitory effect of Zn2+ on Hist-5 antifungal activity. Imaging by confocal microscopy showed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. We found that modulation of extracellular Zn2+ concentration by metal chelating molecules or proteins reversed Zn-induced surface adhesion of Hist-5, leading us to propose a dynamic role for Zn2+ as an inhibitory switch to regulate Hist-5 fungicidal activity. We next present data to support the hypothesis that Hist-5 interacts with intracellular Cu to increase the fungicidal activity Hist-5. Combined fluorescence spectroscopy and microscopy experiments showed reversible Cu-dependent quenching of Hist-5* fluorescence, indicating a direct interaction between Hist-5 and intracellular Cu. X-ray fluorescence microscopy images revealed peptide-induced changes to cellular Cu distribution and cell-associated Cu content. Finally, we present progress towards expanding the scope in which we understand and assess Hist-5 biological activity by investigating the activity of the peptide under biologically relevant conditions and testing its fungicidal activity against other fungal species.
Item Open Access Isolation, speciation and antifungal susceptibility testing of Candida isolates from various clinical specimens at a tertiary care hospital, Nepal.(BMC research notes, 2017-06) Khadka, Sundar; Sherchand, Jeevan Bahadur; Pokhrel, Bharat Mani; Parajuli, Keshab; Mishra, Shyam Kumar; Sharma, Sangita; Shah, Niranjan; Kattel, Hari Prasad; Dhital, Subhash; Khatiwada, Sulochana; Parajuli, Narayan; Pradhan, Manoj; Rijal, Basista PrasadBackground
Candida species are responsible for various clinical infections ranging from mucocutaneous infection to life threatening invasive diseases along with increased resistance to antifungal drugs has made a serious concern. Resistance to antifungal agents has increased during the last decade. Thus, identification of Candida up to species level and its antifungal susceptibility testing has a paramount significance in the management of Candidal infections. The aim of the study was to speciate Candida species and to determine antifungal susceptibility pattern of Candida species to antifungal agents.Methods
A total of 100 consecutive Candida species were isolated from 1248 clinical specimens over 7 months period. Growths on Sabouraud dextrose agar were evaluated for colony appearance, macroscopic examination, Gram staining, germ tube test and urea hydrolysis test. Further, they were processed for Candida speciation on CHROMagar. Antifungal susceptibility testing was performed as recommended by Clinical and Laboratory Standards Institute (CLSI) M44-A document.Results
Out of 100 Candida isolates, Candida albicans (56%) was the most common species. Among the non-albicans Candida species, Candida tropicalis (20%) was the predominant isolate followed by Candida glabrata (14%). Regarding antifungal susceptibility pattern, Candida species were more susceptible to clotrimazole (82%) followed by fluconazole (64%) and miconazole (44%).Conclusions
Candida albicans was the predominant species responsible for various Candidal infections. Among commonly used antifungal drugs clotrimazole, miconazole and fluconazole were most effective.Item Open Access PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.(PLoS Pathog, 2010-08-26) LaFayette, Shantelle L; Collins, Cathy; Zaas, Aimee K; Schell, Wiley A; Betancourt-Quiroz, Marisol; Gunatilaka, AA Leslie; Perfect, John R; Cowen, Leah EFungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.Item Open Access Skewing of the population balance of lymphoid and myeloid cells by secreted and intracellular osteopontin.(Nature immunology, 2017-09) Kanayama, Masashi; Xu, Shengjie; Danzaki, Keiko; Gibson, Jason R; Inoue, Makoto; Gregory, Simon G; Shinohara, Mari LThe balance of myeloid populations and lymphoid populations must be well controlled. Here we found that osteopontin (OPN) skewed this balance during pathogenic conditions such as infection and autoimmunity. Notably, two isoforms of OPN exerted distinct effects in shifting this balance through cell-type-specific regulation of apoptosis. Intracellular OPN (iOPN) diminished the population size of myeloid progenitor cells and myeloid cells, and secreted OPN (sOPN) increase the population size of lymphoid cells. The total effect of OPN on skewing the leukocyte population balance was observed as host sensitivity to early systemic infection with Candida albicans and T cell-mediated colitis. Our study suggests previously unknown detrimental roles for two OPN isoforms in causing the imbalance of leukocyte populations.Item Open Access The Hsp90 co-chaperone Sgt1 governs Candida albicans morphogenesis and drug resistance.(PLoS One, 2012) Shapiro, Rebecca S; Zaas, Aimee K; Betancourt-Quiroz, Marisol; Perfect, John R; Cowen, Leah EThe molecular chaperone Hsp90 orchestrates regulatory circuitry governing fungal morphogenesis, biofilm development, drug resistance, and virulence. Hsp90 functions in concert with co-chaperones to regulate stability and activation of client proteins, many of which are signal transducers. Here, we characterize the first Hsp90 co-chaperone in the leading human fungal pathogen, Candida albicans. We demonstrate that Sgt1 physically interacts with Hsp90, and that it governs C. albicans morphogenesis and drug resistance. Genetic depletion of Sgt1 phenocopies depletion of Hsp90, inducing yeast to filament morphogenesis and invasive growth. Sgt1 governs these traits by bridging two morphogenetic regulators: Hsp90 and the adenylyl cyclase of the cAMP-PKA signaling cascade, Cyr1. Sgt1 physically interacts with Cyr1, and depletion of either Sgt1 or Hsp90 activates cAMP-PKA signaling, revealing the elusive link between Hsp90 and the PKA signaling cascade. Sgt1 also mediates tolerance and resistance to the two most widely deployed classes of antifungal drugs, azoles and echinocandins. Depletion of Sgt1 abrogates basal tolerance and acquired resistance to azoles, which target the cell membrane. Depletion of Sgt1 also abrogates tolerance and resistance to echinocandins, which target the cell wall, and renders echinocandins fungicidal. Though Sgt1 and Hsp90 have a conserved impact on drug resistance, the underlying mechanisms are distinct. Depletion of Hsp90 destabilizes the client protein calcineurin, thereby blocking crucial responses to drug-induced stress; in contrast, depletion of Sgt1 does not destabilize calcineurin, but blocks calcineurin activation in response to drug-induced stress. Sgt1 influences not only morphogenesis and drug resistance, but also virulence, as genetic depletion of C. albicans Sgt1 leads to reduced kidney fungal burden in a murine model of systemic infection. Thus, our characterization of the first Hsp90 co-chaperone in a fungal pathogen establishes C. albicans Sgt1 as a global regulator of morphogenesis and drug resistance, providing a new target for treatment of life-threatening fungal infections.