scholarly journals The HIF-1α/LC3-II Axis Impacts Fungal Immunity in Human Macrophages

2019 ◽  
Vol 87 (7) ◽  
Author(s):  
Dirk Friedrich ◽  
Dorinja Zapf ◽  
Björn Lohse ◽  
Roger A. Fecher ◽  
George S. Deepe ◽  
...  
Keyword(s):  
Mitochondrial Respiration ◽  
Histoplasma Capsulatum ◽  
Hypoxia Inducible Factor ◽  
Peripheral Blood Monocyte ◽  
Yeast Cells ◽  
Content Type ◽  
Human Macrophages ◽  
Disseminated Disease ◽  
Fungal Immunity ◽  
Pathogen Exposure

ABSTRACTThe fungal pathogenHistoplasma capsulatumcauses a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity toH. capsulatumin mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced byH. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery ofH. capsulatumfrom macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested thatH. capsulatumexploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth ofH. capsulatum. Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.

scholarly journals Identification of an Aminothiazole with Antifungal Activity against Intracellular Histoplasma capsulatum

2013 ◽  
Vol 57 (9) ◽  
pp. 4349-4359 ◽  
Author(s):  
Jessica A. Edwards ◽  
Megan M. Kemski ◽  
Chad A. Rappleye
Keyword(s):  
Drug Targets ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Host Cells ◽  
Yeast Cells ◽  
Aromatic Substituent ◽  
Content Type ◽  
Fungistatic Activity ◽  
Phenotypic Screen ◽  
Cell Components

ABSTRACTAs eukaryotes, fungi possess relatively few molecules sufficiently unique from mammalian cell components to be used as drug targets. Consequently, most current antifungals have significant host cell toxicity. Primary fungal pathogens (e.g.,Histoplasma) are of particular concern, as few antifungals are effective in treating them. To identify additional antifungal candidates for the treatment of histoplasmosis, we developed a high-throughput platform for monitoringHistoplasmagrowth and employed it in a phenotypic screen of 3,600 commercially available compounds. Seven hit compounds that inhibitedHistoplasmayeast growth were identified. Compound 41F5 has fungistatic activity againstHistoplasmayeast at micromolar concentrations, with a 50% inhibitory concentration (IC50) of 0.87 μM, and has the greatest selectivity for yeast (at least 62-fold) relative to host cells. Structurally, 41F5 consists of an aminothiazole core with an alicyclic substituent at the 2-position and an aromatic substituent at the 5-position. 41F5 inhibitsHistoplasmagrowth in liquid culture and similarly inhibits yeast cells within macrophages, the actual host environment of this fungal pathogen during infection. Importantly, 41F5 protects infected host cells fromHistoplasma-induced macrophage death, making this aminothiazole hit compound an excellent candidate for development as an antifungal forHistoplasmainfections.

scholarly journals The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

2011 ◽  
Vol 10 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Milene C. Vallejo ◽  
Alisson L. Matsuo ◽  
Luciane Ganiko ◽  
Lia C. Soares Medeiros ◽  
Kildare Miranda ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungus ◽  
Yeast Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dimorphic Fungus ◽  
Content Type

ABSTRACTExosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such asCryptococcus neoformansandHistoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes inParacoccidioides brasiliensis. P. brasiliensisis a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ×g. P. brasiliensisantigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, withMarasmius oreadesagglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. InP. brasiliensiscells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role playedin vivoby vesicle components and α-galactosyl epitopes.

scholarly journals Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen

2014 ◽  
Vol 82 (10) ◽  
pp. 4414-4425 ◽  
Author(s):  
Eric D. Holbrook ◽  
Megan M. Kemski ◽  
Sarah M. Richer ◽  
L. Joseph Wheat ◽  
Chad A. Rappleye
Keyword(s):  
Insertional Mutagenesis ◽  
Histoplasma Capsulatum ◽  
Lung Infection ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Phylogenetic Groups ◽  
Content Type ◽  
The North ◽  
Fitness Advantage ◽  
Yeast Phase

ABSTRACTThe yeast phase ofHistoplasma capsulatumis the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome ofHistoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically byHistoplasmayeast cells, suggesting its role in pathogenesis. We have generated three monoclonal antibodies as tools for characterization and detection of Cfp4 and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 inHistoplasmapathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages or during lung infection in a murine model of histoplasmosis. Coinfection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage toHistoplasmayeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well as a strain from Panama. In addition, human immune sera recognize theHistoplasmaCfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.

scholarly journals The Yeast-Phase Virulence Requirement for α-Glucan Synthase Differs among Histoplasma capsulatum Chemotypes

2010 ◽  
Vol 10 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Jessica A. Edwards ◽  
Elizabeth A. Alore ◽  
Chad A. Rappleye
Keyword(s):  
Cell Wall ◽  
Histoplasma Capsulatum ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Glucan Synthase ◽  
Content Type ◽  
I Factor ◽  
Yeast Phase

ABSTRACTHistoplasma capsulatumstrains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype IIHistoplasmavirulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucanin vitro, yet they remain fully virulentin vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishesAGS1expression. Nonetheless,AGS1mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced duringin vivogrowth despite its absencein vitro. To directly test whetherAGS1contributes to chemotype I strain virulence, we preventedAGS1function by RNA interference and by insertional mutation. Loss ofAGS1function in chemotype I does not impair the cytotoxicity ofags1(−) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, theags1(−) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate thatAGS1expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a uniqueHistoplasmachemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.

scholarly journals Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

2016 ◽  
Vol 60 (4) ◽  
pp. 2052-2062 ◽  
Author(s):  
Ky V. Hoang ◽  
Heather Curry ◽  
Michael A. Collier ◽  
Hassan Borteh ◽  
Eric M. Bachelder ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Lethal Challenge ◽  
Content Type ◽  
Human Macrophages ◽  
Gentamicin Treatment ◽  
Significant Toxicity ◽  
Serovar Typhimurium ◽  
Spectrum Activity

ABSTRACTFrancisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

scholarly journals Identification and Characterization of Novel Rat Polyomavirus 2 in a Colony of X-SCID Rats by P-PIT assay

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Lora H. Rigatti ◽  
Tuna Toptan ◽  
Joseph T. Newsome ◽  
Patrick S. Moore ◽  
Yuan Chang
Keyword(s):  
T Cell ◽  
Potential Model ◽  
Treatment Options ◽  
Pneumocystis Carinii ◽  
Preclinical Studies ◽  
Content Type ◽  
Laboratory Rats ◽  
Wide Range ◽  
Disseminated Disease ◽  
Human Polyomaviruses

ABSTRACT Although P-PIT was developed to detect diseases associated with known human polyomaviruses, the identification of a new polyomavirus in rats suggests that it may have utility as a broad-based screen for new, as well as known polyomaviruses. Our findings suggest that RatPyV2 may be a commensal infection of laboratory rats that can lead to disseminated disease in T cell immune-deficient rats. Infection of the X-SCID rats with RatPyV2 and Pneumocystis carinii is a potential model for coinfection pathogenesis and treatment options during transplant preclinical studies. Polyomaviruses (PyVs) are known to infect a wide range of vertebrates and invertebrates and are associated with a broad spectrum of diseases, including cancers, particularly in immune-suppressed hosts. A novel polyomavirus, designated rat polyomavirus 2 (RatPyV2), was identified from a breeding colony of rats having X-linked severe combined immunodeficiency. Using a human panpolyomavirus immunohistochemistry test (P-PIT), RatPyV2 was initially detected in the parotid salivary gland of a colony member. Rolling circle amplification using DNA from harderian and parotid glands identified a novel 5.1-kb polyomavirus genome closely related to human Washington University (WU) and Karolinska Institute (KI) and vole polyomaviruses but notably divergent from Rattus norvegicus PyV1 (RnorPyV1; also designated RatPyV1). Further screening showed RatPyV2 inclusion body infection in the lung epithelium and variably in other respiratory, reproductive, and glandular tissues of 12/12 (100%) rats. IMPORTANCE Although P-PIT was developed to detect diseases associated with known human polyomaviruses, the identification of a new polyomavirus in rats suggests that it may have utility as a broad-based screen for new, as well as known polyomaviruses. Our findings suggest that RatPyV2 may be a commensal infection of laboratory rats that can lead to disseminated disease in T cell immune-deficient rats. Infection of the X-SCID rats with RatPyV2 and Pneumocystis carinii is a potential model for coinfection pathogenesis and treatment options during transplant preclinical studies.

scholarly journals Adaptation of Cryptococcus neoformans to Mammalian Hosts: Integrated Regulation of Metabolism and Virulence

2011 ◽  
Vol 11 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Jim Kronstad ◽  
Sanjay Saikia ◽  
Erik David Nielson ◽  
Matthias Kretschmer ◽  
Wonhee Jung ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Cryptococcus Neoformans ◽  
Central Carbon Metabolism ◽  
Yeast Cells ◽  
Phagocytic Cells ◽  
Content Type ◽  
The Central Nervous System ◽  
Mechanisms Of Adaptation ◽  
Complex Integration

ABSTRACTThe basidiomycete fungusCryptococcus neoformansinfects humans via inhalation of desiccated yeast cells or spores from the environment. In the absence of effective immune containment, the initial pulmonary infection often spreads to the central nervous system to result in meningoencephalitis. The fungus must therefore make the transition from the environment to different mammalian niches that include the intracellular locale of phagocytic cells and extracellular sites in the lung, bloodstream, and central nervous system. Recent studies provide insights into mechanisms of adaptation during this transition that include the expression of antiphagocytic functions, the remodeling of central carbon metabolism, the expression of specific nutrient acquisition systems, and the response to hypoxia. Specific transcription factors regulate these functions as well as the expression of one or more of the major known virulence factors ofC. neoformans. Therefore, virulence factor expression is to a large extent embedded in the regulation of a variety of functions needed for growth in mammalian hosts. In this regard, the complex integration of these processes is reminiscent of the master regulators of virulence in bacterial pathogens.

scholarly journals O-Antigen-Deficient Francisella tularensis Live Vaccine Strain Mutants Are Ingested via an Aberrant Form of Looping Phagocytosis and Show Altered Kinetics of Intracellular Trafficking in Human Macrophages

2011 ◽  
Vol 80 (3) ◽  
pp. 952-967 ◽  
Author(s):  
Daniel L. Clemens ◽  
Bai-Yu Lee ◽  
Marcus A. Horwitz
Keyword(s):  
Vaccine Strain ◽  
Intracellular Trafficking ◽  
Live Vaccine ◽  
Live Vaccine Strain ◽  
Complement Components ◽  
Content Type ◽  
Human Macrophages ◽  
O Antigen ◽  
Kinetics Of ◽  
Terminal Complement

We examined the uptake and intracellular trafficking ofF. tularensisLive Vaccine Strain (LVS) and LVS with disruptions ofwbtDEFandwbtIgenes essential for synthesis of the O antigen of lipopolysaccharide. Unlike parental bacteria, O-antigen-deficient LVS is efficiently killed by serum with intact complement but not by serum lacking terminal complement components. Opsonization of O-antigen-deficient LVS in serum lacking terminal complement components allows efficient uptake of these live bacteria by macrophages. In the presence of complement, whereas parentalF. tularensisLVS is internalized within spacious pseudopod loops, mutant LVS is internalized within tightly juxtaposed multiple onion-like layers of pseudopodia. Without complement, both parental and mutant LVSs are internalized within spacious pseudopod loops. Thus, molecules other than O antigen are important in triggering dramatic pseudopod extensions and uptake by spacious pseudopod loops. Following uptake, both parental and mutant LVSs enter compartments that show limited staining for the lysosomal membrane glycoprotein CD63 and little fusion with secondary lysosomes. Subsequently, both parental and mutant LVSs lose their CD63 staining. Whereas the majority of parental LVS escapes into the cytosol by 6 h after uptake, mutant LVS shows a marked lag but does escape by 1 day after uptake. Despite the altered kinetics of phagosome escape, both mutant and parental strains grow to high levels within human macrophages. Thus, the O antigen plays a role in the morphology of uptake in the presence of complement and the kinetics of intracellular growth but is not essential for escape, survival, altered membrane trafficking, or intramacrophage growth.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

Sign in / Sign up

Export Citation Format

Share Document