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Chad A Rappleye - One of the best experts on this subject based on the ideXlab platform.
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Histoplasma Responses to Nutritional Immunity Imposed by Macrophage Activation
Journal of fungi (Basel Switzerland), 2019Co-Authors: Peter J. Brechting, Chad A RappleyeAbstract:The fungal pathogen Histoplasma capsulatum resides within the phagosome of host phagocytic cells. Within this intracellular compartment, Histoplasma yeast replication requires the acquisition of several essential nutrients, including metal ions. Recent work has shown that while iron, zinc, and copper are sufficiently abundant in resting macrophages, cytokine activation of these host cells causes restriction of these metals from intracellular yeasts as a form of nutritional immunity. Faced with limited iron availability in the phagosome following macrophage activation by IFN-γ, Histoplasma yeasts secrete iron-scavenging siderophores and employ multiple strategies for reduction of ferric iron to the more physiologically useful ferrous form. IFN-γ activation of macrophages also limits availability of copper in the phagosome, forcing Histoplasma reliance on the high affinity Ctr3 copper importer for copper acquisition. GM-CSF activation stimulates macrophage production of zinc-chelating metallothioneins and zinc transporters to sequester zinc from Histoplasma yeasts. In response, Histoplasma yeasts express the Zrt2 zinc importer. These findings highlight the dynamics of phagosomal metal ion concentrations in host-pathogen interactions and explain one mechanism by which macrophages become a less permissive environment for Histoplasma replication with the onset of adaptive immunity.
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Differentiation of the fungus Histoplasma capsulatum into a pathogen of phagocytes.
Current opinion in microbiology, 2017Co-Authors: Qian Shen, Chad A RappleyeAbstract:Mammalian body temperature triggers differentiation of the fungal pathogen Histoplasma capsulatum into yeast cells. The Drk1 regulatory kinase and an interdependent network of Ryp transcription factors establish the yeast state. Beyond morphology, the differentiation-dependent expression program equips yeasts for invasion and survival within phagosomes. Yeast cells produce α-glucan and the Eng1 endoglucanase which hide yeasts from immune detection. Secretion of yeast phase-specific Sod3 and CatB detoxify phagocyte-derived reactive oxygen molecules. Histoplasma cells adapt to iron and zinc limitation in activated macrophages by production of siderophores and the Zrt2 transporter, respectively. Yeasts also respond to inflammation-associated hypoxia. Histoplasma pathogenicity thus relies on factors controlled by yeast differentiation as well as environment-dependent responses.
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eng1 and exg8 are the major β glucanases secreted by the fungal pathogen Histoplasma capsulatum
Journal of Biological Chemistry, 2017Co-Authors: Andrew L Garfoot, Kacey L Dearing, Andrew D Vanschoiack, Vicki H Wysocki, Chad A RappleyeAbstract:Abstract Fungal cell walls contain β-glucan polysaccharides which stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of β-glucan by host cells through at least two mechanisms: concealment of β-glucans beneath α-glucans and enzymatic removal of any exposed β-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed β-glucans from the yeast cell surface. Here, we characterize the enzymatic specificity of the Eng1 and Exg8 proteins and show that Exg8 is an exo-β1,3-glucanse and Eng1 is an endo-β1,3-glucanase. Together, Eng1 and Exg8 account for nearly all of the total secreted glucanase activity of Histoplasma yeasts. Both Eng1 and Exg8 proteins are secreted through a conventional secretion signal and modified post-translationally by O-linked glycosylation. Both glucanases have near maximal activity at temperature and pH conditions experienced during infection of host cells, supporting roles in Histoplasma pathogenesis. Exg8 has a higher specific activity than Eng1 for β1,3-glucans, yet despite this, Exg8 does not reduce detection of yeasts by the host β-glucan receptor Dectin-1. Exg8 is largely dispensable for virulence in vivo, in contrast to Eng1. These results show that Histoplasma yeasts secrete two β1,3-glucanases and that Eng1 endoglucanase activity is the predominant factor responsible for removal of exposed cell wall β-glucans to minimize host detection of Histoplasma yeasts.
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eng1 and exg8 are the major β glucanases secreted by the fungal pathogen Histoplasma capsulatum
Journal of Biological Chemistry, 2017Co-Authors: Andrew L Garfoot, Kacey L Dearing, Andrew D Vanschoiack, Vicki H Wysocki, Chad A RappleyeAbstract:Fungal cell walls contain β-glucan polysaccharides that stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of β-glucan by host cells through at least two mechanisms: concealment of β-glucans beneath α-glucans and enzymatic removal of any exposed β-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed β-glucans from the yeast cell surface. Here, we characterize the enzymatic specificity of the Eng1 and Exg8 proteins and show that Exg8 is an exo-β1,3-glucanase and Eng1 is an endo-β1,3-glucanase. Together, Eng1 and Exg8 account for nearly all of the total secreted glucanase activity of Histoplasma yeasts. Both Eng1 and Exg8 proteins are secreted through a conventional secretion signal and are modified post-translationally by O-linked glycosylation. Both glucanases have near maximal activity at temperature and pH conditions experienced during infection of host cells, supporting roles in Histoplasma pathogenesis. Exg8 has a higher specific activity than Eng1 for β1,3-glucans; yet despite this, Exg8 does not reduce detection of yeasts by the host β-glucan receptor Dectin-1. Exg8 is largely dispensable for virulence in vivo, in contrast to Eng1. These results show that Histoplasma yeasts secrete two β1,3-glucanases and that Eng1 endoglucanase activity is the predominant factor responsible for removal of exposed cell wall β-glucans to minimize host detection of Histoplasma yeasts.
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the eng1 β glucanase enhances Histoplasma virulence by reducing β glucan exposure
Mbio, 2016Co-Authors: Andrew L Garfoot, Qian Shen, Marcel Wuthrich, Bruce S Klein, Chad A RappleyeAbstract:ABSTRACT The fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes β-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo , particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall β-glucans, which results in enhanced binding to the Dectin-1 β-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed β-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In α-glucan-producing Histoplasma strains, Eng1 acts in concert with α-glucan to minimize β-glucan exposure: α-glucan provides a masking function by covering the β-glucan-rich cell wall, while Eng1 removes any remaining exposed β-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed β-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes. IMPORTANCE The success of Histoplasma capsulatum as an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed that Histoplasma pathogenic yeast cells, but not avirulent mycelia, secrete a β-glucanase, Eng1, which reduces recognition of fungal cell wall β-glucans. We demonstrated that the Eng1 β-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed β-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host β-glucan receptor, Dectin-1. As a consequence, phagocyte recognition of Histoplasma yeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control of Histoplasma infection in vivo . Thus, Histoplasma yeasts express two mechanisms to avoid phagocyte detection: masking of cell wall β-glucans by α-glucan and enzymatic removal of exposed β-glucans by the Eng1 β-glucanase.
William E. Goldman - One of the best experts on this subject based on the ideXlab platform.
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genome sequences reveal cryptic speciation in the human pathogen Histoplasma capsulatum
Mbio, 2017Co-Authors: Victoria E Sepulveda, William E. Goldman, Roberto Marquez, David A Turissini, Daniel R MatuteAbstract:Histoplasma capsulatum is a pathogenic fungus that causes life-threatening lung infections. About 500,000 people are exposed to H. capsulatum each year in the United States, and over 60% of the U.S. population has been exposed to the fungus at some point in their life. We performed genome-wide population genetics and phylogenetic analyses with 30 Histoplasma isolates representing four recognized areas where histoplasmosis is endemic and show that the Histoplasma genus is composed of at least four species that are genetically isolated and rarely interbreed. Therefore, we propose a taxonomic rearrangement of the genus.IMPORTANCE The evolutionary processes that give rise to new pathogen lineages are critical to our understanding of how they adapt to new environments and how frequently they exchange genes with each other. The fungal pathogen Histoplasma capsulatum provides opportunities to precisely test hypotheses about the origin of new genetic variation. We find that H. capsulatum is composed of at least four different cryptic species that differ genetically and also in virulence. These results have implications for the epidemiology of histoplasmosis because not all Histoplasma species are equivalent in their geographic range and ability to cause disease.
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rna interference in Histoplasma capsulatum demonstrates a role for α 1 3 glucan in virulence
Molecular Microbiology, 2004Co-Authors: Chad A Rappleye, Jacquelyn T Engle, William E. GoldmanAbstract:Histoplasma capsulatum is a fungal pathogen that causes respiratory and systemic disease by proliferating within macrophages. While much is known about histoplasmosis, only a single virulence factor has been defined, in part because of the inefficiency of Histoplasma reverse genetics. As an alternative to allelic replacement, we have developed a telomeric plasmid-based system for silencing gene expression in Histoplasma by RNA interference (RNAi). Episomal expression of long RNAs that form stem-loop structures triggered gene silencing. To test the effectiveness of RNAi in Histoplasma, we depleted expression of a gfp transgene as well as two endogenous genes, ADE2 and URA5, and showed significant reductions in corresponding gene function. Silencing was target gene specific, stable during macrophage infection and reversible. We used RNAi targeting AGS1 (encoding alpha-(1,3)-glucan synthase) to deplete levels of alpha-(1,3)-glucan, a cell wall polysaccharide. Loss of alpha-(1,3)-glucan by RNAi yielded phenotypes indistinguishable from an AGS1 deletion: attenuation of the ability to kill macrophages and colonize murine lungs. This demonstrates for the first time that alpha-(1,3)-glucan is an important contributor to Histoplasma virulence.
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RNA interference in Histoplasma capsulatum demonstrates a role for α‐(1,3)‐glucan in virulence
Molecular microbiology, 2004Co-Authors: Chad A Rappleye, Jacquelyn T Engle, William E. GoldmanAbstract:Histoplasma capsulatum is a fungal pathogen that causes respiratory and systemic disease by proliferating within macrophages. While much is known about histoplasmosis, only a single virulence factor has been defined, in part because of the inefficiency of Histoplasma reverse genetics. As an alternative to allelic replacement, we have developed a telomeric plasmid-based system for silencing gene expression in Histoplasma by RNA interference (RNAi). Episomal expression of long RNAs that form stem-loop structures triggered gene silencing. To test the effectiveness of RNAi in Histoplasma, we depleted expression of a gfp transgene as well as two endogenous genes, ADE2 and URA5, and showed significant reductions in corresponding gene function. Silencing was target gene specific, stable during macrophage infection and reversible. We used RNAi targeting AGS1 (encoding alpha-(1,3)-glucan synthase) to deplete levels of alpha-(1,3)-glucan, a cell wall polysaccharide. Loss of alpha-(1,3)-glucan by RNAi yielded phenotypes indistinguishable from an AGS1 deletion: attenuation of the ability to kill macrophages and colonize murine lungs. This demonstrates for the first time that alpha-(1,3)-glucan is an important contributor to Histoplasma virulence.
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Histoplasma capsulatum modulates the acidification of phagolysosomes
Journal of Experimental Medicine, 1993Co-Authors: Linda Eissenberg, William E. Goldman, Paul H SchlesingerAbstract:The phagolysosome is perhaps the most effective antimicrobial site within macrophages due both to its acidity and to its variety of hydrolytic enzymes. Few species of pathogens survive and multiply in these vesicles. However, one strategy for microbial survival would be to induce a higher pH within these organelles, thus interfering with the activity of many lysosomal enzymes. Altering the intravesicular milieu might also profoundly influence antigen processing, antimicrobial drug delivery, and drug activity. Here we report the first example of an organism proliferating within phagolysosomes that maintain a relatively neutral pH for a sustained period of time. We inoculated P388D1 macrophages with fluorescein isothiocyanate (FITC)-labeled Histoplasma capsulatum or zymosan. Using the ratio of fluorescence excitations at 495 and 450 nm, we determined that vesicles containing either virulent or avirulent FITC-labeled H. capsulatum yeasts had a pH one to two units higher than vesicles containing either zymosan or methanol-killed H. capsulatum. The difference in pH remained stable for at least 5.5 h postinoculation. Longer-term studies using cells preincubated with acridine orange indicated that phagolysosomes containing live Histoplasma continued to maintain a relatively neutral pH for at least 30 h. Many agents raise the pH of multiple vesicles within the same cell. In contrast, H. capsulatum affects only the phagolysosome in which it is located; during coinoculation of cells with unlabeled Histoplasma and labeled zymosan, organelles containing zymosan still acidified normally. Similarly, unlabeled zymosan had no influence on the elevated pH of vesicles housing labeled Histoplasma. Thus, zymosan and Histoplasma were segregated into separate phagolysosomes that responded independently to their phagocytized contents. This localized effect might reflect an intrinsic difference between phagosomes housing the two particle types, active buffering by the microbe, or altered ion transport across the phagolysosomal membrane such that acidification is inhibited.
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In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum
Molecular microbiology, 1992Co-Authors: J P Woods, William E. GoldmanAbstract:Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis. We previously developed a transformation system for Histoplasma and demonstrated chromosomal integration of transforming plasmid sequences. In this study, we describe another Histoplasma mechanism for maintaining transforming DNA i.e. the generation of modified, multicopy linear plasmids carrying DNA from the transforming Escherichia coli plasmid. Under selective conditions, these linear plasmids were stable and capable of retransforming Histoplasma without further modification. In vivo modification of the transforming DNA included duplication of plasmid sequence and telomeric addition at the termini of linear DNA. Apparently Histoplasma telomerase, like that of other organisms such as humans and Tetrahymena, is able to act on non-telomeric substrates. The terminus of a Histoplasma linear plasmid was cloned and shown to contain multiple repeats of GGGTTA, the telomeric repeat unit also found in vertebrates, trypanosomes, and slime moulds.
Jessica A. Edwards - One of the best experts on this subject based on the ideXlab platform.
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Histoplasma yeast and mycelial transcriptomes reveal pathogenic phase and lineage specific gene expression profiles
BMC Genomics, 2013Co-Authors: Jessica A. Edwards, Megan M Kemski, Chenxi Chen, Thomas K Mitchell, Chad A RappleyeAbstract:Background The dimorphic fungus Histoplasma capsulatum causes respiratory and systemic disease in mammalian hosts by expression of factors that enable survival within phagocytic cells of the immune system. Histoplasma’s dimorphism is distinguished by growth either as avirulent mycelia or as pathogenic yeast. Geographically distinct strains of Histoplasma differ in their relative virulence in mammalian hosts and in production of and requirement for specific virulence factors. The close similarity in the genome sequences of these diverse strains suggests that phenotypic variations result from differences in gene expression rather than gene content. To provide insight into how the transcriptional program translates into morphological variation and the pathogenic lifestyle, we compared the transcriptional profile of the pathogenic yeast phase and the non-pathogenic mycelial phase of two clinical isolates of Histoplasma.
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identification of an aminothiazole with antifungal activity against intracellular Histoplasma capsulatum
Antimicrobial Agents and Chemotherapy, 2013Co-Authors: Jessica A. Edwards, Megan M Kemski, Chad A RappleyeAbstract:As 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 monitoring Histoplasma growth and employed it in a phenotypic screen of 3,600 commercially available compounds. Seven hit compounds that inhibited Histoplasma yeast growth were identified. Compound 41F5 has fungistatic activity against Histoplasma yeast 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 inhibits Histoplasma growth 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 from Histoplasma-induced macrophage death, making this aminothiazole hit compound an excellent candidate for development as an antifungal for Histoplasma infections.
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Histoplasma mechanisms of pathogenesis one portfolio doesn t fit all
Fems Microbiology Letters, 2011Co-Authors: Jessica A. Edwards, Chad A RappleyeAbstract:Histoplasma capsulatum is the leading cause of endemic mycosis in the world. Analyses of clinical isolates from different endemic regions show important diversity within the species. Recent molecular studies of two isolates, the Chemotype I NAm2 strain G217B and the Chemotype II Panamanian strain G186A, reveal significant genetic, structural, and molecular differences between these representative Histoplasma strains. Some of these variations have functional consequences, representing distinct molecular mechanisms that facilitate Histoplasma pathogenesis. The realization of Histoplasma strain diversity highlights the importance of characterizing Histoplasma virulence factors in the context of specific clinical strain isolates.
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Histoplasma mechanisms of pathogenesis – one portfolio doesn't fit all
FEMS microbiology letters, 2011Co-Authors: Jessica A. Edwards, Chad A RappleyeAbstract:Histoplasma capsulatum is the leading cause of endemic mycosis in the world. Analyses of clinical isolates from different endemic regions show important diversity within the species. Recent molecular studies of two isolates, the Chemotype I NAm2 strain G217B and the Chemotype II Panamanian strain G186A, reveal significant genetic, structural, and molecular differences between these representative Histoplasma strains. Some of these variations have functional consequences, representing distinct molecular mechanisms that facilitate Histoplasma pathogenesis. The realization of Histoplasma strain diversity highlights the importance of characterizing Histoplasma virulence factors in the context of specific clinical strain isolates.
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definition of the extracellular proteome of pathogenic phase Histoplasma capsulatum
Journal of Proteome Research, 2011Co-Authors: Eric D Holbrook, Jessica A. Edwards, Brian H. Youseff, Chad A RappleyeAbstract:The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease. Within the mammalian host, pathogenic Histoplasma yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to Histoplasma virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC−MS/MS analysis of the proteins in pathogenic-phase culture filtrates of Histoplasma. In addition to secreted Cbp1, the extracellular proteome of pathogenic Histoplasma yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp’s). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic Histoplasma mycelia, suggesting that these proteins are linked to Histoplasma pathogenesis. In addition, Histoplasma yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how Histoplasma alters the host-pathogen interaction to its advantage.
Eric D Holbrook - One of the best experts on this subject based on the ideXlab platform.
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Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen
Infection and immunity, 2014Co-Authors: Eric D Holbrook, Megan M Kemski, L. Joseph Wheat, Sarah M. Richer, Chad A RappleyeAbstract:The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the secreted proteome of Histoplasma, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast 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 in Histoplasma pathogenesis, 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 to Histoplasma yeasts 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 the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.
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redundant catalases detoxify phagocyte reactive oxygen and facilitate Histoplasma capsulatum pathogenesis
Infection and Immunity, 2013Co-Authors: Eric D Holbrook, Brian H. Youseff, Katherine A. Smolnycki, Chad A RappleyeAbstract:Histoplasma capsulatum is a respiratory pathogen that infects phagocytic cells. The mechanisms allowing Histoplasma to overcome toxic reactive oxygen molecules produced by the innate immune system are an integral part of Histoplasma's ability to survive during infection. To probe the contribution of Histoplasma catalases in oxidative stress defense, we created and analyzed the virulence defects of mutants lacking CatB and CatP, which are responsible for extracellular and intracellular catalase activities, respectively. Both CatB and CatP protected Histoplasma from peroxide challenge in vitro and from antimicrobial reactive oxygen produced by human neutrophils and activated macrophages. Optimal protection required both catalases, as the survival of a double mutant lacking both CatB and CatP was lower than that of single-catalase-deficient cells. Although CatB contributed to reactive oxygen species defenses in vitro, CatB was dispensable for lung infection and extrapulmonary dissemination in vivo. Loss of CatB from a strain also lacking superoxide dismutase (Sod3) did not further reduce the survival of Histoplasma yeasts. Nevertheless, some catalase function was required for pathogenesis since simultaneous loss of both CatB and CatP attenuated Histoplasma virulence in vivo. These results demonstrate that Histoplasma's dual catalases comprise a system that enables Histoplasma to efficiently overcome the reactive oxygen produced by the innate immune system.
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Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress
PLoS pathogens, 2012Co-Authors: Brian H. Youseff, Eric D Holbrook, Katherine A. Smolnycki, Chad A RappleyeAbstract:In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.
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definition of the extracellular proteome of pathogenic phase Histoplasma capsulatum
Journal of Proteome Research, 2011Co-Authors: Eric D Holbrook, Jessica A. Edwards, Brian H. Youseff, Chad A RappleyeAbstract:The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease. Within the mammalian host, pathogenic Histoplasma yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to Histoplasma virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC−MS/MS analysis of the proteins in pathogenic-phase culture filtrates of Histoplasma. In addition to secreted Cbp1, the extracellular proteome of pathogenic Histoplasma yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp’s). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic Histoplasma mycelia, suggesting that these proteins are linked to Histoplasma pathogenesis. In addition, Histoplasma yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how Histoplasma alters the host-pathogen interaction to its advantage.
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Histoplasma capsulatum pathogenesis making a lifestyle switch
Current Opinion in Microbiology, 2008Co-Authors: Eric D Holbrook, Chad A RappleyeAbstract:The dimorphism of Histoplasma reflects a developmental switch in morphology and lifestyle that is necessary for virulence. The dimorphism regulating kinase DRK1 and the Histoplasma WOR1 homolog RYP1 mediate the thermally induced transition to the pathogenic yeast-phase program. The genes expressed as part of this regulon influence the host–pathogen interaction to favor Histoplasma virulence. While surface localized HSP60 supports yeast attachment to host macrophages, yeast α-glucan polysaccharides conceal immunostimulatory cell wall β-glucans from detection by macrophage receptors. Intramacrophage growth of yeast cells is facilitated by CBP a secreted, protease-resistant calcium-binding protein tailored to function within the phagolysosomal environment. In some Histoplasma strains, YPS3 promotes dissemination of yeast from pulmonary infection sites. The Histoplasma yeast-phase program includes additional cell surface and extracellular molecules that potentially function in further aspects of Histoplasma virulence.
Gary W. Procop - One of the best experts on this subject based on the ideXlab platform.
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Clinical Significance and Histologic Characterization of Histoplasma Granulomas.
American journal of clinical pathology, 2020Co-Authors: Ryan Demkowicz, Gary W. ProcopAbstract:OBJECTIVES To clarify the clinical significance and degree of resolution (ie, grade) of Histoplasma granulomas in routinely reviewed surgical pathology specimens and the clinical outcomes of patients with this diagnosis, with an emphasis on those not receiving antifungal therapy. METHODS We performed a retrospective medical record, laboratory data, and surgical pathology slide review of patients with Histoplasma granulomas following institutional review board approval. RESULTS Clinical, pathologic, and laboratory data from 62 patients with Histoplasma granulomas were available for review. Of these, 1 of 19 (5%) fungal cultures, 4 of 12 (33%) fungal serologic studies, 0 of 9 Histoplasma urinary antigen tests, and 0 of 2 Histoplasma serum antigen tests were positive. All but 3 of the Histoplasma granulomas were either in the resolving (grade 2) or resolved (grade 3) stage of resolution. None of the patients, including those who did not receive antifungal therapy after the histologic diagnosis, developed progressive or disseminated histoplasmosis. CONCLUSIONS These findings, which are supportive of clinical guidelines, suggest that patients with old, hyalinized Histoplasma granulomas do not benefit from further laboratory studies or antifungal therapy. The proposed grading of Histoplasma granulomas informs clinicians of the stage of resolution of an excised lesion, which informs therapeutic decisions and thus is recommended.
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Rapid Diagnosis of Histoplasma capsulatum Endocarditis Using the AccuProbe on an Excised Valve
Journal of Clinical Microbiology, 2001Co-Authors: Roy F. Chemaly, Mary Sholtis, Gerri S. Hall, J. W. Tomford, Jimmy Chua, Gary W. ProcopAbstract:Histoplasma capsulatum is an infrequent but serious cause of endocarditis. The definitive diagnosis requires culture, which may require a long incubation. We demonstrated the ability of the Histoplasma capsulatum AccuProbe to accurately identify this organism when applied directly on an excised valve that contained abundant yeast forms consistent with H. capsulatum.