The influence of a copper efflux pump in Histoplasma capsulatum virulence.

During the infectious process, pathogenic microorganisms must obtain nutrients from the host in order to survive and proliferate. These nutritional sources include the metallic nutrient copper. Despite its essentiality, copper in large amounts is toxic. Host defense mechanisms use high copper poisoning as a fungicidal strategy to control infection. Transcriptional analyses showed that yeast cultured in the presence of copper or inside macrophages (24 h) had elevated expression of CRP1, a copper efflux pump, suggesting that Histoplasma capsulatum could be exposed to a high copper environment in macrophages during the innate immune stage of infection. Accordingly, macrophages cultured in high copper are more efficient in controlling H. capsulatum growth. Also, silencing of ATP7a, a copper pump that promotes the copper influx in phagosomes, increases fungal survival in macrophages. The rich copper environment faced by the fungus is not dependent on IFN-γ, since fungal CRP1 expression is induced in untreated macrophages. Appropriately, CRP1 knockdown fungal strains are more susceptible to macrophage control than wild-type yeasts. Additionally, CRP1 silencing decreases fungal burden in mice during the phase of innate immune response (4-day postinfection) and CRP1 is required for full virulence in a macrophage cell lines (J774 A.1 and RAW 264.7), as well as primary cells (BMDM). Thus, induction of fungal copper detoxifying genes during innate immunity and the attenuated virulence of CRP1-knockdown yeasts suggest that H. capsulatum is exposed to a copper-rich environment at early infection, but circumvents this condition to establish infection.

Histoplasma capsulatum requires peroxisomes for multiple virulence functions including siderophore biosynthesis.

Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.

Targeted gene deletions in the dimorphic fungal pathogen Histoplasma using an optimized episomal CRISPR/Cas9 system.

The rapid development of CRISPR/CRISPR-associated (Cas) systems has revolutionized the ability to produce genetic mutations in a desired locus, particularly in organisms with low rates of homologous recombination. Histoplasma is an important respiratory and systemic fungal pathogen that has few reverse genetic options. We describe an optimized CRISPR/Cas system for the efficient generation of mutations in desired genes. The limited requirements for CRISPR/Cas, namely a gene-targeting guide RNA (gRNA) and expression of a Cas endonuclease, enabled both the gRNA and the Streptococcus pyogenes Cas9 gene to be expressed from a single episomal vector. The gRNAs are expressed from a strong Pol(II) promoter, a critical parameter for increasing the recovery of mutated genes, and processed into the mature gRNA by ribozymes in the mRNA. Expression of dual-tandem gRNAs facilitates the generation of gene deletions at a good frequency which can be detected by PCR-based screening of pooled isolates resulting in the isolation of marker-less deletion mutants. The CRISPR/Cas system is encoded on an episomal telomeric vector facilitating curing strains of the CRISPR/Cas vector upon generation of the mutant. We demonstrate the successful application of this CRISPR/Cas system in diverse Histoplasma species and applicable for multiple genes. The optimized system shows promise for accelerating reverse genetic studies in Histoplasma spp. IMPORTANCE The ability to eliminate gene product functions is central to understanding molecular mechanisms. In the fungal pathogen Histoplasma, methods to inactivate or deplete gene products are inefficient, which hampers progress in defining Histoplasma's virulence mechanisms. We describe an efficient CRISPR/Cas-based system for generating gene deletions in Histoplasma and show its validation on multiple genes with selectable and non-selectable phenotypes.

Histoplasma capsulatum Relies on Tryptophan Biosynthesis To Proliferate within the Macrophage Phagosome.

Histoplasma capsulatum yeasts reside and proliferate within the macrophage phagosome during infection. This nutrient-depleted phagosomal environment imposes challenges to Histoplasma yeasts for nutrition acquisition. Histoplasma yeasts require all 20 amino acids, which can be formed by de novo biosynthesis and/or acquired directly from the phagosomal environment. We investigated how Histoplasma obtains aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) within the phagosome during infection of macrophages. Depletion of key enzymes of the phenylalanine or tyrosine biosynthetic pathway neither impaired Histoplasma's ability to proliferate within macrophages nor resulted in attenuated virulence in vivo. However, loss of tryptophan biosynthesis resulted in reduced growth within macrophages and severely attenuated virulence in vivo. Together, these results indicate that phenylalanine and tyrosine, but not tryptophan, are available to Histoplasma within the macrophage phagosome. The herbicide glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase of the aromatic amino acid biosynthetic pathway, inhibited Histoplasma yeast growth, and this growth inhibition was partially reversed by aromatic amino acid supplementation or overexpression of ARO1. These results suggest that the aromatic amino acid biosynthetic pathway is a candidate drug target to develop novel antifungal therapeutics.

Antifungal Susceptibility Testing and Drug Discovery in the Dimorphic Fungus Histoplasma Capsulatum.

The thermal dimorphism of the fungal pathogen Histoplasma is linked to its virulence in mammalian hosts. Mammalian body temperature triggers differentiation of the fungus into virulent yeasts which successfully infect host phagocytes. Accurate determination of antifungal susceptibility with relevance to infection requires that the tests be performed specifically using the yeast form, not the filamentous environmental form. However, traditional CLSI methodology for antifungal susceptibility testing of yeasts with Histoplasma is in adequate. We present optimized methodology for performing antifungal susceptibility assays on Histoplasma yeasts with an emphasis on quantitative yeast growth determination. Colorimetric and fluorometric assays for Histoplasma growth overcome challenges associated with quantifying some Histoplasma strains which grow as aggregates of yeasts. We also describe antifungal susceptibility testing of Histoplasma yeasts within macrophages to provide improved accuracy and better physiological relevance of antifungal susceptibility profiles.

Mac1-Dependent Copper Sensing Promotes Histoplasma Adaptation to the Phagosome during Adaptive Immunity.

Intracellular pathogens residing within macrophage phagosomes are challenged with recognizing the phagosomal environment and appropriately responding to changing host defense strategies imposed in this organelle. One such phagocyte defense is the restriction of available copper as a form of nutritional immunity during the adaptive immune response to fungal pathogens. The intracellular fungal pathogen Histoplasma capsulatum adapts to this decreased copper through upregulation of the Ctr3 copper transporter. In this study, we show that Histoplasma recognizes the characteristic low-copper phagosomal environment of activated macrophages through the copper-dependent transcriptional regulator Mac1. Multiple cis-acting regulatory sequences in the CTR3 promoter control upregulation of CTR3 transcription under low-copper conditions, and the loss of Mac1 function prevents induction of Ctr3 under low-copper conditions. During adaptive immunity, this loss of copper sensing by Mac1 attenuates Histoplasma virulence more severely than loss of Ctr3 alone, indicating that Mac1 controls the expression of additional mechanisms important for pathogenesis. Transcriptional profiling of Histoplasma yeasts identified a small regulon of Mac1-dependent genes, with the most strongly regulated genes encoding proteins linked to copper, iron, and zinc homeostasis and defenses against reactive oxygen (iron-requiring catalase [CatB] and superoxide dismutase [Sod4]). Accordingly, the loss of Mac1 function increased sensitivity to copper and iron restriction and blocked low-copper-induced expression of extracellular catalase activity. Thus, Mac1-mediated sensing of low-copper signals to Histoplasma yeasts their residence within the activated macrophage phagosome, and in response, Histoplasma yeasts produce factors, including non-copper-dependent factors, to combat the enhanced defenses of activated macrophages. IMPORTANCE Histoplasma capsulatum is a fungal pathogen that survives and grows within host macrophages. For successful infection, Histoplasma must sense and adapt to a dynamic intracellular environment over the course of an infection. We demonstrate that the copper-dependent transcription factor, Mac1, enables Histoplasma sensing of low copper that characterizes the phagosome environment of activated macrophages. Histoplasma recognition of this state leads not only to upregulation of copper acquisition mechanisms but also to other non-copper-related pathogenesis strategies, including scavenging of other metals and detoxification of reactive oxygen produced by host cells. The limited set of genes regulated by Histoplasma Mac1 compared to those of other fungal pathogens suggests a response that has been tailored specifically for Histoplasma's life inside the phagosome. Thus, low levels of phagosomal copper serve as a signal to Histoplasma, enabling responses to the enhanced antimicrobial defenses resulting from immune activation of macrophages.

Diversification of Fungal Chitinases and Their Functional Differentiation in Histoplasma capsulatum.

Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the "C" clade are not resolved as distinct from the "A" clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the "B" clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.

Living Within the Macrophage: Dimorphic Fungal Pathogen Intracellular Metabolism.

Histoplasma and Paracoccidioides are related thermally dimorphic fungal pathogens that cause deadly mycoses (i.e., histoplasmosis and paracoccidioidomycosis, respectively) primarily in North, Central, and South America. Mammalian infection results from inhalation of conidia and their subsequent conversion into pathogenic yeasts. Macrophages in the lung are the first line of defense, but are generally unable to clear these fungi. Instead, Histoplasma and Paracoccidioides yeasts survive and proliferate within the phagosomal compartment of host macrophages. Growth within macrophages requires strategies for acquisition of sufficient nutrients (e.g., carbon, nitrogen, and essential trace elements and co-factors) from the nutrient-depleted phagosomal environment. We review the transcriptomic and recent functional genetic studies that are defining how these intracellular fungal pathogens tune their metabolism to the resources available in the macrophage phagosome. In addition, recent studies have shown that the nutritional state of the macrophage phagosome is not static, but changes upon activation of adaptive immune responses. Understanding the metabolic requirements of these dimorphic pathogens as they thrive within host cells can provide novel targets for therapeutic intervention.

Metabolism of Gluconeogenic Substrates by an Intracellular Fungal Pathogen Circumvents Nutritional Limitations within Macrophages.

Microbial pathogens exploit host nutrients to proliferate and cause disease. Intracellular pathogens, particularly those exclusively living in the phagosome such as Histoplasma capsulatum, must adapt and acquire nutrients within the nutrient-limited phagosomal environment. In this study, we investigated which host nutrients could be utilized by Histoplasma as carbon sources to proliferate within macrophages. Histoplasma yeasts can grow on hexoses and amino acids but not fatty acids as the carbon source in vitro Transcriptional analysis and metabolism profiling showed that Histoplasma yeasts downregulate glycolysis and fatty acid utilization but upregulate gluconeogenesis within macrophages. Depletion of glycolysis or fatty acid utilization pathways does not prevent Histoplasma growth within macrophages or impair virulence in vivo However, loss of function in Pck1, the enzyme catalyzing the first committed step of gluconeogenesis, impairs Histoplasma growth within macrophages and severely attenuates virulence in vivo, indicating that Histoplasma yeasts rely on catabolism of gluconeogenic substrates (e.g., amino acids) to proliferate within macrophages.IMPORTANCEHistoplasma is a primary human fungal pathogen that survives and proliferates within host immune cells, particularly within the macrophage phagosome compartment. The phagosome compartment is a nutrient-limited environment, requiring Histoplasma yeasts to be able to assimilate available carbon sources within the phagosome to meet their nutritional needs. In this study, we showed that Histoplasma yeasts do not utilize fatty acids or hexoses for growth within macrophages. Instead, Histoplasma yeasts consume gluconeogenic substrates to proliferate in macrophages. These findings reveal the phagosome composition from a nutrient standpoint and highlight essential metabolic pathways that are required for a phagosomal pathogen to proliferate in this intracellular environment.

Transcriptomic response to soybean meal-based diets as the first formulated feed in juvenile yellow perch (Perca flavescens).

With increasing levels of fish meal (FM) protein in aquafeeds being replaced with soybean meal (SBM) protein, understanding the molecular mechanisms involved in response to alternative diets has become a critical concern. Thus, the goal of this study was to examine transcriptional differences in the intestine of juvenile yellow perch through RNA-sequencing (RNA-seq), after their initial introduction to a formulated diet with 75% SBM protein inclusion for 61 days, compared to those fed a traditional FM-based diet. Transcriptomic analysis revealed a concise set of differentially expressed genes in juveniles fed the SBM-based diet, the majority of which were intrinsic to the cholesterol biosynthesis pathway. Analysis of total body lipid and cholesterol levels were also investigated, with no between-treatment differences detected. Results of this study demonstrate that in response to SBM-based diets, yellow perch juveniles up-regulate the cholesterol biosynthesis pathway in order to maintain homeostasis. These findings suggest that the upregulation of the cholesterol biosynthesis pathway may negatively impact fish growth due to its large energy expenditure, and future studies are warranted.

Morphology and quantification of fungal growth in residential dust and carpets.

Mold growth indoors is associated with negative human health effects, and this growth is limited by moisture availability. Dust deposited in carpet is an important source of human exposure due to potential elevated resuspension compared to hard floors. However, we need an improved understanding of fungal growth in dust and carpet to better estimate human exposure. The goal of this study was to compare fungal growth quantity and morphology in residential carpet under different environmental conditions, including equilibrium relative humidity (ERH) (50%, 85%, 90%, 95%, 100%), carpet fiber material (nylon, olefin, wool) and presence/absence of dust. We analyzed incubated carpet and dust samples from three Ohio homes for total fungal DNA, fungal allergen Alt a 1, and fungal morphology. Dust presence and elevated ERH (≥85%) were the most important variables that increased fungal growth. Elevated ERH increased mean fungal DNA concentration (P < 0.0001), for instance by approximately 1000 times at 100% compared to 50% ERH after two weeks. Microscopy also revealed more fungal growth at higher ERH. Fungal concentrations were up to 100 times higher in samples containing house dust compared to no dust. For fiber type, olefin had the least total fungal growth, and nylon had the most total fungi and A. alternata growth in unaltered dust. Increased ERH conditions were associated with increased Alt a 1 allergen concentration. The results of this study demonstrate that ERH, presence/absence of house dust, and carpet fiber type influence fungal growth and allergen production in residential carpet, which has implications for human exposure.

α-Pyrone and Sterol Constituents of Penicillium aurantiacobrunneum, a Fungal Associate of the Lichen Niebla homalea.

Four new metabolites, 4-epi-citreoviridin (1), auransterol (3), and two analogues (2 and 4) of paxisterol (6), together with two known metabolites (15R*,20S*)-dihydroxyepisterol (5) and (6), were isolated from cultures of the fungal associate, Penicillium aurantiacobrunneum, of the lichen Niebla homalea, endemic to California and Baja California. The structures of all compounds were determined by comprehensive spectroscopic and spectrometric methods, as well as single-crystal X-ray diffraction for the determination of the absolute configuration of 3. Compound 1 showed selective cytotoxicity toward MCF-7 breast and A2780 ovarian cells with IC50 values of 4.2 and 5.7 µM, respectively.

Histoplasma Responses to Nutritional Immunity Imposed by Macrophage Activation.

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.

Flying under the radar: Histoplasma capsulatum avoidance of innate immune recognition.

The dimorphic fungal pathogen Histoplasma capsulatum takes advantage of the innate immune system, utilizing host macrophages as a proliferative niche while largely avoiding stimulation of signaling host receptors. As a result, innate immune cells are unable to control H. capsulatum on their own. Not all host phagocytes respond to H. capsulatum in the same way, with neutrophils and dendritic cells playing important roles in impeding fungal growth and initiating a protective TH1 response, respectively. Dendritic cells prime T-cell differentiation after internalization of yeasts via VLA-5 receptors and subsequent degradation of the yeasts. Dendritic cell-expressed TLR7 and TLR9 promote a type I interferon response for TH1 polarization. In contrast to dendritic cells, macrophages provide a hospitable intracellular environment. H. capsulatum yeasts enter macrophages via binding to phagocytic receptors. Simultaneously, α-glucan masks immunostimulatory cell wall ß-glucans and a secreted endoglucanase removes exposed ß-glucans to minimize recognition of yeasts by Dectin-1. This review highlights how phagocytes interact with H. capsulatum yeasts and the mechanisms H. capsulatum uses to limit the innate immune response.

Macrophage activation by IFN-γ triggers restriction of phagosomal copper from intracellular pathogens.

Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.

Characterization of the APSES-family transcriptional regulators of Histoplasma capsulatum.

The fungal APSES protein family of transcription factors is characterized by a conserved DNA-binding motif facilitating regulation of gene expression in fungal development and other biological processes. However, their functions in the thermally dimorphic fungal pathogen Histoplasma capsulatum are unexplored. Histoplasma capsulatum switches between avirulent hyphae in the environment and virulent yeasts in mammalian hosts. We identified five APSES domain-containing proteins in H. capsulatum homologous to Swi6, Mbp1, Stu1 and Xbp1 proteins and one protein found in related Ascomycetes (APSES-family protein 1; Afp1). Through transcriptional analyses and RNA interference-based functional tests we explored their roles in fungal biology and virulence. Mbp1 serves an essential role and Swi6 contributes to full yeast cell growth. Stu1 is primarily expressed in mycelia and is necessary for aerial hyphae development and conidiation. Xbp1 is the only factor enriched specifically in yeast cells. The APSES proteins do not regulate conversion of conidia into yeast and hyphal morphologies. The APSES-family transcription factors are not individually required for H. capsulatum infection of cultured macrophages or murine infection, nor do any contribute significantly to resistance to cellular stresses including cell wall perturbation, osmotic stress, oxidative stress or antifungal treatment. Further studies of the downstream genes regulated by the individual APSES factors will be helpful in revealing their functional roles in H. capsulatum biology.

Synthesis and biological evaluation of aminothiazoles against Histoplasma capsulatum and Cryptococcus neoformans.

The design and synthesis of a library of forty novel 2-aminoazole analogues as well as their evaluation as antifungal compounds against Histoplasma capsulatum and Cryptococcus neoformans is described. These structures were derived from N-[5-(1-naphthalenylmethyl)-2-thiazolyl]cyclohexanecarboxamide (41F5), a fungistatic agent previously identified through phenotypic screening (Antimicrob Agents Chemother. 2013;57:4349). Modifications to improve potency and water-solubility of 41F5 focused primarily on the 5-naphthalenyl group, the thiazole core, and the methylene linker between these two structural elements. In general, compounds with lipophilic [5+6] bicyclic ring systems, such as the 7-benzothiophenyl- and 4-indanyl groups, at the 5-position were 2-3 times more active against both fungal species as compared to 41F5. Also, introduction of a carbonyl group at the methylene linker of 41F5 resulted in a 2-3-fold increase in potency. These highly active compounds also showed generally low toxicities against murine P388D1 macrophages resulting in selectivity indices ranging from 63 to >200. Compounds that were highly active against fluconazole-sensitive C. neoformans strains had almost identical activity against fluconazole-resistant variants of this fungus indicating that 14α-demethylase is not their molecular target. Highly active compounds also retained activity against H. capsulatum phagocytosed into P388D1 macrophages.

O-Mannosylation of Proteins Enables Histoplasma Yeast Survival at Mammalian Body Temperatures.

The ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen Histoplasma capsulatum, elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection. Histoplasma yeast cells lacking the Pmt1 and Pmt2 protein mannosyltransferases, which catalyze O-linked mannosylation of proteins, are severely attenuated during infection of mammalian hosts. Cells lacking Pmt2 have altered surface characteristics that increase recognition of yeast cells by the macrophage mannose receptor and reduce recognition by the ß-glucan receptor Dectin-1. Despite these changes, yeast cells lacking these factors still associate with and survive within phagocytes. Depletion of macrophages or neutrophils in vivo does not recover the virulence of the mutant yeast cells. We show that yeast cells lacking Pmt functions are more sensitive to thermal stress in vitro and consequently are unable to productively infect mice, even in the absence of fever. Treatment of mice with cyclophosphamide reduces the normal core body temperature of mice, and this decrease is sufficient to restore the infectivity of O-mannosylation-deficient yeast cells. These findings demonstrate that O-mannosylation of proteins increases the thermotolerance of Histoplasma yeast cells, which facilitates infection of mammalian hosts.IMPORTANCE For dimorphic fungal pathogens, mammalian body temperature can have contrasting roles. Mammalian body temperature induces differentiation of the fungal pathogen Histoplasma capsulatum into a pathogenic state characterized by infection of host phagocytes. On the other hand, elevated temperatures represent a significant barrier to infection by many microbes. By functionally characterizing cells lacking O-linked mannosylation enzymes, we show that protein mannosylation confers thermotolerance on H. capsulatum, enabling infection of mammalian hosts.

Differentiation of the fungus Histoplasma capsulatum into a pathogen of phagocytes.

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.