Dimorphic Fungal Pathogen Histoplasma Capsulatum Research Articles

Deletion of the Stress Response Gene DDR48 from Histoplasma capsulatum Increases Sensitivity to Oxidative Stress, Increases Susceptibility to Antifungals, and Decreases Fitness in Macrophages.

The stress response gene DDR48 has been characterized in Saccharomyces cerevisiae and Candida albicans to be involved in combating various cellular stressors, from oxidative agents to antifungal compounds. Surprisingly, the biological function of DDR48 has yet to be identified, though it is likely an important part of the stress response. To gain insight into its function, we characterized DDR48 in the dimorphic fungal pathogen Histoplasma capsulatum. Transcriptional analyses showed preferential expression of DDR48 in the mycelial phase. Induction of DDR48 in Histoplasma yeasts developed after treatment with various cellular stress compounds. We generated a ddr48∆ deletion mutant to further characterize DDR48 function. Loss of DDR48 alters the transcriptional profile of the oxidative stress response and membrane synthesis pathways. Treatment with ROS or antifungal compounds reduced survival of ddr48∆ yeasts compared to controls, consistent with an aberrant cellular stress response. In addition, we infected RAW 264.7 macrophages with DDR48-expressing and ddr48∆ yeasts and observed a 50% decrease in recovery of ddr48∆ yeasts compared to wild-type yeasts. Loss of DDR48 function results in numerous negative effects in Histoplasma yeasts, highlighting its role as a key player in the global sensing and response to cellular stress by fungi.

Cysteine Dioxygenase Enzyme Activity and Gene Expression in the Dimorphic Pathogenic Fungus Histoplasma capsulatum Is in both the Mold and Yeast Morphotypes and Exhibits Substantial Strain Variation.

In the dimorphism (mold/yeast) Histoplasma capsulatum (Hc) literature are reports that yeast (the so-called pathogenic form) uniquely expresses a cysteine dioxygenase (CDO, approx. 10,500 dal) activity which the mold morphotype (the so-called saprophytic soil form) does not express (C.F., Kumar et al., Biochem 22, 762, 1983). This yeast-specific CDO activity is postulated to play a critical role in the mold-to-yeast shift. A number of years ago, our lab isolated the gene encoding the Hc cysteine dioxygenase (CDO1, Genbank accession AY804144) and noted significant expression in the mold morphotype of several Histoplasma strains and also determined that the predicted protein would be over double the 10,500 dal reported by Kumar et al. Our report demonstrates (in the class 1 Downs strain, the class 2 G271B strain and two Panamanian strains, 184AS and 186AS) that the CDO1 gene is expressed in both the mold and yeast morphotypes and both morphotypes show significant CDO activity. Furthermore, we show via a FLAG-tag analysis that the expressed protein is approximately 24.7 ± 2.4 kd, in agreement with the putative protein sequence (determined from cDNA sequence) which yields 23.8 kd and is consistent with most other eukaryotic CDO enzymes. Additionally, we demonstrate that intracellular cysteine levels are actually significantly higher in the mold form of the two Panamanian strains, 184AS and 186AS, equal in both mold and yeast in the class 1 Downs strain and significantly higher in yeast of the more pathogenic class 2 G217B strain.

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.

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

ABSTRACTThe 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 β-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.

Definition of the extracellular proteome of pathogenic-phase Histoplasma capsulatum.

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.

Surface architecture of Histoplasma capsulatum

The dimorphic fungal pathogen Histoplasma capsulatum is the most frequent cause of clinically significant fungal pneumonia in humans. H. capsulatum virulence is achieved, in part, through diverse and dynamic alterations to the fungal cell surface. Surface components associated with H. capsulatum pathogenicity include carbohydrates, lipids, proteins, and melanins. Here, we describe the various structures comprising the cell surface of H. capsulatum that have been associated with virulence and discuss their involvement in the pathobiology of disease.

Reverse genetics through random mutagenesis in Histoplasma capsulatum

BackgroundThe dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease in humans and other mammals. Progress in understanding the mechanisms underlying the biology and the pathogenesis of Histoplasma has been hindered by a shortage of methodologies for mutating a gene of interest.ResultsWe describe a reverse genetics process that combines the random mutagenesis of Agrobacterium-mediated transformation with screening techniques to identify targeted gene disruptions in a collection of insertion mutants. Isolation of the desired mutant is accomplished by arraying individual clones from a pool and employing a PCR-addressing method. Application of this procedure facilitated the isolation of a cbp1 mutant in a North American type 2 strain, a Histoplasma strain recalcitrant to gene knock-outs through homologous recombination. Optimization of cryopreservation conditions allows pools of mutants to be banked for later analysis and recovery of targeted mutants.ConclusionThis methodology improves our ability to isolate mutants in targeted genes, thereby facilitating the molecular genetic analysis of Histoplasma biology. The procedures described are widely applicable to many fungal systems and will be of particular interest to those for which homologous recombination techniques are inefficient or do not currently exist.

Isolation and characterization of a calmodulin-encoding cDNA from the pathogenic fungus Histoplasma capsulatum.

We describe in this paper the isolation and complete sequence of a calmodulin (CaM) encoding cDNA from the dimorphic pathogenic fungus Histoplasma capsulatum (GenBank accession U12505). The deduced amino acid sequence was identical to the CaM of Aspergillus nidulans and had only one amino acid difference from the CaM of Neurospora crassa. Saccharomyces cerevisiae CaM, however, has only about 60% amino acid identity compared with H. capsulatum. These data further support the close relationship of Histoplasma to the filamentous ascomycetes. Histoplasma CaM was expressed as a single major transcript of approximately 1200 nt. in both the yeast and mould growth forms. CaM mRNA levels were approximately two-fold greater in the yeast than in the mould form of the organism. Southern blot analysis indicated that the H. capsulatum CaM gene is present in a single copy.

Autonomous replication of foreign DNA in Histoplasma capsulatum: role of native telomeric sequences.

Genetic transformation of the dimorphic pathogenic fungus Histoplasma capsulatum can result in chromosomal integration of the transforming DNA or the generation of multicopy linear plasmids carrying the transforming DNA. We showed previously that Escherichia coli plasmids do not replicate autonomously in H. capsulatum without significant modifications, one of which is the in vivo addition of Histoplasma telomeres at the termini of linear DNA. To address the requirements for autonomous replication in H. capsulatum, we constructed a circular E. coli plasmid containing adjacent inverted stretches of Histoplasma telomeric repeats separated by a unique restriction site. The linearized plasmid bearing telomeric termini was maintained in H. capsulatum without modification other than the addition of more telomeric sequence. We recovered the original plasmid in E. coli after removal of the telomeric termini by using engineered restriction sites. Thus, no special Histoplasma modification or sequence other than the telomeres was needed for autonomous replication in H. capsulatum. Additionally, this plasmid provides a shuttle vector that replicates autonomously in E. coli (as a circular plasmid) and in H. capsulatum (as a linear plasmid).

Molecular cloning and expression of hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum

We have cloned a nucleotide sequence from Histoplasma capsulatum G222B corresponding to a heat inducible hsp82 gene, and determined its entire sequence and the flanking regions. During the temperature-controlled mycelium-to-yeast phase transition the gene is more actively transcribed at 37°C in the temperature tolerant and mouse-virulent G222B strain, while 34°C is the optimum for transcription in the temperature sensitive and mouse-avirulent Downs strain.

The intron-containing hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum is properly spliced in severe heat shock conditions.

We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.

The Intron-Containing hsp82 Gene of the Dimorphic Pathogenic Fungus Histoplasma capsulatum Is Properly Spliced in Severe Heat Shock Conditions

We have isolated and characterized a heat-inducible gene, hsp82, from the dimorphic pathogenic fungus Histoplasma capsulatum, which is a filamentous mold at 25 degrees C and a unicellular yeast at 37 degrees C. This gene, which has a high degree of homology with other members of the hsp82 gene family, is split into three exons and two introns of 122 and 86 nucleotides, respectively. Contrary to what has been demonstrated in Drosophila melanogaster, Saccharomyces cerevisiae, and other organisms, hsp82 mRNA in H. capsulatum is properly spliced during the severe heat conditions of 37 to 40 degrees C in the temperature-sensitive Downs strain. Splicing accuracy was also observed at 42 degrees C in the temperature-tolerant G222B strain, which showed no evidence of accumulation of primary transcripts. Furthermore, the intron containing the beta-tubulin gene is also properly spliced at the upper temperature range, suggesting that the lack of a block in splicing may be a general phenomenon in this organism.

Variable expression of a yeast-phase-specific gene in Histoplasma capsulatum strains differing in thermotolerance and virulence

By means of differential hybridization techniques, several yeast-phase-specific DNA sequences were identified in the dimorphic pathogenic fungus Histoplasma capsulatum. A 1.85-kilobase (kb) HindIII fragment from one genomic clone, yps-3, hybridized to at least three distinct yeast poly(A)+ RNAs of 1.3, 1.05, and 0.95 kb from the virulent strain G217B. These mRNAs were not detected in mycelia. When mycelia from G217B were induced to become yeast by transfer from 25 to 37 degrees C, a process requiring approximately 9 days, expression of yps-3 was detected within 24 h, although not in the initial 2 h following the temperature shift. In contrast, a low-virulence strain (Downs) which completes the transition in approximately 2 weeks failed to express the yps-3 gene during phase transitions. A third isolate, G186B, intermediate in its virulence properties and in the time required for the transition (11 days), expressed a single 1.25-kb mRNA but only at low levels in the yeast phase and only after 3 days following the 25-to-37 degrees C temperature shift. Although yps-3 expression does not appear to be essential for the transformation to the yeast phase, it may facilitate the early adaptive processes which permit the mycelium-to-yeast transition and survival of the yeast phase of H. capsulatum at elevated host temperatures. The phase-specific yps-3 nuclear gene is carried on highly polymorphic restriction fragments in all three strains, suggesting that this probe may provide a sensitive diagnostic tool for the classification of H. capsulatum isolates.

Purification of membranes and identification of phase-specific proteins of the dimorphic pathogenic fungus Histoplasma capsulatum

Plasma membrane vesicles from the yeast and mycelial phases of Histoplasma capsulatum have been purified and characterized. The method of purification involved differential centrifugation of ballistically fractured cells followed by sedimentation through discontinuous sucrose density gradient and equilibrium centrifugation. Purity of the preparation was assessed by electron microscopy. The protein composition of the membrane preparations from the yeast and mycelial phases of the fungus was analyzed by polyacrylamide gels. A comparison of the two morphologic phases revealed quantitative and qualitative differences in the expressions of several membrane-specific proteins. Physical differences in the appearance of the membranes were also observed by electron micrography of membrane preparations. Alteration in membrane fluidity may be one of the many causes for differences in the appearance of membrane vesicles in the two phases.

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.

Yeastlike to mycelial phase transformation of Histoplasma capsulatum as observed by scanning electron microscopy.

Details of the sequential events occurring during the critical phases of yeast to mold morphogenesis of the dimorphic fungal pathogenHistoplasma capsulatum as seen by the new technique of scanning electron microscopy are described and illustrated by electron micrographs.