Blastomycosis.

Blastomycosis is the fungal disease caused by thermally dimorphic fungi in the genus Blastomyces, with B dermatitidis complex causing most cases. It is considered hyperendemic in areas adjacent to the Great Lakes and along the St. Lawrence, Mississippi, and Ohio rivers, but definitive geographic distribution of blastomycoses remains obscure. Clinical presentation is variable. Disseminated blastomycosis with extrapulmonary manifestations is more common in immunosuppressed individuals. Culture positivity is required for definitive diagnosis, but compatible histology is often sufficient for presumptive diagnosis and initiation of treatment. Treatment should be provided to all symptomatic cases to prevent progression or recurrence.

Molecular detection of Blastomyces in an air sample from an outbreak associated residence.

Based on epidemiologic data during a blastomycosis outbreak, exposure within the home was suspected for two case patients that resided together. Soil and air samples were collected from the basement of their residence. Samples were tested for Blastomyces by culture and polymerase chain reaction (PCR) to compare with an available clinical isolate. An air sample from the basement of the residence was PCR positive for Blastomyces. Sequence data from the air sample and the outbreak clinical isolate were identified as different Blastomyces spp. Despite this, our findings suggest that the basement was suitable for the growth of Blastomyces and airborne organism was circulating.

CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis.

Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivoIMPORTANCEBlastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.

Outcomes of persons with blastomycosis involving the central nervous system.

Blastomyces dermatitidis is a dimorphic fungus which is potentially life-threatening if central nervous system (CNS) dissemination occurs. Sixteen patients with proven or probable CNS blastomycosis are presented. Median duration of symptoms was 90 days; headache and focal neurologic deficit were the most common presenting symptoms. Magnetic resonance imaging (MRI) consistently demonstrated an abnormality, compared to 58% of computed tomography scans. Tissue culture yielded the pathogen in 71% of histology-confirmed cases. All patients who completed treatment of an amphotericin B formulation and extended azole-based therapy did not relapse. Initial nonspecific symptoms lead to delayed diagnosis of CNS blastomycosis. A high index of suspicion is necessary if there is history of contact with an area where B. dermatitidis is endemic. Diagnostic tests should include MRI followed by biopsy for tissue culture and pathology. Optimal treatment utilizes a lipid-based amphotericin B preparation with an extended course of voriconazole.

Discordant influence of Blastomyces dermatitidis yeast-phase-specific gene BYS1 on morphogenesis and virulence.

Blastomyces dermatitidis is a thermally induced dimorphic fungus capable of causing lung and systemic infections in immunocompetent animal hosts. With the publication of genomic sequences from three different strains of B. dermatitidis and the development of RNA interference as a gene-silencing tool, it has become possible to easily ascertain the virulence and morphological effects of knocking down the expression of candidate genes of interest. BYS1 (Blastomyces yeast-phase-specific 1), first identified by Burg and Smith, is expressed at high levels in yeast cells and is undetectable in mold. The deduced protein sequence of BYS1 has a putative signal sequence at its N terminus, opening the possibility that the BYS1-encoded protein is associated with the yeast cell wall. Herein, strains of B. dermatitidis with silenced expression of BYS1 were engineered and tested for morphology and virulence. The silenced strains produced rough-surfaced cultures on agar medium and demonstrated a propensity to form pseudohyphal cells on prolonged culture in vitro and in vivo, as measured in the mouse lung. Tests using a mouse model of blastomycosis with either yeast or spore inocula showed that the bys1-silenced strains were as virulent as control strains. Thus, although silencing of BYS1 alters morphology at 37 degrees C, it does not appear to impair the pathogenicity of B. dermatitidis.

Classic pyomyositis of the extremities as an unusual manifestation of Blastomyces dermatitidis: a report of two cases.

Pyomyositis is an infection of skeletal muscle that, by definition, arises intramuscularly rather than secondarily from adjacent infection. It is usually associated with bacterial infection, particularly Staphylcococcus aureus. Fungi are rare causes, and Blastomyces dermatitidis has not been reported previously. In this case series, we report two cases of pyomyositis caused by B. dermatitidis. Cases were prospectively identified through routine clinical care at a single academic referral hospital. Two patients with complaints of muscle pain and subacute cough were treated at our hospital in 2007. Both patients were found to have pyomyositis caused by B. dermatitidis- in the quadriceps muscles in one patient, and in the calf muscle in another - by radiological imaging and fungal culture. Both were also diagnosed with pneumonia caused by B. dermatitidis (presumptive in one, confirmed in the other). There was no evidence of infection of adjacent structures, suggesting that the route of infection was likely direct haematogenous seeding of the muscle. A review of the literature confirmed that although B. dermatitidis has been described as causing axial muscle infection secondary to adjacent infection such as vertebral osteomyelitis, our description of isolated muscle involvement (classic pyomyositis) caused by B. dermatitidis, particularly of the extremity muscles, is unique. We conclude that B. dermatitidis is a potential cause of classic pyomyositis.

Microecology of Blastomyces dermatitidis: the ammonia hypothesis.

The precise microecology of Blastomyces dermatitidis is unknown, but the fungus has been associated with nitrogenous waste products and rapidly changing environmental conditions. Ammonia accumulates in certain microenvironments, is toxic to most fungi, but may not be identified in processed soil samples. Ammonia tolerance of B. dermatitidis was investigated with two strains recovered in Wisconsin, one from a dog and the other from an environmental source. The samples were grown on phosphate and HEPES buffered agar media supplemented with mineral salts, low (1 g/l) and high (20 g/l) dextrose and increasing amounts of ammonium sulfate, at pH 7-8.2, in gas-impermeable bags at 20 degrees C. Moderate mold growth and sporulation of the strains were observed at low glucose concentration and calculated ammonia concentrations of 4.2 mmol/l when plates were inoculated with either mold or yeast forms. Three recent B. dermatitidis human clinical isolates also exhibited similar growth on this media, and 4/5 strains tolerated ammonia concentrations of 42-62 mmol/l. Growth of virtually all soil fungi from 206 aqueous slurries of fresh and frozen soil from the northern USA and Canada was inhibited at ammonia concentrations of 2.1-4.2 mmol/l. The ability of B. dermatitidis to survive and grow in organic carbon-poor, high ammonia microenvironments may be important to the competitive success of this fungus. These findings may have implications for other dimorphic fungi.

Development and application of a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation.

A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, "X," next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.

Identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species by repetitive-sequence-based PCR.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of Coccidioides species, Blastomyces dermatitidis, and Histoplasma capsulatum was assessed by comparing data obtained to colony morphology and microscopic characteristics and to nucleic acid probe results. DNA from cultures of 23 Coccidioides, 24 B. dermatitidis, 24 H. capsulatum, 3 Arthrographis, and 2 Malbranchea isolates was extracted using a microbial DNA isolation kit as recommended by Bacterial Barcodes, Inc. Rep-PCR and probe results agreed for 97.2% of the dimorphic fungi when > or =85% similarity was used as the criterion for identification. Two H. capsulatum isolates were not identified, but no isolates were misidentified. From 43 of those cultures (15 Coccidioides, 14 B. dermatitidis, 14 H. capsulatum, 3 Arthrographis, and 2 Malbranchea), DNA also was extracted using an IDI lysis kit, a simpler method. Rep-PCR and probe results agreed for 97.7% of the dimorphic fungi when a criterion of > or =90% similarity was used for identification. One H. capsulatum isolate could not be identified; no isolates were misidentified. Using > or =85% similarity for identification resulted in one misidentification. These data suggest that the DiversiLab system can be used to identify Coccidioides and B. dermatitidis and, possibly, H. capsulatum isolates.

Geographic distribution of human blastomycosis cases in Milwaukee, Wisconsin, USA: association with urban watersheds.

Most studies of endemic blastomycosis and outbreaks have involved rural areas. Case homesites in rural Northern Wisconsin have been associated with waterways and sand soils. ARC-GIS was used to geocode addresses and to observe geographic features of homesites from 45 State-mandated reports of human blastomycosis in urban Milwaukee County, Southeastern Wisconsin 2000-2004. Each case property was directly observed, and houses and duplexes (N = 38) were compared with 151 same-street control homesites. Categorical data was analyzed using a chi-square or Fisher's exact test; continuous variables by Kruskal-Wallis test. One case cluster was seen on Milwaukee's North side where the estimated annual incidence was 2.8/100,000 compared to 0.96/100,000 for the entire county. Cases were less common in the most urbanized watersheds (0.49/100,000/yr) versus Lake Michigan shores (0.85) versus remaining three open watersheds (1.4) [P<0.01]. Case homesites averaged 1067 m to waterways and none were on sand soils. (Comparison is made to a Northern Wisconsin community where case homesites averaged 354 m to waterways, 24/25 were on sand soils and annual incidence was 74/100,000.) No unique features of case homesites were identified in Milwaukee County. In this urban area of Wisconsin, relatively low incidence rates may be explained, in part, by lower density of inland waterways and lack of sand soils, however, blastomycosis cases appear to be associated with open watersheds.

Blastomyces dermatitidis produces melanin in vitro and during infection.

Melanin is made by several important pathogenic fungi and is implicated in the pathogenesis of a number of mycoses. This study investigates whether the thermally dimorphic fungal pathogen Blastomyces dermatitidis produces melanin. Using techniques developed to study melanization in other fungi, we demonstrate that B. dermatitidis conidia and yeast produce melanin in vitro and that yeast cells synthesize melanin or melanin-like pigment in vivo. Melanization reduced susceptibility to amphotericin B, but not to itraconazole or voriconazole. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may affect the pathogenesis of blastomycosis.

Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii.

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii. Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.

Extracellular calcium and magnesium, but not iron, are needed for optimal growth of Blastomyces dermatitidis yeast form cells in vitro.

In the present study, we demonstrate that the yeast form of Blastomyces dermatitidis can proliferate for short periods of time in the absence of ferric iron but not in the absence of calcium or magnesium. The results of this study shed light on the resistance of B. dermatitidis to chelating agents, such as deferoxamine, and may explain how B. dermatitidis resists the iron-binding activity of serum transferrin.

An imported case of Blastomyces dermatitidis infection in Mexico.

Blastomycosis is an acute or chronic primary infection of the respiratory system, endemic in North America (United States of America and Canada), Africa and Asia. We report a case in Mexico, in a three years old child who had been born in California and lived in Chicago, U.S.A. The patient presented pulmonary symptoms prior to development of a skin ulcer. Blastomyces dermatitidis was identified by mycological and molecular procedures. The patient was successfully treated with amphotericin B, oral ketoconazole and itraconazole.

Novel role for albumin in innate immunity: serum albumin inhibits the growth of Blastomyces dermatitidis yeast form in vitro.

In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

Molecular genetic analysis of Blastomyces dermatitidis reveals new insights about pathogenic mechanisms.

Fungal pathogens have emerged as a public health menace owing to the expanding population of vulnerable patients and to a heightened exposure to fungi in our environment, particularly for the systemic dimorphic fungi that inhabit soil worldwide. A better understanding of these microbes and their pathogenic mechanisms is badly needed to further research into therapeutic options. Advances in the molecular tools for genetic manipulation of Blastomyces dermatitidis have enhanced our ability to study this poorly understood dimorphic fungal pathogen. Recent refinements in gene-transfer technique, new selection markers, reliable reporter fusions and successes in gene targeting have shed light upon the importance of the mycelium-to-yeast transition and the crucial and complex role the BAD1 adhesin plays in pathogenesis.

Transferrin independent serum inhibition of Blastomyces dermatitidis.

Human serum has been reported to inhibit the growth of several fungal pathogens. Serum inhibitory activity has predominantly been associated with transferrin-mediated iron binding. We found that serum from several animal species (human, dog, mouse and fetal bovine) inhibited the growth of the dimorphic fungal pathogen, Blastomyces dermatitidisin vitro. In initial studies, we found no correlation between the total iron-binding capacity of various sera and their inhibitory activity against B. dermatitidis yeast. In addition, we were unable to abrogate the inhibitory activity of human serum against B. dermatitidis yeast by the addition of ferric chloride (10-200 microM). We found that apo-transferrin had little or no inhibitory activity against B. dermatitidis yeast. Furthermore, serum from hypotransferrinemic mice (hpx/hpx) had inhibitory activity against B. dermatitidis yeast that was equivalent to that of normal mouse serum. These findings are consistent with our initial findings and suggest that serum inhibitory activity against B. dermatitidis yeast is transferrin independent. Although high concentrations (3.5-5 mg/ml) of lactoferrin did have inhibitory activity against B. dermatitidis yeast, these concentrations were orders of magnitude greater than those found in serum under normal physiological conditions, suggesting that lactoferrin was not likely to contribute to serum inhibitory activity against B. dermatitidis yeast. Our findings suggest that host iron-binding proteins may be relatively ineffective in innate host defense against infection with B. dermatitidis yeast.

Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism.

Most dimorphic fungal pathogens grow as non-pathogenic moulds in soil and convert to pathogenic yeast in the host, suggesting that virulence factors are upregulated during phase transition. Such factors have been difficult to identify. We analysed BAD1 (formerly WI-1), a virulence factor in the dimorphic fungus Blastomyces dermatitidis, for expression in yeast and mycelial morphotypes. BAD1 was expressed in yeast but not in mycelia of North American strains of B. dermatitidis, and this expression pattern was confirmed for BAD1 transcript. BAD1 under the control of its promoter was transferred into African B. dermatitidis lacking a native BAD1 locus, and phase-specific expression was conserved. Sequence similarity was identified between the BAD1 promoter and the promoters of two yeast phase-specific genes in Histoplasma capsulatum. In H. capsulatum BAD1 transformants, yeast phase-specific expression of BAD1 was conserved, and no transcript was detected in mycelia. BAD1 beta-galactosidase reporter fusions analysed in B. dermatitidis and H. capsulatum confirmed that BAD1 is transcriptionally regulated in both fungi. BAD1 promoter activity and surface BAD1 expression were detected 6 h after shifting mycelia to 37 degrees C. Thus, BAD1 is expressed after transition to the pathogenic yeast morphotype and is regulated by a mechanism for phase-specific gene expression that appears to be conserved.

Genetic diversity and transcriptional analysis of the bys1 gene from Blastomyces dermatitidis.

Blastomyces dermatitidis, a pathogenic fungal organism, is able to exist in two different morphologies, a multicellular mycelium or a unicellular yeast, according to temperature, 25 degrees C and 37 degrees C respectively. The switching between morphologies must be accompanied by a cascade of signaling events in which expression of genes responsible for the change of morphology is increased or decreased. bys1, a gene from B. dermatitidis isolate #58, is expressed at high levels in the unicellular yeast, but gradually diminishes as the temperature is lowered and the organism converts to the mycelial phase where there is no transcription of bys1. We explored if bys1 homologs are found in other B. dermatitidis isolates and if the transcription of the homologs were regulated by temperature. bys1 was identified in all B. dermatitidis isolates tested and could be grouped into two classes by Southern blot, PCR, and DNA sequence. Although the bys1 transcripts of both classes were regulated by temperature, transcription rates varied between the three isolates tested.

Studies on the molecular ecology of Blastomyces dermatitidis.

The microecology of Blastomyces dermatitidis, the dimorphic etiologic agent of the potentially fatal systemic fungal infection, blastomycosis, is not well defined. Blastomyces dermatitidis may occur periodically at natural sites, perhaps aided by rotting organic material, animal droppings and physical changes. Semi-quantitative growth studies of B. dermatitidis on 2% agar plates determined the ability to utilize or tolerate a variety of substrates including simple and complex molecules as carbon source, and organic and inorganic nitrogen sources. Allantoin, creatinine, quanidoacetic acid, guanidine and cysteine may be used as sole nitrogen source. Allantoin in combination with dextrose, glycerol, lichenen, celloboise and other wood by-products support growth of B. dermatitidis at room temperature. The nutritional conversion of the fungus to the yeast form at room temperature, well demonstrated on allantoin/glycerol/yeast extract media, appears to be affected by certain inorganic compounds. The organism tolerates low to moderate levels of alpha-pinene, tannic acid, shikimic acid, veratryl alcohol, vanillic acid, and polyethyleneglycol-200. There are significant differences among isolates regarding growth on various substances at 20 degrees and 37 degrees centigrade. It appears that a variety of wood by-products and animal waste substrates, in combination, support the growth of B. dermatitidis. Their role in the ecological niche of B. dermatitidis, and the importance of nutritional dimorphism in the natural environment warrants further investigation.