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BMC Microbiology Nov 2009The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease in humans and other mammals. Progress in understanding the mechanisms...
BACKGROUND
The 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.
RESULTS
We 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.
CONCLUSION
This 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.
Topics: Genetic Techniques; Histoplasma; Mutagenesis
PubMed: 19919692
DOI: 10.1186/1471-2180-9-236 -
The Medical Journal of Australia
Topics: Animals; Australia; Histoplasma; Histoplasmosis; Humans; Skin Tests
PubMed: 7162437
DOI: 10.5694/j.1326-5377.1953.tb81577.x -
Antimicrobial Agents and Chemotherapy Oct 1975We have found that amphotericin B is unstable in two commonly employed fungal culture media. This instability leads to inaccuracies in determining the actual level of...
We have found that amphotericin B is unstable in two commonly employed fungal culture media. This instability leads to inaccuracies in determining the actual level of susceptibility of slow-growing strains that require prolonged incubation for growth. To help compensate for this problem, we have described two rapid methods of susceptibility testing.
Topics: Amphotericin B; Culture Media; Drug Stability; Histoplasma; Saccharomyces cerevisiae
PubMed: 1103723
DOI: 10.1128/AAC.8.4.426 -
Molecular Ecology Dec 2003Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human... (Comparative Study)
Comparative Study
Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein-coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein-coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.
Topics: Cluster Analysis; Evolution, Molecular; Geography; Histoplasma; Models, Genetic; Phylogeny; Sequence Analysis, DNA; Species Specificity
PubMed: 14629354
DOI: 10.1046/j.1365-294x.2003.01995.x -
Pathogens and Global Health Feb 2020: Histoplasmosis is a fungal infection acquired through inhalation of microconidia, mostly present in the Americas. Both immunocompetent and immunocompromised patients...
: Histoplasmosis is a fungal infection acquired through inhalation of microconidia, mostly present in the Americas. Both immunocompetent and immunocompromised patients can present a wide spectrum of signs/symptoms, ranging from mild disease to a severe, disseminated infection. The aim of this observational study is to describe histoplasmosis cases diagnosed in travelers and their clinical/radiological and therapeutic pattern.: Retrospective study at the Department of Infectious - Tropical Diseases and Microbiology (DITM) of Negrar, Verona, Italy, between January 2005 and December 2015.: Twenty-three cases of acute histoplasmosis were diagnosed, 17 of which belong to the same cluster. Seven of the 23 patients (30.4%) were admitted to hospital, four of whom underwent invasive diagnostic procedures. Thirteen patients (56.5%) received oral itraconazole. All patients recovered, although nine (39.1%) had radiological persisting lung nodules at 12 month follow up.: Clinical, laboratory and radiological features of histoplasmosis can mimic other conditions, resulting in unnecessary invasive diagnostic procedures. However, a history of travel to endemic areas and of exposure to risk factors (such as visits to caves and presence of bats) should trigger the clinical suspicion of histoplasmosis. Treatment may be indicated in severe or prolonged disease.
Topics: Acute Disease; Adult; Aged; Female; Histoplasma; Histoplasmosis; Humans; Italy; Male; Middle Aged; Retrospective Studies; Travel; Young Adult
PubMed: 31959091
DOI: 10.1080/20477724.2020.1716517 -
Journal of Proteomics Feb 2017Histoplasma capsulatum, the causative agent of histoplasmosis (also called "Darling's disease"), can affect both immunocompetent and immunocompromised hosts....
UNLABELLED
Histoplasma capsulatum, the causative agent of histoplasmosis (also called "Darling's disease"), can affect both immunocompetent and immunocompromised hosts. Post-translational protein modification by lysine succinylation (Ksuc) is a frequent occurrence in eukaryote and prokaryote. Recently, the roles of succinylation and its regulatory enzymes in regulating metabolic pathway in bacteria, mammalian and fungus were highlighted. Here, we report the first global profiling of lysine succinylation, with 463 modification sites in 202 proteins from H. capsulatum NAM1 identified, coupling immune-affinity enrichment using an anti-succinyllysine antibody with mass spectrometry. The bioinformatics results including GO functional and enrichment analysis showed that these succinylated proteins are mainly involved in central metabolism and protein synthesis, consistent with previous reports. 13 lysine succinylation sites on histones including H2A, H2B, H3 and H4 in H. capsulatum were firstly reported. The data is a good resource for further functional characterization of lysine succinylation in H. capsulatum.
BIOLOGICAL SIGNIFICANCE
H. capsulatum is the causative agent of lung disease histoplasmosis. The ability of H. capsulatum yeasts to infect and proliferate within macrophages as an intracellular pathogen can be contributed to several virulence factors and metabolic regulation. Lysine succinylation was recently shown to play a critical role in the metabolism regulation of Candida albicans. H. capsulatum succinylated proteins were firstly characterized in this work, and bioinformatics results showed that this modification may also be relevant with central metabolism in H. capsulatum. New succinylation sites on histones were reported. This represents an important resource to address the function of H. capsulatum lysine succinylation.
Topics: Computational Biology; Fungal Proteins; Histones; Histoplasma; Humans; Lysine; Metabolic Networks and Pathways; Protein Biosynthesis; Protein Processing, Post-Translational; Proteomics; Succinic Acid; Tandem Mass Spectrometry
PubMed: 28063982
DOI: 10.1016/j.jprot.2016.12.020 -
FEMS Immunology and Medical Microbiology Sep 2005This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic...
This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.
Topics: Animals; Brazil; DNA, Fungal; Genetic Variation; Histoplasma; Histoplasmosis; Humans; Mycological Typing Techniques; Polymerase Chain Reaction; Random Amplified Polymorphic DNA Technique; Rats; Soil Microbiology
PubMed: 16055317
DOI: 10.1016/j.femsim.2005.05.018 -
Journal of Medical and Veterinary... 1994Oral histoplasmosis in a 30-year-old male with no history of travel outside India is described. An ulcerating lesion was located on the hard palate. A chest X-ray was... (Review)
Review
Oral histoplasmosis in a 30-year-old male with no history of travel outside India is described. An ulcerating lesion was located on the hard palate. A chest X-ray was normal. Based on physical examination, regional lymph nodes, liver and spleen were not involved. The diagnosis was established by demonstrating yeast-like budding cells in a biopsy of the lesion and by isolating Histoplasma capsulatum in pure culture. The identity of the isolate was confirmed by a chemiluminescent DNA-probe assay and the exoantigen test. A review of the Indian literature from 1968 to 1992 revealed the occurrence of 25 authentic cases of histoplasmosis in India. In 19 cases, lesions were confined to the oral cavity confirming prior observation that histoplasmosis in Indian patients tends to occur primarily in extrapulmonary sites, particularly the oral cavity.
Topics: Adult; Histoplasma; Histoplasmosis; Humans; India; Male; Mouth Diseases
PubMed: 8064548
DOI: 10.1080/02681219480000141 -
Cellular Microbiology Sep 2020Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum...
Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule-degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non-conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.
Topics: Culture Media; Extracellular Vesicles; Fungal Proteins; Histoplasma; Nutrients
PubMed: 32406582
DOI: 10.1111/cmi.13217 -
PLoS Neglected Tropical Diseases Aug 2019Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly...
BACKGROUND
Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis.
METHODOLOGY
Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction.
RESULTS
Both assays were able to detect down to 1 fg/μL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL.
CONCLUSION
ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.
Topics: Blood; Bone Marrow; DNA, Ribosomal Spacer; Histoplasma; Histoplasmosis; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Prospective Studies; Sensitivity and Specificity
PubMed: 31449526
DOI: 10.1371/journal.pntd.0007692