-
MSphere Aug 2023The rapid development of CRISPR/CRISPR-associated (Cas) systems has revolutionized the ability to produce genetic mutations in a desired locus, particularly in organisms...
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. 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 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 species and applicable for multiple genes. The optimized system shows promise for accelerating reverse genetic studies in spp. IMPORTANCE The ability to eliminate gene product functions is central to understanding molecular mechanisms. In the fungal pathogen , methods to inactivate or deplete gene products are inefficient, which hampers progress in defining 's virulence mechanisms. We describe an efficient CRISPR/Cas-based system for generating gene deletions in and show its validation on multiple genes with selectable and non-selectable phenotypes.
Topics: CRISPR-Cas Systems; Gene Editing; Histoplasma; Gene Deletion; Plasmids
PubMed: 37389430
DOI: 10.1128/msphere.00178-23 -
Journal of Fungi (Basel, Switzerland) Jan 2024Histoplasmosis is a widespread systemic disease caused by , prevalent in the Americas. Despite its significant morbidity and mortality rates, no vaccines are currently...
Histoplasmosis is a widespread systemic disease caused by , prevalent in the Americas. Despite its significant morbidity and mortality rates, no vaccines are currently available. Previously, five vaccine targets and specific epitopes for were identified. Immunoinformatics has emerged as a novel approach for determining the main immunogenic components of antigens through in silico methods. Therefore, we predicted the main helper and cytotoxic T lymphocytes and B-cell epitopes for these targets to create a potential multi-epitope vaccine known as HistoVAC-TSFM. A total of 38 epitopes were found: 23 common to CTL and B-cell responses, 11 linked to HTL and B cells, and 4 previously validated epitopes associated with the B subunit of cholera toxin, a potent adjuvant. In silico evaluations confirmed the stability, non-toxicity, non-allergenicity, and non-homology of these vaccines with the host. Notably, the vaccine exhibited the potential to trigger both innate and adaptive immune responses, likely involving the TLR4 pathway, as supported by 3D modeling and molecular docking. The designed HistoVAC-TSFM appears promising against , with the ability to induce important cytokines, such as IFN-γ, TNF-α, IL17, and IL6. Future studies could be carried out to test the vaccine's efficacy in in vivo models.
PubMed: 38248954
DOI: 10.3390/jof10010043 -
BioRxiv : the Preprint Server For... Nov 2023is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into...
UNLABELLED
is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte-MΦ colony stimulating factor (GM-CSF) induce metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the high affinity zinc importer, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under the control of this importer. This reporter accurately responds to medium devoid of Zn. ZRT2 expression increased (∼5-fold) in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with reporter yeasts and assessed ZRT2 expression at 0-, 3-, 7-, and 14-days post-infection (dpi). ZRT2 expression minimally increased at 3-dpi and peaked on 7-dpi, corresponding with onset of adaptive immunity. We discovered that the major phagocyte populations that restrict Zn to the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control replication, M-CSF. These findings illustrate the reporter's ability to sense Zn and and correlate ZRT2 activity with GM-CSF and M-CSF activation of MΦ.
IMPORTANCE
Phagocytes use an arsenal of defenses to control replication of yeasts, one of which is limitation of trace metals. On the other hand, combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 reporter to probe Zn sensing during infection and exposed a role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to studying how intracellular pathogens sense and respond to the changing environments of the host.
PubMed: 38014056
DOI: 10.1101/2023.11.14.567133 -
Journal of Infection in Developing... Jan 2024Fungi play a vital role in ensuring a physiological balance in the surrounding environments, interacting closely with humans, plants, and animals. While most of the time... (Review)
Review
Fungi play a vital role in ensuring a physiological balance in the surrounding environments, interacting closely with humans, plants, and animals. While most of the time their contribution is beneficial, occasionally, they can become harmful, especially in patients with weakened immune systems. The work at hand aims to present the most common fungal pathogens involved in invasive infections, focusing on fungi that are present in the tropical and temperate areas of the world. While in the former, due to the humid climate, most fungal infections are caused by dimorphic fungi such as Coccidioides spp., Blastomyces spp., Histoplasma spp., Emergomyces spp. and Paracoccidioides spp., in the latter, after Candida spp., the most frequent fungi that are involved in disseminated mycosis are Aspergillus spp., Fusarium spp. and species from the order Mucorales. Nowadays, the etiology, severity, and number of cases of fungal diseases are starting to rise significantly. There are no exact reasons reported for this increase, but several factors are thought to be incriminated: the expansion of the range of medical conditions that constitute risk factors for developing the disease, an improvement in the available diagnostic methods, the commodity offered by modern traveling services associated with the lack of an available vaccine against fungal infections, as well as climatic influences. All the above-mentioned aspects consequently caused infections that used to be endemic to be spread worldwide. Therefore, it is of critical importance to understand the epidemiology, clinical manifestations of fungi induced diseases, virulence factors, and diagnosis for each of those pathogens.
Topics: Animals; Humans; Fungi; Mycoses; Aspergillus; Candida
PubMed: 38377080
DOI: 10.3855/jidc.18206 -
MSphere Jun 2024Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported...
Histoplasmosis is an endemic mycosis that often presents as a respiratory infection in immunocompromised patients. Hundreds of thousands of new infections are reported annually around the world. The etiological agent of the disease, is a dimorphic fungus commonly found in the soil where it grows as mycelia. Humans can become infected by through inhalation of its spores (conidia) or mycelial particles. The fungi transition into the yeast phase in the lungs at 37°C. Once in the lungs, yeast cells reside and proliferate inside alveolar macrophages. Genomic work has revealed that is composed of at least five cryptic phylogenetic species that differ genetically. Three of those lineages have received new names. Here, we evaluated multiple phenotypic characteristics (colony morphology, secreted proteolytic activity, yeast size, and growth rate) of strains from five of the phylogenetic species of to identify phenotypic traits that differentiate between these species: , , , , and an African lineage. We report diagnostic traits for three species. The other two species can be identified by a combination of traits. Our results suggest that (i) there are significant phenotypic differences among the cryptic species of and (ii) those differences can be used to positively distinguish those species in a clinical setting and for further study of the evolution of this fungal pathogen.IMPORTANCEIdentifying species boundaries is a critical component of evolutionary biology. Genome sequencing and the use of molecular markers have advanced our understanding of the evolutionary history of fungal pathogens, including , and have allowed for the identification of new species. This is especially important in organisms where morphological characteristics have not been detected. In this study, we revised the taxonomic status of the four named species of the genus (), , , and and propose the use of species-specific phenotypic traits to aid their identification when genome sequencing is not available. These results have implications not only for evolutionary study of but also for clinicians, as the species could determine the outcome of disease and treatment needed.
Topics: Histoplasma; Phenotype; Phylogeny; Histoplasmosis; Humans; Genome, Fungal
PubMed: 38771035
DOI: 10.1128/msphere.00009-24 -
Microbiology Spectrum Dec 2023Histoplasmosis is considered one of the most important mycoses due to the increasing number of individuals susceptible to develop severe clinical forms, particularly...
Histoplasmosis is considered one of the most important mycoses due to the increasing number of individuals susceptible to develop severe clinical forms, particularly those with HIV/AIDS or receiving immunosuppressive biological therapies, the high mortality rates reported when antifungal treatment is not initiated in a timely manner, and the limitations of conventional diagnostic methods. In this context, there is a clear need to improve the capacity of diagnostic tools to specifically detect the fungal pathogen, regardless of the patient's clinical condition or the presence of other co-infections. The proposed novel pathogen-specific biomarkers have the potential to be used in immunodiagnostic platforms and antifungal treatment monitoring in histoplasmosis. In addition, the bioinformatics strategy used in this study could be applied to identify potential diagnostic biomarkers in other models of fungal infection of public health importance.
Topics: Humans; Histoplasmosis; Antifungal Agents; Mycoses; Biomarkers; Histoplasma
PubMed: 37882565
DOI: 10.1128/spectrum.00939-23 -
PLoS Neglected Tropical Diseases Sep 2023Histoplasma capsulatum exposure is rarely suspected in Indonesia. Pulmonary histoplasmosis can occur simultaneously with pulmonary tuberculosis (TB) or as an alternative...
BACKGROUND
Histoplasma capsulatum exposure is rarely suspected in Indonesia. Pulmonary histoplasmosis can occur simultaneously with pulmonary tuberculosis (TB) or as an alternative diagnosis in clinically-diagnosed TB patients with no microbiological evidence of TB. This study aimed to determine the seroprevalence of anti-H. capsulatum IgG antibody among pulmonary TB patients.
METHODOLOGY
This was a sub-study of 306 participants from a prospective cohort pulmonary TB study conducted at seven TB referral hospitals in Indonesia. The study population was presumptive pulmonary TB adult patients who underwent microbiological TB examinations and were categorized as drug-sensitive (DS), drug-resistant (DR), and clinically-diagnosed TB. Anti-H. capsulatum IgG antibody levels at baseline were measured using MVista Histoplasma Ab enzyme immunoassays. Data were summarized using descriptive statistics. Bivariate and multivariate logistic regression analysis were performed to assess factors associated with anti-H. capsulatum IgG antibody positive result.
RESULTS
12.7% (39/306) of pulmonary TB patients were positive for anti-H. capsulatum IgG antibodies (DR-TB patients (15.9%, 18/114), DS-TB (13.0%, 15/115), and clinically-diagnosed TB (7.8%, 6/77)). The median unit value of anti-H. capsulatum IgG antibody for all positive samples was 15.7 (IQR 10.2-28.9) EU. This median unit value was higher in clinically-diagnosed TB patients compared to DS-TB or DR-TB patients (38.1 (IQR 25.6-46.6) EU, 19.7 (IQR 12.3-28.9) EU, and 10.9 (IQR 9.2-15.4), respectively). There were 10 patients (3.3%) with anti-H. capsulatum IgG antibody levels above 30 EU. Factors associated with the anti-H. capsulatum IgG antibody positive result were malignancies (OR 4.88, 95% CI 1.09-21.69, p = 0.037) and cavitary lesions (OR 2.27, 95% CI 1.09-4.70, p = 0.028).
CONCLUSIONS
Our results provide evidence of exposure to H. capsulatum among pulmonary TB patients in Indonesia. Further studies are needed to provide a comprehensive picture of this fungal disease in other populations and regions to enhance awareness among clinicians and public health officials.
Topics: Adult; Humans; Indonesia; Seroepidemiologic Studies; Hospitals, Chronic Disease; Prospective Studies; Immunoglobulin G; Antibodies, Fungal; Histoplasma
PubMed: 37729126
DOI: 10.1371/journal.pntd.0011575 -
Scientific Reports Mar 2024Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and...
Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and structural integrity. This study analyses seventeenth century handwritten historical letters stored at the Correr Museum Library in Venice, Italy, exhibiting pronounced signs of biodeterioration. The techniques used encompassed traditional colony isolation on agar plates and proteomics analyses, employing nanoscale liquid chromatography coupled with high-resolution mass spectrometry (nano-LC-MS). Fluorescence microscopy was used for the first time in the historical paper biodeterioration context to supplement the conventional stereoscopic, optical, and scanning electron microscopic imaging techniques. This method enables the visualisation of microorganisms beyond and beneath the paper's surface through their natural intrinsic autofluorescence in a non-invasive and non-destructive way. The results demonstrate a diverse, complex, and abundant microbiota composed of coexisting fungal and bacterial species (Ascomycota, Mucoromycota, Basidiomycota, Proteobacteria, and Actinobacteria), along with mite carcasses, insects, parasites, and possibly protists. Furthermore, this study reveals certain species that were not previously documented in the biodeterioration of historical paper, including human pathogens, such as Histoplasma capsulatum, Brucella, Candida albicans, and species of Aspergillus (A. flavus, A. fumigatus, A. oryzae, A. terreus, A. niger) known to cause infections or produce mycotoxins, posing substantial risk to both artefacts and humans.
Topics: Humans; Aspergillus; Bacteria; Candida albicans; Mycotoxins; Italy
PubMed: 38523163
DOI: 10.1038/s41598-024-57228-2 -
Open Forum Infectious Diseases Mar 2024Invasive fungal disease caused by dimorphic fungi is associated with significant morbidity and mortality. Super-bioavailability itraconazole (SUBA-itra) is a novel...
BACKGROUND
Invasive fungal disease caused by dimorphic fungi is associated with significant morbidity and mortality. Super-bioavailability itraconazole (SUBA-itra) is a novel antifungal agent with pharmacokinetic advantages over currently available formulations. In this prospective comparative study, we report the outcomes of patients with endemic fungal infections (histoplasmosis, blastomycosis, coccidioidomycosis, and sporotrichosis).
METHODS
This open-label randomized trial evaluated the efficacy, safety, and pharmacokinetics SUBA-itra compared with conventional itraconazole (c-itra) treatment for endemic fungal infections. An independent data review committee determined responses on treatment days 42 and 180.
RESULTS
Eighty-eight patients were enrolled for IFD (SUBA-itra, n = 42; c-itra, n = 46) caused by (n = 51), (n = 18), (n = 13), or (n = 6). On day 42, clinical success was observed with SUBA-itra and c-itra on day 42 (in 69% and 67%, respectively, and on day 180 (in 60% and 65%). Patients treated with SUBA-itra exhibited less drug-level variability at days 7 ( = .03) and 14 ( = .06) of randomized treatment. The concentrations of itraconazole and hydroxyitraconazole were comparable between the 2 medications ( = .77 and = .80, respectively). There was a trend for fewer adverse events (AEs; 74% vs 87%, respectively; = .18) and serious AEs (10% vs 26%; = .06) in the SUBA-itra-treated patients than in those receiving c-itra. Serious treatment-emergent AEs were less common in SUBA-itra-treated patients (12% vs 50%, respectively; < .001).
CONCLUSIONS
SUBA-itra was bioequivalent, well tolerated, and efficacious in treating endemic fungi, with a more favorable safety profile than c-itra.
CLINICAL TRIALS REGISTRATION
NCT03572049.
PubMed: 38440302
DOI: 10.1093/ofid/ofae010 -
Journal of Feline Medicine and Surgery Jun 2024The aim of the present study was to evaluate minimally invasive diagnostic techniques, such as the semi-quantitative indirect IgG antibody enzyme immunoassay (EIA) using...
OBJECTIVES
The aim of the present study was to evaluate minimally invasive diagnostic techniques, such as the semi-quantitative indirect IgG antibody enzyme immunoassay (EIA) using blood serum and the urinary lateral flow assay (LFA), for the detection of in cats with histoplasmosis.
METHODS
Eight client-owned domestic cats diagnosed with histoplasmosis were selected based on cytological, histopathological, mycological, molecular or antigenic techniques. The blood serum of these animals was tested in a semi-quantitative indirect IgG antibody EIA for the detection of . Urine samples were tested for antigen using LFA.
RESULTS
Five cats were seropositive on IgG EIA (5/8, with diagnostic sensitivity equal to 62.5%; 95% confidence interval [CI] 24.5-91.5) and five cats were positive on antigen LFA (5/7, with diagnostic sensitivity equal to 71.4%; 95% CI 29.0-96.3). The combined diagnostic sensitivity when interpreted in parallel was 87.5% (7/8, 95% CI 47.3-99.7). The specificity for the anti- IgG EIA was 100% (95% CI 71.5-100) and for the antigen LFA it was also 100% (95% CI 71.5-100).
CONCLUSIONS AND RELEVANCE
The semi-quantitative indirect IgG antibody EIA for the detection of in blood serum and the urinary LFA for the detection of the same agent emerge as new minimally invasive diagnostic techniques that can assist in the approach to disseminated and pulmonary feline histoplasmosis, especially when both techniques are considered together.
Topics: Cats; Animals; Histoplasmosis; Cat Diseases; Histoplasma; Sensitivity and Specificity; Male; Female; Antibodies, Fungal; Immunoenzyme Techniques; Immunoglobulin G
PubMed: 38857445
DOI: 10.1177/1098612X241248984