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1.
JCI Insight ; 7(22)2022 11 22.
Article in English | MEDLINE | ID: mdl-36166305

ABSTRACT

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, less than 1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled patients with DCM and performed whole-exome sequencing. In an exploratory set of 67 patients with DCM, 2 had haploinsufficient STAT3 mutations, and defects in ß-glucan sensing and response were seen in 34 of 67 cases. Damaging CLEC7A and PLCG2 variants were associated with impaired production of ß-glucan-stimulated TNF-α from PBMCs compared with healthy controls. Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were overrepresented in DCM, including CLEC7A Y238* and PLCG2 R268W. A validation cohort of 111 patients with DCM confirmed the PLCG2 R268W, CLEC7A I223S, and CLEC7A Y238* variants. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived hydrogen peroxide [H2O2] in transfected cells. Heterozygous DUOX1 or DUOXA1 variants that impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired ß-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.


Subject(s)
Coccidioidomycosis , beta-Glucans , Humans , Tumor Necrosis Factor-alpha/genetics , Hydrogen Peroxide , Coccidioidomycosis/genetics , Coccidioidomycosis/epidemiology , Coccidioidomycosis/microbiology , Coccidioides/genetics
2.
Immunohorizons ; 6(2): 130-143, 2022 02 11.
Article in English | MEDLINE | ID: mdl-35149520

ABSTRACT

STAT4 plays a critical role in the generation of both innate and adaptive immune responses. In the absence of STAT4, Th1 responses, critical for resistance to fungal disease, do not occur. Infection with the dimorphic fungus, Coccidioides, is a major cause of community-acquired pneumonia in the endemic regions of Arizona and California. In some people and often for unknown reasons, coccidioidal infection results in hematogenous dissemination and progressive disease rather than the typical self-limited pneumonia. Members of three generations in a family developed disseminated coccidioidomycosis, prompting genetic investigation. All affected family members had a single heterozygous base change in STAT4, c.1877A>G, causing substitution of glycine for glutamate at AA626 (STAT4E626G/+ ). A knockin mouse, heterozygous for the substitution, developed more severe experimental coccidioidomycosis than did wild-type mice. Stat4E626G/+ T cells were deficient in production of IFN-γ after anti-CD3/CD28 stimulation. Spleen cells from Stat4E626G mice showed defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early postinfection, mutant Stat4E626G/+ mice failed to produce IFN-γ and related cytokines in the lung and to accumulate activated adaptive immune cells in mediastinal lymph nodes. Therefore, defective early induction of IFN-γ and adaptive responses by STAT4 prevents normal control of coccidioidomycosis in both mice and humans.


Subject(s)
Coccidioidomycosis , STAT4 Transcription Factor , Animals , Coccidioidomycosis/genetics , Genetic Predisposition to Disease , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Point Mutation , STAT4 Transcription Factor/genetics
3.
G3 (Bethesda) ; 12(4)2022 04 04.
Article in English | MEDLINE | ID: mdl-35137016

ABSTRACT

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.


Subject(s)
Coccidioides , Coccidioidomycosis , Base Sequence , Coccidioides/genetics , Coccidioidomycosis/diagnosis , Coccidioidomycosis/epidemiology , Coccidioidomycosis/genetics , Humans , Phylogeny
4.
G3 (Bethesda) ; 11(7)2021 07 14.
Article in English | MEDLINE | ID: mdl-33871031

ABSTRACT

Fungal mitochondrial genomes encode genes involved in crucial cellular processes, such as oxidative phosphorylation and mitochondrial translation, and the molecule has been used as a molecular marker for population genetics studies. Coccidioides immitis and C. posadasii are endemic fungal pathogens that cause coccidioidomycosis in arid regions across both American continents. To date, approximately 150 Coccidioides isolates have been sequenced to infer patterns of variation in nuclear genomes. However, less attention has been given to the mitochondrial genomes of Coccidioides. In this report, we describe the assembly and annotation of mitochondrial reference genomes for two representative strains of C. posadasii and C. immitis, as well as assess population variation among 77 selected genomes. The sizes of the circular-mapping molecules are 68.2 Kb in C. immitis and 75.1 Kb in C. posadasii. We identify 14 mitochondrial protein-coding genes common to most fungal mitochondria, which are largely syntenic across different populations and species of Coccidioides. Both Coccidioides species are characterized by a large number of group I and II introns, harboring twice the number of elements as compared to closely related Onygenales. The introns contain complete or truncated ORFs with high similarity to homing endonucleases of the LAGLIDADG and GIY-YIG families. Phylogenetic comparisons of mitochondrial and nuclear genomes show extensive phylogenetic discordance suggesting that the evolution of the two types of genetic material is not identical. This work represents the first assessment of mitochondrial genomes among isolates of both species of Coccidioides, and provides a foundation for future functional work.


Subject(s)
Coccidioidomycosis , Genome, Mitochondrial , Humans , Coccidioides/genetics , Phylogeny , Coccidioidomycosis/epidemiology , Coccidioidomycosis/genetics , Coccidioidomycosis/microbiology
5.
Respir Res ; 21(1): 321, 2020 Dec 04.
Article in English | MEDLINE | ID: mdl-33276795

ABSTRACT

RATIONALE: Despite the availability of multi-"omics" strategies, insights into the etiology and pathogenesis of sarcoidosis have been elusive. This is partly due to the lack of reliable preclinical models and a paucity of validated biomarkers. As granulomas are a key feature of sarcoidosis, we speculate that direct genomic interrogation of sarcoid tissues, may lead to identification of dysregulated gene pathways or biomarker signatures. OBJECTIVE: To facilitate the development sarcoidosis genomic biomarkers by gene expression profiling of sarcoidosis granulomas in lung and lymph node tissues (most commonly affected organs) and comparison to infectious granulomas (coccidiodomycosis and tuberculosis). METHODS: Transcriptomic profiles of immune-related gene from micro-dissected sarcoidosis granulomas within lung and mediastinal lymph node tissues and compared to infectious granulomas from paraffin-embedded blocks. Differentially-expressed genes (DEGs) were profiled, compared among the three granulomatous diseases and analyzed for functional enrichment pathways. RESULTS: Despite histologic similarities, DEGs and pathway enrichment markedly differed in sarcoidosis granulomas from lymph nodes and lung. Lymph nodes showed a clear immunological response, whereas a structural regenerative response was observed in lung. Sarcoidosis granuloma gene expression data corroborated previously reported genomic biomarkers (STAB1, HBEGF, and NOTCH4), excluded others and identified new genomic markers present in lung and lymph nodes, ADAMTS1, NPR1 and CXCL2. Comparisons between sarcoidosis and pathogen granulomas identified pathway divergences and commonalities at gene expression level. CONCLUSION: These findings suggest the importance of tissue and disease-specificity evaluation when exploring sarcoidosis genomic markers. This relevant translational information in sarcoidosis and other two histopathological similar infections provides meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis.


Subject(s)
Coccidioidomycosis/genetics , Gene Expression Profiling , Granuloma/genetics , Lymphatic Diseases/genetics , Sarcoidosis, Pulmonary/genetics , Transcriptome , Tuberculosis/genetics , Adult , Aged , Coccidioidomycosis/diagnosis , Coccidioidomycosis/immunology , Coccidioidomycosis/microbiology , Diagnosis, Differential , Female , Genetic Markers , Granuloma/diagnosis , Granuloma/immunology , Granuloma/microbiology , Humans , Lymphatic Diseases/diagnosis , Lymphatic Diseases/immunology , Male , Middle Aged , Sarcoidosis, Pulmonary/diagnosis , Sarcoidosis, Pulmonary/immunology , Tuberculosis/diagnosis , Tuberculosis/immunology , Tuberculosis/microbiology , Young Adult
6.
Methods Mol Biol ; 1625: 45-64, 2017.
Article in English | MEDLINE | ID: mdl-28584982

ABSTRACT

Coccidioidomycosis is a potentially life-threatening mycosis endemic to the Southwestern USA and some arid regions of Central and South America. A vaccine against Coccidioides infection would benefit over 30-million people who reside in or visit the endemic regions. Vaccine candidates against systemic fungal infections come in many forms. Live attenuated vaccines are derived from disease-causing pathogens and generally stimulate excellent protective immunity. Since attenuated vaccines contain living microbes, there is a degree of unpredictability raising concerns regarding safety and stability. Generation of a subunit vaccine has initiated efforts to design a safe reagent suitable for administration to humans at risk of coccidioidomycosis. Epitope-based vaccines allow for eliciting specific protective immune responses and removal of potentially detrimental sequences to improve safety. This chapter describes methods for the identification of T cell epitopes derived from Coccidioides antigens, design, and production of a recombinant vaccine containing multiple T cell epitopes, and evaluation of its protective efficacy and vaccine immunity against pulmonary Coccidioides infection using a strain of transgenic mice that express a human MHC II molecule.


Subject(s)
Coccidioides/immunology , Coccidioidomycosis/immunology , Epitopes, T-Lymphocyte/immunology , Fungal Vaccines/immunology , Vaccines, Subunit/immunology , Amino Acid Sequence , Animals , Antigens, Fungal/chemistry , Antigens, Fungal/immunology , Coccidioides/drug effects , Coccidioidomycosis/genetics , Coccidioidomycosis/prevention & control , Computational Biology/methods , Cytokines/metabolism , Databases, Genetic , Disease Models, Animal , Enzyme-Linked Immunospot Assay , Epitopes, T-Lymphocyte/chemistry , Formaldehyde/pharmacology , HLA-DR4 Antigen/genetics , Humans , Mice , Mice, Transgenic , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Vaccine Potency , Vaccines, Inactivated/immunology , Web Browser
7.
Infect Immun ; 84(10): 3007-16, 2016 10.
Article in English | MEDLINE | ID: mdl-27481239

ABSTRACT

The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of <1,000 CFU from an otherwise lethal C. posadasii intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to C. posadasii infection when used as a vaccine, the Δcps1 strain is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals.


Subject(s)
Coccidioides/physiology , Coccidioidomycosis/genetics , Sequence Deletion , Virulence Factors/genetics , Virulence/physiology , Animals , Coccidioides/genetics , Coccidioidomycosis/prevention & control , Disease Models, Animal , Female , Fungal Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Vaccination/methods
8.
Rev Iberoam Micol ; 31(1): 49-53, 2014.
Article in English | MEDLINE | ID: mdl-24270069

ABSTRACT

The prevalence of coccidioidomycosis in endemic areas has been observed to increase daily. To understand the causes of the spread of the disease and design strategies for fungal detection in clinical and environmental samples, scientists have resorted to molecular tools that allow fungal detection in a natural environment, reliable identification in clinical cases and the study of biological characteristics, such as reproductive and genetic structure, demographic history and diversification. We conducted a review of the most important molecular markers in the epidemiology of Coccidioides spp. and the diagnosis of coccidioidomycosis. A literature search was performed for scientific publications concerning the application of molecular tools for the epidemiology and diagnosis of coccidioidomycosis. The use of molecular markers in the epidemiological study and diagnosis of coccidioidomycosis has allowed for the typing of Coccidioides spp. isolates, improved understanding of their mode of reproduction, genetic variation and speciation and resulted in the development specific, rapid and sensitive strategies for detecting the fungus in environmental and clinical samples. Molecular markers have revealed genetic variability in Coccidioides spp. This finding influences changes in the epidemiology of coccidioidomycosis, such as the emergence of more virulent or antifungal resistant genotypes. Furthermore, the molecular markers currently used to identify Coccidioides immitis and Coccidioides posadasii are specific and sensitive. However, they must be validated to determine their application in diagnosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).


Subject(s)
Coccidioidomycosis/diagnosis , Molecular Diagnostic Techniques , Mycology/methods , Biomarkers , California/epidemiology , Coccidioides/classification , Coccidioides/genetics , Coccidioides/isolation & purification , Coccidioidomycosis/epidemiology , Coccidioidomycosis/genetics , Coccidioidomycosis/microbiology , DNA, Fungal/genetics , Endemic Diseases , Genetic Variation , Humans , Latin America/epidemiology , Mycological Typing Techniques/methods , Reproduction , Species Specificity
9.
J Allergy Clin Immunol ; 131(6): 1624-34, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23541320

ABSTRACT

BACKGROUND: Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. OBJECTIVE: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. METHODS: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. RESULTS: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation. CONCLUSION: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation.


Subject(s)
Coccidioidomycosis/genetics , Histoplasmosis/genetics , Mutation , STAT1 Transcription Factor/genetics , Adolescent , Adult , Cell Line, Transformed , Child , Coccidioidomycosis/diagnosis , Coccidioidomycosis/immunology , Cytokines/biosynthesis , Female , Gene Expression Regulation , Histoplasmosis/diagnosis , Histoplasmosis/immunology , Humans , Male , Phosphorylation , Protein Inhibitors of Activated STAT/metabolism , STAT1 Transcription Factor/metabolism , Th17 Cells/immunology , Transcriptional Activation , Young Adult
10.
BMC Microbiol ; 12: 218, 2012 Sep 24.
Article in English | MEDLINE | ID: mdl-23006927

ABSTRACT

BACKGROUND: Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2) or susceptible (e.g. C57BL/6) to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized. RESULTS: Microarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG) and the signal transducer and activator of transcription 1 (STAT1) contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A), possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA), may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection. CONCLUSION: These results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection.


Subject(s)
Coccidioides/immunology , Coccidioidomycosis/genetics , Coccidioidomycosis/immunology , Host-Pathogen Interactions , Hypoxia-Inducible Factor 1, alpha Subunit/immunology , Interferon-gamma/immunology , Animals , Coccidioides/pathogenicity , Disease Models, Animal , Gene Expression Profiling , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Microarray Analysis
11.
J Immunol ; 187(3): 1421-31, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21705621

ABSTRACT

Dimorphic fungi collectively account for 5-10 million new infections annually worldwide. Ongoing efforts seek to clarify mechanisms of cellular resistance to these agents and develop vaccines. A major limitation in studying the development of protective T cells in this group of organisms is the lack of tools to detect, enumerate, and characterize fungus-specific T cells during vaccination and infection. We generated a TCR transgenic mouse (Bd 1807) whose CD4(+) T cells respond to a native epitope in Blastomyces dermatitidis and also in Histoplasma capsulatum. In this study, we characterize the mouse, reveal its applications, and extend our analysis showing that 1807 cells also respond to the related dimorphic fungi Coccidioides posadasii and Paracoccidioides lutzii. On adoptive transfer into vaccinated wild-type mice, 1807 cells become activated, proliferate, and expand in the draining lymph nodes, and they differentiate into T1 effectors after trafficking to the lung upon lethal experimental challenge. Bd 1807 cells confer vaccine-induced resistance against B. dermatitidis, H. capsulatum, and C. posadasii. Transfer of naive 1807 cells at serial intervals postvaccination uncovered the prolonged duration of fungal Ag presentation. Using 1807 cells, we also found that the administration of vaccine only once induced a maximal pool of effector/memory CD4(+) cells and protective immunity by 4 wk after vaccination. The autologous adoptive transfer system described in this study reveals novel features of antifungal immunity and offers a powerful approach to study the differentiation of Ag-specific T cells responsive to multiple dimorphic fungi and the development of CD4(+) T cell memory needed to protect against fungal infection.


Subject(s)
Blastomyces/genetics , Blastomyces/immunology , Fungal Vaccines/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Coccidioidomycosis/genetics , Coccidioidomycosis/immunology , Coccidioidomycosis/pathology , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Fungal Vaccines/administration & dosage , Fungal Vaccines/genetics , Immunodominant Epitopes/administration & dosage , Immunodominant Epitopes/genetics , Immunodominant Epitopes/metabolism , Immunologic Memory/genetics , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Paracoccidioidomycosis/genetics , Paracoccidioidomycosis/immunology , Paracoccidioidomycosis/pathology
12.
PLoS Pathog ; 7(4): e1002013, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21552326

ABSTRACT

For many pathogens with environmental stages, or those carried by vectors or intermediate hosts, disease transmission is strongly influenced by pathogen, host, and vector movements across complex landscapes, and thus quantitative measures of movement rate and direction can reveal new opportunities for disease management and intervention. Genetic assignment methods are a set of powerful statistical approaches useful for establishing population membership of individuals. Recent theoretical improvements allow these techniques to be used to cost-effectively estimate the magnitude and direction of key movements in infectious disease systems, revealing important ecological and environmental features that facilitate or limit transmission. Here, we review the theory, statistical framework, and molecular markers that underlie assignment methods, and we critically examine recent applications of assignment tests in infectious disease epidemiology. Research directions that capitalize on use of the techniques are discussed, focusing on key parameters needing study for improved understanding of patterns of disease.


Subject(s)
Communicable Diseases/transmission , Disease Vectors , Host-Parasite Interactions/genetics , Administration, Oral , Animal Migration , Animals , Chagas Disease/prevention & control , Coccidioidomycosis/genetics , Coccidioidomycosis/prevention & control , Genetics, Population , Geography , Humans , Insecticides , Rabies Vaccines/administration & dosage , Raccoons/parasitology , Sciuridae/parasitology , Siphonaptera/parasitology , Triatoma , Yersinia pestis/genetics
14.
Med Mycol ; 48(3): 470-5, 2010 May.
Article in English | MEDLINE | ID: mdl-20370361

ABSTRACT

Coccidioides posadasii is one of the two fungal pathogens that cause coccidioidomycosis. The inhalation of air-borne arthroconidia leads to the formation of endospore-forming spherules in the lungs and pulmonary infection. In severe condition, the endospores are disseminated to other non-pulmonary organs in the body. The Toll-like receptors (TLR) expressed by a number of immune and non-immune cells can significantly impact the host defense and susceptibility to C. posadasii infection. In this study, we infected TLR4-defective C3H/HeJ mice with a sublethal dose of C. posadasii and studied fungal dissemination, mortality and humoral response. We also measured IL-12 cytokine secreted by C. posadasii-infected dendritic cells. We found that the C3H/HeJ mice were equally susceptible to C. posadasii as compared to C3H/OuJ mice which have intact TLR4. No significant changes were observed in pulmonary fungal load, survival and humoral response. The blockade of TLR4 did not affect C. posadasii-induced IL-12 secretion. However, the fungal counts were 10 times less in spleens of C3H/HeJ mice as compared to C3H/OuJ mice (P<0.05). Our results suggest that the TLR4 may not be involved in inducing protective host defense against C. posadasii, but it appears to be critical for fungal dissemination.


Subject(s)
Coccidioides/immunology , Coccidioidomycosis/immunology , Disease Susceptibility , Toll-Like Receptor 4/deficiency , Animals , Antibodies, Fungal/blood , Coccidioides/pathogenicity , Coccidioidomycosis/genetics , Colony Count, Microbial , Dendritic Cells/immunology , Female , Interleukin-12/metabolism , Mice , Mice, Inbred C3H , Mice, Knockout , Spleen/microbiology , Survival Analysis
16.
Mycopathologia ; 167(4): 173-80, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19083122

ABSTRACT

BACKGROUND: Mannose-binding lectin (MBL) is a circulating collectin that is part of the innate immune response. We explored the serum levels of MBL in persons with different forms of coccidioidomycosis. METHODS: Serum MBL was measured by ELISA from samples obtained from healthy donors with immunity to Coccidioides, and those with various forms of active coccidioidomycosis. Blood cell specimens from a subgroup of subjects with active coccidioidomycosis were examined for single nucleotide polymorphisms of the MBL gene and promoter regions. RESULTS: The control group comprised 29 healthy immune subjects. Patient groups with active coccidioidomycosis consisted of 20 patients with symptomatic primary pulmonary coccidioidomycosis, 26 with non-meningeal disseminated coccidioidomycosis, and nine with coccidioidal meningitis. The group with active coccidioidomycosis was significantly older and more likely to be male than the control group (for both, P < 0.001). The mean +/- SEM level of serum MBL in the healthy controls was 169.4 +/- 28.6 ng/ml, significantly higher than the 79.2 +/- 10.9 ng/ml for all active groups (P < 0.001). Moreover, the active coccidioidomycosis group was significantly more likely to have serum MBL level

Subject(s)
Coccidioidomycosis , Mannose-Binding Lectin/blood , Adult , Aged , Coccidioides/immunology , Coccidioidomycosis/ethnology , Coccidioidomycosis/genetics , Coccidioidomycosis/immunology , Coccidioidomycosis/microbiology , Female , Genetic Predisposition to Disease , Humans , Lung Diseases, Fungal/ethnology , Lung Diseases, Fungal/genetics , Lung Diseases, Fungal/immunology , Lung Diseases, Fungal/microbiology , Male , Mannose-Binding Lectin/genetics , Meningitis, Fungal/ethnology , Meningitis, Fungal/genetics , Meningitis, Fungal/immunology , Meningitis, Fungal/microbiology , Middle Aged , Polymerase Chain Reaction , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics
17.
Ann N Y Acad Sci ; 1111: 198-207, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17344520

ABSTRACT

Coccidioidomycosis is a mild to life-threatening disease in otherwise healthy humans and other mammals caused by the fungus Coccidioides spp. Understanding the development of the unique dimorphic life cycle of Coccidioides spp. and its role in pathogenesis has been an area of research focus. However, nuclear behavior during the saprobic and parasitic life cycle has not been studied intensively. In this study, green fluorescent protein (GFP) was fused to histone H1 and introduced into Coccidioides posadasii (C. posadasii) strain Silveira to monitor the nuclear behavior of the fungus during the saprobic and parasitic stages of the life cycle. We constructed an Agrobacterium tumefaciens-mediated transformation (ATMT) vector that had in its T-DNA region a hygromycin-resistance gene as well as the fused histone H1-GFP gene under the control of the histone H3 promoter of C. posadasii. More than 30 hygromycin-resistant transformants were obtained and 23 were purified to homozygosity through multiple passages of the original transformants on hygromycin-containing media. One strain (VFC1420) transformed with a single copy of the fusion histone H1-GFP gene was selected for cytological studies. Strong nuclear-localized GFP signals were observed in arthroconidia, hyphae, as well as in spherules and endospores developed in vitro. Thus GFP can be used to study the expression pattern of potential virulence genes identified in serial analysis of gene expression (SAGE) or expressed sequence tags (EST) libraries, and could be a useful tool to monitor disease development in the murine model.


Subject(s)
Cell Nucleus/metabolism , Coccidioides/metabolism , Coccidioidomycosis/genetics , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/instrumentation , Agrobacterium tumefaciens/metabolism , Coccidioidomycosis/metabolism , Expressed Sequence Tags , Genetic Techniques , Genetic Vectors , Humans , Microscopy, Fluorescence/methods , Promoter Regions, Genetic
18.
Ann N Y Acad Sci ; 1111: 236-44, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17363443

ABSTRACT

Epidemiological and clinical studies have confirmed that coccidioidomycosis is more severe in African American and Filipino patients than in Caucasians, suggesting a genetic basis for susceptibility in humans. We discovered that inbred strains of mice also vary greatly in their susceptibility to Coccidioides immitis infections, and although resistance is the dominant phenotype, it is a multigenic trait in mice. We found a strong direct correlation between susceptibility in mice and the amount of IL-10 made in response to infection. We then showed that IL-10-deficient mice are much more resistant to infection than the parent C57BL/6 strain. Finally, we showed that genetically resistant mice that are transgenic for IL-10 and so overproduce that cytokine are more susceptible to C. immitis. This is in part due to suppression of NOS2 expression by IL-10.


Subject(s)
Coccidioidomycosis/genetics , Coccidioidomycosis/microbiology , Genetic Predisposition to Disease , Interleukin-10/physiology , Animals , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Transgenic , Phenotype , Species Specificity , Time Factors , Tissue Distribution
19.
Infect Immun ; 73(10): 7011-3, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16177382

ABSTRACT

We investigated secondary immunity against coccidioidomycosis by using gene expression microarrays. Surprisingly, a high percentage of B-cell-related genes were associated with protective immunity. A functional confirmation of the importance of B cells against coccidioidomycosis was achieved by demonstrating that vaccination was not fully protective in B-cell-deficient MuMT mice.


Subject(s)
B-Lymphocytes/immunology , Coccidioidomycosis/immunology , Fungal Vaccines/immunology , Gene Expression Regulation , Animals , Coccidioidomycosis/genetics , Coccidioidomycosis/prevention & control , Female , Fungal Vaccines/therapeutic use , Gene Expression Profiling , Genes, Immunoglobulin , Immunization , Male , Mice , Mice, Mutant Strains , Oligonucleotide Array Sequence Analysis
20.
Cell Immunol ; 231(1-2): 49-55, 2004.
Article in English | MEDLINE | ID: mdl-15919369

ABSTRACT

Coccidioides posadasii is a soil fungus that causes coccidioidomycosis or Valley Fever in the endemic regions of the southwestern US and Central America. Persons with decreased T cells reactivity and immune deficiency are at increased risk of developing severe disseminated infection. Among different mouse strains, DBA/2 mice are relatively resistant to C. posadasii whereas BALB/c mice are highly susceptible, and this discrepancy has been attributed to the difference in the development and expression of their Th1 cellular response. Dendritic cells (DC) are the most potent antigen-presenting cells that are activated after taking up pathogens or pathogens-derived antigens and regulate the immune response in the host, including Th1 cellular response. However, the DC responses against C. posadasii are not characterized. In the present study, we cultured bone-marrow derived DC (BMDC) from BALB/c and DBA/2 mice and infected with C. posadasii arthroconidia. The activation of BMDC was characterized by studying expression of cell surface co-stimulatory molecules (CD11c, MHC class II, CD40, CD80, and CD86), expression of genes encoding Toll-like receptors and release of IL-12. We found that the BMDC from DBA/2 mice showed significant upregulation of Toll-like receptor-2 and 4 genes expression, secretion of IL-12 (p<0.05) and modest increase in T cell co-stimulatory molecules as compared to BMDC from BALB/c mice. The data suggest that the differences in the activation status of DC in DBA/2 and BALB/c mice may be responsible for the discrepancy in their susceptibility to C. posadasii.


Subject(s)
Bone Marrow Cells/cytology , Coccidioides/physiology , Dendritic Cells/metabolism , Disease Susceptibility , Interleukin-12/metabolism , Receptors, Immunologic/genetics , Animals , Cell Differentiation , Cells, Cultured , Coccidioides/immunology , Coccidioidomycosis/genetics , Coccidioidomycosis/immunology , Coccidioidomycosis/metabolism , Dendritic Cells/cytology , Dendritic Cells/immunology , Female , Gene Expression Regulation , Mice , Mice, Inbred DBA , Phenotype , Toll-Like Receptor 2 , Toll-Like Receptor 4
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