Transcriptional regulatory features associated with Coccidioides immitis phase transition

Coccidioidomycosis (Valley Fever) is an emerging endemic fungal infection with a rising incidence and an expanding geographic range. It is caused by Coccidiodes, which are thermally dimorphic fungi that grow as mycelia in soil but transition in the lung to form pathogenic spherules. The regulatory mechanisms underlying this transition are not understood. Exploiting capped small (cs)RNA-seq, which identifies actively initiated stable and unstable transcripts and thereby detects acute changes in gene regulation with remarkable sensitivity, here we report the changes in architectural organization and key sequence features underlying phase transition of this highly pathogenic fungus. Spherule transition was accompanied by large-scale transcriptional reprogramming, functional changes in transcript isoforms, and a massive increase in promoter-distal transcription of ncRNAs. Analysis of spherule-activated regulatory elements revealed a motif predicted to recruit a WOPR family transcription factor, which are known regulators of virulence in other fungi. We identify CIMG_02671 as a C. immitis WOPR homologue and show that it activates transcription in a WOPR motif-dependent manner, suggesting it is an important regulator of pathogenic phase transition. Collectively, this also highlights csRNA-seq as a powerful means to identify transcriptional mechanisms that control pathogenesis.

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The Known Unknowns of the Immune Response to Coccidioides

Journal of Fungi ◽

10.3390/jof7050377 ◽

2021 ◽

Vol 7 (5) ◽

pp. 377

Author(s):

Rebecca A. Ward ◽

George R. Thompson ◽

Alexandra-Chloé Villani ◽

Bo Li ◽

Michael K. Mansour ◽

...

Keyword(s):

Immune Responses ◽

Pulmonary Infection ◽

Prolonged Treatment ◽

Coccidioides Immitis ◽

Dimorphic Fungi ◽

Valley Fever ◽

Adaptive Immune ◽

Immunological Mechanisms ◽

Known Unknowns ◽

Insight Into

Coccidioidomycosis, otherwise known as Valley Fever, is caused by the dimorphic fungi Coccidioides immitis and C. posadasii. While most clinical cases present with self-limiting pulmonary infection, dissemination of Coccidioides spp. results in prolonged treatment and portends higher mortality rates. While the structure, genome, and niches for Coccidioides have provided some insight into the pathogenesis of disease, the underlying immunological mechanisms of clearance or inability to contain the infection in the lung are poorly understood. This review focuses on the known innate and adaptive immune responses to Coccidioides and highlights three important areas of uncertainty and potential approaches to address them. Closing these gaps in knowledge may enable new preventative and therapeutic strategies to be pursued.

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Lifecycle dominates the volatilome character of the dimorphic fungus Coccidioides spp

10.1101/2021.01.15.426916 ◽

2021 ◽

Author(s):

Emily A. Higgins Keppler ◽

Heather L. Mead ◽

Bridget M. Barker ◽

Heather D. Bean

Keyword(s):

Solid Phase ◽

Initial Study ◽

Fungal Species ◽

Diagnostic Tools ◽

Coccidioides Immitis ◽

Dimorphic Fungus ◽

Dimorphic Fungi ◽

Valley Fever ◽

The North

ABSTRACTValley fever (coccidioidomycosis) is an endemic fungal pneumonia of the North and South American deserts. The causative agents of Valley fever are the dimorphic fungi Coccidioides immitis and C. posadasii, which grow as mycelia in the environment and spherules within the lungs of vulnerable hosts. The current diagnostics for Valley fever are severely lacking due to poor sensitivity and invasiveness, contributing to a 23-day median time-to-diagnosis, and therefore new diagnostic tools are needed. We are working toward the development of a breath-based diagnostic for coccidioidomycosis, and in this initial study we characterized the volatile metabolomes (or volatilomes) of in vitro cultures of Coccidioides. Using solid-phase microextraction and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC–TOFMS), we characterized the VOCs produced by six strains of each species during mycelial or spherule growth. We detected a total of 353 VOCs that were at least two-fold more abundant in a Coccidioides culture versus medium controls and found the volatile metabolome of Coccidioides is more dependent on growth phase (spherule versus mycelia) than on the species. The volatile profiles of C. immitis and C. posadasii have strong similarities, indicating that a single suite of Valley fever breath biomarkers can be developed to detect both species.IMPORTANCECoccidioidomycosis, or Valley fever, causes up to 30% of community-acquired pneumonias in endemic and highly populated areas of the US desert southwest. The infection is difficult to diagnose by standard serological and histopathological methods, which delays an appropriate treatment. Therefore, we are working toward the development of breath-based diagnostics for Valley fever. In this study, we characterized the volatile metabolomes of six strains each of Coccidioides immitis and C. posadasii, the dimorphic fungal species that cause Valley fever. By analyzing the volatilomes during the two modes of growth for the fungus—mycelia and spherules—we observed that the lifecycle plays a significant role in the volatiles produced by Coccidioides. In contrast, we observed no significant differences in the C. immitis versus C. posadasii volatilomes. These data suggest that lifecycle, rather than species, should guide the selection of putative biomarkers for a Valley fever breath test.

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Coccidioides immitisCervical Lymphadenitis Complicated by Esophageal Fistula

Case Reports in Infectious Diseases ◽

10.1155/2016/8715405 ◽

2016 ◽

Vol 2016 ◽

pp. 1-4

Author(s):

Michael Loudin ◽

Daniel R. Clayburgh ◽

Morgan Hakki

Keyword(s):

Lymphatic Spread ◽

Fistula Formation ◽

Esophageal Fistula ◽

Coccidioides Immitis ◽

Coccidioides Posadasii ◽

Dimorphic Fungi ◽

Valley Fever ◽

Exposure History ◽

Extrapulmonary Manifestation ◽

Mediastinal Extension

Coccidioidomycosis (valley fever) is caused by the dimorphic fungiCoccidioides immitisorCoccidioides posadasii. Most infections are asymptomatic or result in self-limited pneumonia; extrapulmonary dissemination via either hematogenous or lymphatic spread is rare. Here, we present a case of cervicalC. immitislymphadenitis that resulted in fistula formation to the esophagus via mediastinal extension. This case highlights a very unusual extrapulmonary manifestation of coccidioidomycosis, the difficulty in diagnosing coccidioidal infection when it is not suspected, and the importance of obtaining a thorough exposure history to assist with diagnosis.

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Regulation of Proto-Oncogenes and Salivary Gland Cell Proliferation

Advances in Dental Research ◽

10.1177/08959374900040010901 ◽

1990 ◽

Vol 4 (1) ◽

pp. 61-68 ◽

Cited By ~ 7

Author(s):

E. Kousvelari ◽

C.-K. Yeh ◽

P.M. Mertz ◽

M. Chinchetru

Keyword(s):

Cell Proliferation ◽

Adrenergic Receptor ◽

Regulatory Elements ◽

Salivary Gland Cell ◽

Dependent Manner ◽

Parotid Glands ◽

Receptor Stimulation ◽

Functional Changes ◽

Rat Parotid

The proto-oncogenes c-fos and c-jun express proteins targeted into the nucleus. The fos and jun proteins form a heterodimeric complex that binds to regulatory elements in the promoter region of specific genes to influence their transcription. Through such a mechanism, the fos and jun proteins have been suggested to link extracellular stimuli to short- and long-term functional changes in cells. Recently we have shown that β-adrenergic receptor stimulation of rat parotid acinar cells in vitro or addition of 8-BrcAMP in a rat submandibular cell line (RSMT-A5) increases the expression of the c-fos gene in a time-dependent manner. Maximal responses were found at 60 min. The expression of the c-fos gene did not correlate with DNA synthesis in either cell type, and c-fos transcripts were undetectable in the glands of animals treated for eight days with isoproterenol. The new data presented here extended our observations to c-jun gene expression in both salivary cell types where a similar pattern of expression for this proto-oncogene was seen. Conversely, treatment of rats with isoproterenol for nine days resulted in the appearance of two c-abl mRNAs of unique size, in addition to the known 5.3-kb c-abl transcripts. The data suggest that β-adrenergic receptor stimulation or exposure to 8-BrcAMP induces the early expression of the "nuclear proto-oncogenes" c-fos and c-jun before changes are noted in salivary epithelial cell proliferation. Differences in c-abl mRNA size, occurring later, may be associated with the morphological and biochemical changes known to occur in rat parotid glands after chronic β-adrenoreceptor stimulation.

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Dectin-1 Is Required for Resistance to Coccidioidomycosis in Mice

mBio ◽

10.1128/mbio.00597-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 42

Author(s):

Suganya Viriyakosol ◽

Maria del Pilar Jimenez ◽

Michael A. Gurney ◽

Mark E. Ashbaugh ◽

Joshua Fierer

Keyword(s):

Immune Response ◽

Pathogenic Fungus ◽

Lavage Fluid ◽

Dependent Manner ◽

Coccidioides Immitis ◽

Necrosis Factor Alpha ◽

Cell Immunity ◽

Th17 Cytokines

ABSTRACTWe assessed the role of Dectin-1 in the immune response to the pathogenic fungusCoccidioides, bothin vitroandin vivo, using mice with a targeted mutation inClec7a. Elicited peritoneal macrophages responded to formalin-killed spherules (FKS) and alkali-treated FKS by secreting proinflammatory cytokines in a Dectin-1- and β-glucan-dependent manner. The responses of bone marrow-derived dendritic cells (BMDC) to the same stimulants were more complex; interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) secretion was independent of Dectin-1, while IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were largely but not entirely dependent on Dectin-1. After intranasal infection, Dectin-1−/−mice had lower concentrations of IL-12p70, gamma interferon (IFN-γ), IL-1β, and the Th17 cytokines IL-22, IL-23, and 17A in the lung lavage fluid, which may explain why they were significantly more susceptible to pulmonary coccidioidomycosis two weeks after infection. The Dectin-1 mutation was even more deleterious in (B6 × DBA/2)F2mice, which are more resistant to coccidioidomycosis than B6 mice by virtue of protective genes from DBA/2, a genetically resistant strain. We also found that two susceptible strains of mice (B6 and BALB/c) expressed much less Dectin-1 in their lungs than did resistant DBA/2 mice. We conclude that Dectin-1 is necessary for resistance toCoccidioides immitis, that Dectin-1 promotes both Th1 and Th17 protective immune responses to this infection, and that there is a correlation between expression of Dectin-1 by the inflammatory infiltrate and resistance to coccidioidomycosis.IMPORTANCECoccidioidomycosis is a fungal infection endemic in the southwestern United States and neighboring Mexico, causing ~150,000 lung infections in people and resulting in ~17,000 hospitalizations annually in California alone. Very little is known about innate immunity to this fungus. This paper shows that Dectin-1, the primary β-glucan receptor on myeloid cells, is required for resistance to this pathogen. Dectin-1 is part of the innate immune system, and it is needed to direct the acquired immune response toward into a pathway that will lead to macrophage activation. Lungs from infected mice lacking Dectin-1 had lower concentrations of Th1 and Th17 cytokines, two cytokine pathways that are very important for acquired T cell immunity toCoccidioidesspp. This is the first demonstration that Dectin-1 is required for host resistance to a dimorphic, primary pathogenic fungus.

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Life Cycle Dominates the Volatilome Character of Dimorphic Fungus Coccidioides spp.

mSphere ◽

10.1128/msphere.00040-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Emily A. Higgins Keppler ◽

Heather L. Mead ◽

Bridget M. Barker ◽

Heather D. Bean

Keyword(s):

Life Cycle ◽

Solid Phase ◽

Initial Study ◽

Diagnostic Tools ◽

Coccidioides Immitis ◽

Dimorphic Fungus ◽

Dimorphic Fungi ◽

Valley Fever ◽

Content Type

ABSTRACT Valley fever (coccidioidomycosis) is an endemic fungal pneumonia of the North and South American deserts. The causative agents of Valley fever are the dimorphic fungi Coccidioides immitis and C. posadasii, which grow as mycelia in the environment and as spherules within the lungs of vulnerable hosts. Current diagnostics for Valley fever are severely lacking due to poor sensitivity and invasiveness, contributing to a 23-day median time to diagnosis, and therefore, new diagnostic tools are needed. We are working toward the development of a breath-based diagnostic for coccidioidomycosis, and in this initial study, we characterized the volatile metabolomes (or volatilomes) of in vitro cultures of Coccidioides. Using solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS), we characterized the volatile organic compounds (VOCs) produced by six strains of each species during mycelial or spherule growth. We detected a total of 353 VOCs that were at least 2-fold more abundant in a Coccidioides culture than in medium controls and found that the volatile metabolome of Coccidioides is more dependent on the growth phase (spherules versus mycelia) than on the species. The volatile profiles of C. immitis and C. posadasii have strong similarities, indicating that a single suite of Valley fever breath biomarkers can be developed to detect both species. IMPORTANCE Coccidioidomycosis, or Valley fever, causes up to 30% of community-acquired pneumonias in highly populated areas of the U.S. desert southwest where the disease is endemic. The infection is difficult to diagnose by standard serological and histopathological methods, which delays appropriate treatment. Therefore, we are working toward the development of breath-based diagnostics for Valley fever. In this study, we characterized the volatile metabolomes (or volatilomes) of six strains each of Coccidioides immitis and C. posadasii, the dimorphic fungal species that cause Valley fever. By analyzing the volatilomes during the two modes of growth of the fungus—mycelia and spherules—we observed that the life cycle plays a significant role in the volatiles produced by Coccidioides. In contrast, we observed no significant differences in the C. immitis versus C. posadasii volatilomes. These data suggest that life cycle, rather than species, should guide the selection of putative biomarkers for a Valley fever breath test.

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The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657715 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1202-1208 ◽

Cited By ~ 18

Author(s):

Marianne Kjalke ◽

Julie A Oliver ◽

Dougald M Monroe ◽

Maureane Hoffman ◽

Mirella Ezban ◽

...

Keyword(s):

Tissue Factor ◽

Active Site ◽

Large Scale ◽

Thrombin Generation ◽

Factor Viia ◽

Dependent Manner ◽

Antithrombotic Agent ◽

Before And After ◽

Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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Atorvastatin impairs liver mitochondrial function in obese Göttingen Minipigs but heart and skeletal muscle are not affected

Scientific Reports ◽

10.1038/s41598-021-81846-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Liselotte Bruun Christiansen ◽

Tine Lovsø Dohlmann ◽

Trine Pagh Ludvigsen ◽

Ewa Parfieniuk ◽

Michal Ciborowski ◽

...

Keyword(s):

Skeletal Muscle ◽

Mitochondrial Function ◽

Citrate Synthase ◽

Obese Group ◽

Control Group ◽

Dependent Manner ◽

Respiratory Capacity ◽

Protein Carbonyl Content ◽

Functional Changes ◽

Mitochondrial Respiratory

AbstractStatins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.

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Potentiating Effect of Mandelate and Lactate on Chemically Induced Germination in Members of Bacillus cereus Sensu Lato

Applied and Environmental Microbiology ◽

10.1128/aem.01722-17 ◽

2017 ◽

Vol 83 (24) ◽

Author(s):

Alistair H. Bishop

Keyword(s):

Bacillus Cereus ◽

Large Scale ◽

Germination Rate ◽

Dependent Manner ◽

Clostridium Sporogenes ◽

Specific Chemical ◽

Content Type ◽

Ph Range ◽

Significant Discovery ◽

The Impact

ABSTRACT Endospores of the genus Bacillus can be triggered to germinate by a limited number of chemicals. Mandelate had powerful additive effects on the levels and rates of germination produced in non-heat-shocked spores of Bacillus anthracis strain Sterne, Bacillus cereus, and Bacillus thuringiensis when combined with l-alanine and inosine. Mandelate had no germinant effect on its own but was active with these germinants in a dose-dependent manner at concentrations higher than 0.5 mM. The maximum rate and extent of germination were produced in B. anthracis by 100 mM l-alanine with 10 mM inosine; this was equaled by just 25% of these germinants when supplemented with 10 mM mandelate. Half the maximal germination rate was produced by 40% of the optimum germinant concentrations or 15% of them when supplemented with 0.8 mM mandelate. Germination rates in B. thuringiensis were highest around neutrality, but the potentiating effect of mandelate was maintained over a wider pH range than was germination with l-alanine and inosine alone. For all species, lactate also promoted germination in the presence of l-alanine and inosine; this was further increased by mandelate. Ammonium ions also enhanced l-alanine- and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant. Mandelate appeared to bind to spores while enhancing germination. There was no effect when mandelate was used in conjunction with nonnutrient germinants. No effect was produced with spores of Bacillus subtilis, Clostridium sporogenes, or C. difficile. IMPORTANCE The number of chemicals that can induce germination in the species related to Bacillus cereus has been defined for many years, and they conform to specific chemical types. Although not a germinant itself, mandelate has a structure that is different from these germination-active compounds, and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of B. cereus in foodborne disease and B. anthracis as a threat agent. The destruction of spores of B. anthracis, for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of l-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of “germinate to decontaminate” strategy may thus become more practicable.

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A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker

Nature Communications ◽

10.1038/s41467-021-23980-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yavor K. Bozhilov ◽

Damien J. Downes ◽

Jelena Telenius ◽

A. Marieke Oudelaar ◽

Emmanuel N. Olivier ◽

...

Keyword(s):

Gene Expression ◽

Genetic Diseases ◽

Regulatory Elements ◽

Base Change ◽

Dependent Manner ◽

Globin Genes ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Super Enhancer ◽

Single Base Change

AbstractMany single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.

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