Single nucleotide polymorphisms and chromosomal copy number variation may impact the Sporothrix brasiliensis antifungal susceptibility and sporotrichosis clinical outcomes.

Feline-transmitted sporotrichosis has garnered attention due to the recent high incidence and the lack of efficient control in the epicenter of the epidemic, Rio de Janeiro, Brazil. Sporothrix brasiliensis is the major pathogen involved in feline-to-human sporotrichosis in Brazil and displays more virulent genotypes than the closely related species S. schenckii. Over the last two decades, several reports of antifungal-resistant strains have emerged. Sequencing and comparison analysis of the outbreak strains allowed us to observe that the azole non-wild-type S. brasiliensis strain CFP 1054 had significant chromosomal variations compared to wild-type strains. One of these variants includes a region of 231 Kb containing 75 duplicated genes, which were overrepresented for lipid and isoprenoid metabolism. We also identified an additional strain (CFP 1055) that was resistant to itraconazole and amphotericin B, which had a single nucleotide polymorphism in the tac1 gene. The patients infected with these two strains showed protracted clinical course and sequelae. Even though our sample size is modest, these results suggest the possibility of identifying specific point mutations and large chromosomal duplications potentially associated with antifungal resistance and clinical outcomes of sporotrichosis.

Genomic characterization of Parengyodontium americanum sp. nov.

Modern genome analysis and phylogenomic methods have increased the number of fungal species, as well as enhanced appreciation of the degree of diversity within the fungal kingdom. In this context, we describe a new Parengyodontium species, P. americanum, which is phylogenetically related to the opportunistic human fungal pathogen P. album. Five unusual fungal isolates were recovered from five unique and confirmed coccidioidomycosis patients, and these isolates were subsequently submitted to detailed molecular and morphological identification procedures to determine identity. Molecular and morphological diagnostic analyses showed that the isolates belong to the Cordycipitaceae. Subsequently, three representative genomes were sequenced and annotated, and a new species, P. americanum, was identified. Using various genomic analyses, gene family expansions related to novel compounds and potential for ability to grow in diverse habitats are predicted. A general description of the genomic composition of this newly described species and comparison of genome content with Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris shows a shared core genome of 6371 genes, and 148 genes that appear to be specific for P. americanum. This work provides the framework for future investigations of this interesting fungal species.

Investigating the Role of Animal Burrows on the Ecology and Distribution of Coccidioides spp. in Arizona Soils.

The lack of knowledge regarding the ecology of Coccidioides spp. makes both modeling the potential for disease outbreaks and predicting the distribution of the organism in the environment challenging. No single ecological parameter explains the biogeography of the pathogen. Previous investigations suggest an association with desert mammals, but these results should be confirmed with modern molecular techniques. Therefore, we used molecular tools to analyze soils associated with animal activity (i.e., burrows) to better define the ecology and biogeography of Coccidioides spp. in Arizona. Soils were collected from locations predicted to have favorable habitat outside of the established endemic regions to better understand the ecological niche of the organism in this state. Our central hypothesis is that soils taken from within animal burrows will have a higher abundance of Coccidioides spp. when compared to soils not directly associated with animal burrows. Our results show that there is a positive relationship with Coccidioides spp. and animal burrows. The organism was detected in two locations in northern Arizona at sites not known previously to harbor the fungus. Moreover, this fungus is able to grow on keratinized tissues (i.e., horse hair). These results provide additional evidence that there is a relationship between Coccidioides spp. and desert animals, which sheds new light on Coccidioides' ecological niche. These results also provide evidence that the geographic range of the organism may be larger than previously thought, and the concept of endemicity should be reevaluated for Coccidioides.

Coccidioidal Meningitis in New York Traced to Texas by Fungal Genomic Analysis.

A child developed hydrocephalus. Sixteen months later, it was discovered to be a complication of coccidioidal meningitis. The infection's source was uncertain until genomic analysis of the fungal isolate identified its origin to be a visit to Beeville, Texas. Improved national reporting of cases of coccidioidomycosis might reduce diagnostic delays.

The endozoan, small-mammal reservoir hypothesis and the life cycle of Coccidioides species.

The prevailing hypothesis concerning the ecology of Coccidioides immitis and C. posadasii is that these human pathogenic fungi are soil fungi endemic to hot, dry, salty regions of the New World and that humans and the local, small-mammal fauna are only accidental hosts. Here we advance an alternative hypothesis that Coccidioides spp. live in small mammals as endozoans, which are kept inactive but alive in host granulomas and which transform into spore-producing hyphae when the mammal dies. The endozoan hypothesis incorporates results from comparative genomic analyses of Coccidioides spp. and related taxa that have shown a reduction in gene families associated with deconstruction of plant cell walls and an increase in those associated with digestion of animal protein, consistent with an evolutionary shift in substrate from plants to animals. If true, the endozoan hypothesis requires that models of the prevalence of human coccidioidomycosis account not only for direct effects of climate and soil parameters on the growth and reproduction of Coccidioides spp. but also consider indirect effects on these fungi that come from the plants that support the growth and reproduction of the small mammals that, in turn, support these endozoic fungi.

Coccidioides ecology and genomics.

Although the natural history and ecology of Coccidioides spp. have been studied for over 100 years, many fundamental questions about this fungus remain unanswered. Two of the most challenging aspects of the study of Coccidioides have been the undefined ecological niche and the outdated geographic distribution maps dating from midcentury. This review details the history of Coccidioides ecological research, and discusses current strategies and advances in understanding Coccidioides genetics and ecology.

Characterizing in vitro spherule morphogenesis of multiple strains of both species of Coccidioides.

The disease San Joaquin Valley Fever (coccidioidomycosis) is caused by the inhalation of Coccidioides arthroconidia. In vivo, arthroconidia transform into pathogenic structures termed spherules. Exposure to the host milieu triggers spherule development; however, the molecular mechanisms responsible for the morphological shift are not well characterized. This study compared the morphogenesis of five strains of both species of Coccidioides in two media types to improve the in vitro model of dimorphism that can be easily reproduced, and is amenable to tissue culture. We also sought to establish a modern record of the morphological switch among commonly used lab strains through a detailed account of growth under various conditions. Spherules from five strains were grown in standard (Converse) and experimental media (RPMI-sph). Strain behavior was quantified by median spherule size and spherule concentration, beginning 3 days after inoculation and followed for 10 days of growth. There were significant differences observed among Coccidioides immitis and C. posadasii strains, as well as differences between the in vitro systems.

Large-Scale Evaluation of In Vitro Amphotericin B, Triazole, and Echinocandin Activity against Coccidioides Species from U.S. Institutions.

Large-scale testing of Coccidioides isolates has not been performed, and the frequency of clinical isolates with elevated amphotericin B or triazole MICs has not been evaluated. Coccidioides isolates (n = 581) underwent antifungal susceptibility testing. Elevated MIC values were observed for fluconazole (≥16 µg/ml, 37.3% of isolates; ≥32 µg/ml, 7.9% of isolates), itraconazole (≥2 µg/ml, 1.0% of isolates), posaconazole (≥1 µg/ml, 1.0% of isolates), and voriconazole (≥2 µg/ml, 1.2% of isolates). However, mold-active triazoles exhibited low MICs for the majority of isolates tested. Additional correlation with patient outcomes to determine the relevance of elevated MICs in Coccidioides isolates is needed.

IL-1α signaling is critical for leukocyte recruitment after pulmonary Aspergillus fumigatus challenge.

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1ß and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1ß was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1ß were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1ß in controlling A. fumigatus infection in the murine lung.

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps.

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.

Use of Population Genetics to Assess the Ecology, Evolution, and Population Structure of Coccidioides.

During the past 20 years, a general picture of the genetic diversity and population structure of Coccidioides, the causal agent of coccidioidomycosis (Valley fever), has emerged. The genus consists of 2 genetically diverse species, C. immitis and C. posadasii, each of which contains 1 or more distinct populations with limited gene flow. Genotypic data indicate that C. immitis is divided into 2 subpopulations (central and southern California populations) and C. posadasii is divided into 3 subpopulations (Arizona, Mexico, and Texas/South America populations). However, admixture within and among these populations and the current paucity of environmental isolates limit our understanding of the population genetics of Coccidioides. We assessed population structure of Coccidioides in Arizona by analyzing 495 clinical and environmental isolates. Our findings confirm the population structure as previously described and indicate a finer scale population structure in Arizona. Environmental isolates appear to have higher genetic diversity than isolates from human patients.

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

Molecular detection of airborne Coccidioides in Tucson, Arizona.

Environmental surveillance of the soil-dwelling fungus Coccidioides is essential for the prevention of Valley fever, a disease primarily caused by inhalation of the arthroconidia. Methods for collecting and detecting Coccidioides in soil samples are currently in use by several laboratories; however, a method utilizing current air sampling technologies has not been formally demonstrated for the capture of airborne arthroconidia. In this study, we collected air/dust samples at two sites (Site A and Site B) in the endemic region of Tucson, Arizona, and tested a variety of air samplers and membrane matrices. We then employed a single-tube nested qPCR assay for molecular detection. At both sites, numerous soil samples (n = 10 at Site A and n = 24 at Site B) were collected and Coccidioides was detected in two samples (20%) at Site A and in eight samples (33%) at Site B. Of the 25 air/dust samples collected at both sites using five different air sampling methods, we detected Coccidioides in three samples from site B. All three samples were collected using a high-volume sampler with glass-fiber filters. In this report, we describe these methods and propose the use of these air sampling and molecular detection strategies for environmental surveillance of Coccidioides.

Differences in Host Innate Responses among Coccidioides Isolates in a Murine Model of Pulmonary Coccidioidomycosis.

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi and the causative agents of coccidioidomycosis, a mycosis endemic to certain semiarid regions in the Americas. The most common route of infection is by inhalation of airborne Coccidioides arthroconidia. Once a susceptible host inhales the conidia, a transition to mature endosporulated spherules can occur within the first 5 days of infection. For this study, we examined the host response in a murine model of coccidioidomycosis during a time period of infection that has not been well characterized. We collected lung tissue and bronchoalveolar lavage fluid (BALF) from BALB/c mice that were infected with a C. immitis pure strain, a C. immitis hybrid strain, or a C. posadasii strain as well as uninfected mice. We compared the host responses to the Coccidioides strains used in this study by assessing the level of transcription of selected cytokine genes in lung tissues and characterized host and fungal proteins present in BALF. Host response varied depending on the Coccidioides strain that was used and did not appear to be overly robust. This study provides a foundation to begin to dissect the host immune response early in infection, to detect abundant Coccidioides proteins, and to develop diagnostics that target these early time points of infection.

Valley fever: finding new places for an old disease: Coccidioides immitis found in Washington State soil associated with recent human infection.

We used real-time polymerase chain reaction and culture to demonstrate persistent colonization of soils by Coccidioides immitis, an agent of valley fever, in Washington State linked to recent human infections and located outside the endemic range. Whole-genome sequencing confirmed genetic identity between isolates from soil and one of the case-patients.

Evaluation of VT-1161 for Treatment of Coccidioidomycosis in Murine Infection Models.

Coccidioidomycosis, or valley fever, is a growing health concern endemic to the southwestern United States. Safer, more effective, and more easily administered drugs are needed especially for severe, chronic, or unresponsive infections. The novel fungal CYP51 inhibitor VT-1161 demonstrated in vitro antifungal activity, with MIC50 and MIC90 values of 1 and 2 µg/ml, respectively, against 52 Coccidioides clinical isolates. In the initial animal study, oral doses of 10 and 50 mg/kg VT-1161 significantly reduced fungal burdens and increased survival time in a lethal respiratory model in comparison with treatment with a placebo (P < 0.001). Oral doses of 25 and 50 mg/kg VT-1161 were similarly efficacious in the murine central nervous system (CNS) model compared to placebo treatment (P < 0.001). All comparisons with the positive-control drug, fluconazole at 50 mg/kg per day, demonstrated either statistical equivalence or superiority of VT-1161. VT-1161 treatment also prevented dissemination of infection from the original inoculation site to a greater extent than fluconazole. Many of these in vivo results can be explained by the long half-life of VT-1161 leading to sustained high plasma levels. Thus, the efficacy and pharmacokinetics of VT-1161 are attractive characteristics for long-term treatment of this serious fungal infection.

SREBP coordinates iron and ergosterol homeostasis to mediate triazole drug and hypoxia responses in the human fungal pathogen Aspergillus fumigatus.

Sterol regulatory element binding proteins (SREBPs) are a class of basic helix-loop-helix transcription factors that regulate diverse cellular responses in eukaryotes. Adding to the recognized importance of SREBPs in human health, SREBPs in the human fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus are required for fungal virulence and susceptibility to triazole antifungal drugs. To date, the exact mechanism(s) behind the role of SREBP in these observed phenotypes is not clear. Here, we report that A. fumigatus SREBP, SrbA, mediates regulation of iron acquisition in response to hypoxia and low iron conditions. To further define SrbA's role in iron acquisition in relation to previously studied fungal regulators of iron metabolism, SreA and HapX, a series of mutants were generated in the ΔsrbA background. These data suggest that SrbA is activated independently of SreA and HapX in response to iron limitation, but that HapX mRNA induction is partially dependent on SrbA. Intriguingly, exogenous addition of high iron or genetic deletion of sreA in the ΔsrbA background was able to partially rescue the hypoxia growth, triazole drug susceptibility, and decrease in ergosterol content phenotypes of ΔsrbA. Thus, we conclude that the fungal SREBP, SrbA, is critical for coordinating genes involved in iron acquisition and ergosterol biosynthesis under hypoxia and low iron conditions found at sites of human fungal infections. These results support a role for SREBP-mediated iron regulation in fungal virulence, and they lay a foundation for further exploration of SREBP's role in iron homeostasis in other eukaryotes.

Large-scale transcriptional response to hypoxia in Aspergillus fumigatus observed using RNAseq identifies a novel hypoxia regulated ncRNA.

We utilized RNAseq analysis of the Aspergillus fumigatus response to early hypoxic condition exposure. The results show that more than 89% of the A. fumigatus genome is expressed under normoxic and hypoxic conditions. Replicate samples were highly reproducible; however, comparisons between normoxia and hypoxia revealed that >23 and 35% of genes were differentially expressed after 30 and 120 min of hypoxia exposure, respectively. Consistent with our previous report detailing transcriptomic and proteomic responses at later time points, the results here show major repression of ribosomal function and induction of ergosterol biosynthesis, as well as activation of alternate respiratory mechanisms at the later time point. RNAseq data were used to define 32 hypoxia-specific genes, which were not expressed under normoxic conditions. Transcripts of a C6 transcription factor and a histidine kinase-response regulator were found only in hypoxia. In addition, several genes involved in the phosphoenylpyruvate and D-glyceraldehyde-3-phosphate metabolism were only expressed in hypoxia. Interestingly, a 216-bp ncRNA Afu-182 in the 3' region of insA (AFUB_064770) was significantly repressed under hypoxia with a 40-fold reduction in expression. A detailed analysis of Afu-182 showed similarity with several genes in the genome, many of which were also repressed in hypoxia. The results from this study show that hypoxia induces very early and widely drastic genome-wide responses in A. fumigatus that include expression of protein-coding and ncRNA genes. The role of these ncRNA genes in regulating the fungal hypoxia response is an exciting future research direction.

Challenges to Implementing a Vaccine for Coccidioidomycosis.

A vaccine for coccidioidomycosis is likely to undergo trials in the near future. In this paper, we raise 4 questions that should be answered before its use and offer our solutions to these questions. These include defining the goals of vaccination, determining who should be vaccinated, how to measure vaccine immunity and protection, and how to address vaccine hesitancy and denial.

Assessing Engraftment Following Fecal Microbiota Transplant.

Fecal Microbiota Transplant (FMT) is an FDA approved treatment for recurrent Clostridium difficile infections, and is being explored for other clinical applications, from alleviating digestive and neurological disorders, to priming the microbiome for cancer treatment, and restoring microbiomes impacted by cancer treatment. Quantifying the extent of engraftment following an FMT is important in determining if a recipient didn't respond because the engrafted microbiome didn't produce the desired outcomes (a successful FMT, but negative treatment outcome), or the microbiome didn't engraft (an unsuccessful FMT and negative treatment outcome). The lack of a consistent methodology for quantifying FMT engraftment extent hinders the assessment of FMT success and its relation to clinical outcomes, and presents challenges for comparing FMT results and protocols across studies. Here we review 46 studies of FMT in humans and model organisms and group their approaches for assessing the extent to which an FMT engrafts into three criteria: 1) Chimeric Asymmetric Community Coalescence investigates microbiome shifts following FMT engraftment using methods such as alpha diversity comparisons, beta diversity comparisons, and microbiome source tracking. 2) Donated Microbiome Indicator Features tracks donated microbiome features (e.g., amplicon sequence variants or species of interest) as a signal of engraftment with methods such as differential abundance testing based on the current sample collection, or tracking changes in feature abundances that have been previously identified (e.g., from FMT or disease-relevant literature). 3) Temporal Stability examines how resistant post-FMT recipient's microbiomes are to reverting back to their baseline microbiome. Individually, these criteria each highlight a critical aspect of microbiome engraftment; investigated together, however, they provide a clearer assessment of microbiome engraftment. We discuss the pros and cons of each of these criteria, providing illustrative examples of their application. We also introduce key terminology and recommendations on how FMT studies can be analyzed for rigorous engraftment extent assessment.