homeRNA: A Self-Sampling Kit for the Collection of Peripheral Blood and Stabilization of RNA.

Gene expression analysis (e.g., targeted gene panels and transcriptomics) from whole blood can elucidate mechanisms of the immune function and aid in the discovery of biomarkers. Conventional venipuncture offers only a small snapshot of our broad immune landscape as immune responses may occur outside of the time and location parameters available for conventional venipuncture. A self-operated method that enables flexible sampling of liquid whole blood coupled with immediate stabilization of cellular RNA is instrumental in facilitating capture and preservation of acute or transient immune fluxes. To this end, we developed homeRNA, a kit for self-collection of peripheral blood (∼0.5 mL) and immediate stabilization of cellular RNA, using the Tasso-SST blood collection device with a specially designed stabilizer tube containing RNAlater. To assess the feasibility of homeRNA for self-collection and stabilization of whole blood RNA, we conducted a pilot study (n = 47 participants) in which we sent homeRNA to participants aged 21-69, located across 10 US states (94% successful blood collections, n = 61/65). Among participants who successfully collected blood, 93% reported no or minimal pain/discomfort using the kit (n = 39/42), and 79% reported very easy/somewhat easy stabilization protocol (n = 33/42). Total RNA yield from the stabilized samples ranged between 0.20 and 5.99 µg (mean = 1.51 µg), and all but one RNA integrity number values were above 7.0 (mean = 8.1), indicating limited RNA degradation. The results from this study demonstrate the self-collection and RNA stabilization of whole blood with homeRNA by participants themselves in their own home.

Miniaturizing Wet Scrubbers for Aerosolized Droplet Capture.

Aerosols dispersed and transmitted through the air (e.g., particulate matter pollution and bioaerosols) are ubiquitous and one of the leading causes of adverse health effects and disease transmission. A variety of sampling methods (e.g., filters, cyclones, and impactors) have been developed to assess personal exposures. However, a gap still remains in the accessibility and ease-of-use of these technologies for people without experience or training in collecting airborne samples. Additionally, wet scrubbers (large non-portable industrial systems) utilize liquid sprays to remove aerosols from the air; the goal is to "scrub" (i.e., clean) the exhaust of industrial smokestacks, not collect the aerosols for analysis. Inspired by wet scrubbers, we developed a device fundamentally different from existing portable air samplers by using aerosolized microdroplets to capture aerosols in personal spaces (e.g., homes, offices, and schools). Our aerosol-sampling device is the size of a small teapot, can be operated without specialized training, and features a winding flow path in a supersaturated relative humidity environment, enabling droplet growth. The integrated open mesofluidic channels shuttle coalesced droplets to a collection chamber for subsequent sample analysis. Here, we present the experimental demonstration of aerosol capture in water droplets. An iterative study optimized the non-linear flow manipulating baffles and enabled an 83% retention of the aerosolized microdroplets in the confined volume of our device. As a proof-of-concept for aerosol capture into a liquid medium, 0.5-3 µm model particles were used to evaluate aerosol capture efficiency. Finally, we demonstrate that the device can capture and keep a bioaerosol (bacteriophage MS2) viable for downstream analysis.

FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection.

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

A Cationic Polymer That Shows High Antifungal Activity against Diverse Human Pathogens.

Invasive fungal diseases are generally difficult to treat and often fatal. The therapeutic agents available to treat fungi are limited, and there is a critical need for new agents to combat these deadly infections. Antifungal compound development has been hindered by the challenge of creating agents that are highly active against fungal pathogens but not toxic to the host. Host defense peptides (HDPs) are produced by eukaryotes as a component of the innate immune response to pathogens and have served as inspiration for the development of many new antibacterial compounds. HDP mimics, however, have largely failed to exhibit potent and selective antifungal activity. Here, we present an HDP-like nylon-3 copolymer that is effective against diverse fungi while displaying only mild to moderate toxicity toward mammalian cells. This polymer is active on its own and in synergy with existing antifungal drugs against multiple species of Candida and Cryptococcus, reaching levels of efficacy comparable to those of the clinical agents amphotericin B and fluconazole in some cases. In addition, the polymer acts synergistically with azoles against different species of Aspergillus, including some azole-resistant strains. These findings indicate that nylon-3 polymers are a promising lead for development of new antifungal therapeutic strategies.

Redundant synthesis of a conidial polyketide by two distinct secondary metabolite clusters in Aspergillus fumigatus.

Filamentous fungi are renowned for the production of bioactive secondary metabolites. Typically, one distinct metabolite is generated from a specific secondary metabolite cluster. Here, we characterize the newly described trypacidin (tpc) cluster in the opportunistic human pathogen Aspergillus fumigatus. We find that this cluster as well as the previously characterized endocrocin (enc) cluster both contribute to the production of the spore metabolite endocrocin. Whereas trypacidin is eliminated when only tpc cluster genes are deleted, endocrocin production is only eliminated when both the tpc and enc non-reducing polyketide synthase-encoding genes, tpcC and encA, respectively, are deleted. EncC, an anthrone oxidase, converts the product released from EncA to endocrocin as a final product. In contrast, endocrocin synthesis by the tpc cluster likely results from incomplete catalysis by TpcK (a putative decarboxylase), as its deletion results in a nearly 10-fold increase in endocrocin production. We suggest endocrocin is likely a shunt product in all related non-reducing polyketide synthase clusters containing homologues of TpcK and TpcL (a putative anthrone oxidase), e.g. geodin and monodictyphenone. This finding represents an unusual example of two physically discrete secondary metabolite clusters generating the same natural product in one fungal species by distinct routes.

Low-volume toolbox for the discovery of immunosuppressive fungal secondary metabolites.

The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new "function-omic" avenues downstream of the metabolomics, identification, and purification phases.

Co-ordination between BrlA regulation and secretion of the oxidoreductase FmqD directs selective accumulation of fumiquinazoline C to conidial tissues in Aspergillus fumigatus.

Aerial spores, crucial for propagation and dispersal of the Kingdom Fungi, are commonly the initial inoculum of pathogenic fungi. Natural products (secondary metabolites) have been correlated with fungal spore development and enhanced virulence in the human pathogen Aspergillus fumigatus but mechanisms for metabolite deposition in the spore are unknown. Metabolomic profiling of A. fumigatus deletion mutants of fumiquinazoline (Fq) cluster genes reveal that the first two products of the Fq cluster, FqF and FqA, are produced to comparable levels in all fungal tissues but the final enzymatically derived product, FqC, predominantly accumulates in the fungal spore. Loss of the sporulation-specific transcription factor, BrlA, yields a strain unable to produce FqA or FqC. Fluorescence microscopy showed FmqD, the oxidoreductase required to generate FqC, was secreted via the Golgi apparatus to the cell wall in an actin-dependent manner. In contrast, all other members of the Fq pathway including the putative transporter, FmqE - which had no effect on Fq biosynthesis - were internal to the hyphae. The co-ordination of BrlA-mediated tissue specificity with FmqD secretion to the cell wall presents a previously undescribed mechanism to direct localization of specific secondary metabolites to spores of the differentiating fungus.

Illumina identification of RsrA, a conserved C2H2 transcription factor coordinating the NapA mediated oxidative stress signaling pathway in Aspergillus.

BACKGROUND: Chemical mutagenesis screens are useful to identify mutants involved in biological processes of interest. Identifying the mutation from such screens, however, often fails when using methodologies involving transformation of the mutant to wild type phenotype with DNA libraries. RESULTS: Here we analyzed Illumina sequence of a chemically derived mutant of Aspergillus nidulans and identified a gene encoding a C2H2 transcription factor termed RsrA for regulator of stress response. RsrA is conserved in filamentous fungal genomes, and upon deleting the gene in three Aspergillus species (A. nidulans, A. flavus and A. fumigatus), we found two conserved phenotypes: enhanced resistance to oxidative stress and reduction in sporulation processes. For all species, rsrA deletion mutants were more resistant to hydrogen peroxide treatment. In depth examination of this latter characteristic in A. nidulans showed that upon exposure to hydrogen peroxide, RsrA loss resulted in global up-regulation of several components of the oxidative stress metabolome including the expression of napA and atfA, the two bZIP transcription factors mediating resistance to reactive oxygen species (ROS) as well as NapA targets in thioredoxin and glutathione systems. Coupling transcriptional data with examination of ΔrsrAΔatfA and ΔrsrAΔnapA double mutants indicate that RsrA primarily operates through NapA-mediated stress response pathways. A model of RsrA regulation of ROS response in Aspergillus is presented. CONCLUSION: RsrA, found in a highly syntenic region in Aspergillus genomes, coordinates a NapA mediated oxidative response in Aspergillus fungi.

Spatial and temporal control of fungal natural product synthesis.

Despite their oftentimes-elusive ecological role, fungal natural products have, for better or worse, impacted our daily lives tremendously owing to their diverse and potent bioactive properties. This Janus-faced nature of fungal natural products inevitably ushered in a field of research dedicated towards understanding the ecology, organisms, genes, enzymes, and biosynthetic pathways that give rise to this arsenal of diverse and complex chemistry. Ongoing research in fungal secondary metabolism has not only increased our appreciation for fungal natural products as an asset but also sheds light on the pivotal role that these once-regarded "metabolic wastes" play in fungal biology, defense, and stress response in addition to their potential contributions towards human mycoses. Full orchestration of secondary metabolism requires not only the seamless coordination between temporal and spatial control of SM-associated machineries (e.g. enzymes, cofactors, intermediates, and end-products) but also integration of these machineries into primary metabolic processes and established cellular mechanisms. An intriguing, but little known aspect of microbial natural product synthesis lies in the spatial organization of both pathway intermediates and enzymes responsible for the production of these compounds. In this highlight, we summarize some major breakthroughs in understanding the genes and regulation of fungal natural product synthesis and introduce the current state of knowledge on the spatial and temporal control of fungal natural product synthesis.

homeRNA self-blood collection enables high-frequency temporal profiling of pre-symptomatic host immune kinetics to respiratory viral infection: a prospective cohort study.

Background: Early host immunity to acute respiratory infections (ARIs) is heterogenous, dynamic, and critical to an individual's infection outcome. Due to limitations in sampling frequency/timepoints, kinetics of early immune dynamics in natural human infections remain poorly understood. In this nationwide prospective cohort study, we leveraged a self-blood collection tool (homeRNA) to profile detailed kinetics of the pre-symptomatic to convalescence host immunity to contemporaneous respiratory pathogens. Methods: We enrolled non-symptomatic adults with recent exposure to ARIs who subsequently tested negative (exposed-uninfected) or positive for respiratory pathogens. Participants self-collected blood and nasal swabs daily for seven consecutive days followed by weekly blood collection for up to seven additional weeks. Symptom burden was assessed during each collection. Nasal swabs were tested for SARS-CoV-2 and common respiratory pathogens. 92 longitudinal blood samples spanning the pre-shedding to post-acute phase of eight SARS-CoV-2-infected participants and 40 interval-matched samples from four exposed-uninfected participants were subjected to high-frequency longitudinal profiling of 773 host immune genes. Findings: Between June 2021 - April 2022, 68 participants across 26 U.S. states completed the study and self-collected a total of 691 and 466 longitudinal blood and nasal swab samples along with 688 symptom surveys. SARS-CoV-2 was detected in 17 out of 22 individuals with study-confirmed respiratory infection. With rapid dissemination of home self-collection kits, two and four COVID-19+ participants started collection prior to viral shedding and symptom onset, respectively, enabling us to profile detailed expression kinetics of the earliest blood transcriptional response to contemporaneous variants of concern. In pre-shedding samples, we observed transient but robust expression of T-cell response signatures, transcription factor complexes, prostaglandin biosynthesis genes, pyrogenic cytokines, and cytotoxic granule genes. This is followed by a rapid induction of many interferon-stimulated genes (ISGs), concurrent to onset of viral shedding and increase in nasal viral load. Finally, we observed increased expression of host defense peptides (HDPs) in exposed-uninfected individuals over the 4-week observational window. Interpretation: We demonstrated that unsupervised self-collection and stabilization of capillary blood can be applied to natural infection studies to characterize detailed early host immune kinetics at a temporal resolution comparable to that of human challenge studies. The remote (decentralized) study framework enables conduct of large-scale population-wide longitudinal mechanistic studies. Expression of cytotoxic/T-cell signatures in pre-shedding samples preceding expansion of innate ISGs suggests a potential role for T-cell mediated pathogen control during early infection. Elevated expression of HDPs in exposed-uninfected individuals warrants further validation studies to assess their potential role in protective immunity during pathogen exposure. Funding: This study was funded by R35GM128648 to ABT for in-lab developments of homeRNA, Packard Fellowship from the David and Lucile Packard Foundation to ABT, and R01AI153087 to AW.