The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of Aspergillus fumigatus biofilms.

IMPORTANCE: PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.

The pan-genome of Aspergillus fumigatus provides a high-resolution view of its population structure revealing high levels of lineage-specific diversity driven by recombination.

Aspergillus fumigatus is a deadly agent of human fungal disease where virulence heterogeneity is thought to be at least partially structured by genetic variation between strains. While population genomic analyses based on reference genome alignments offer valuable insights into how gene variants are distributed across populations, these approaches fail to capture intraspecific variation in genes absent from the reference genome. Pan-genomic analyses based on de novo assemblies offer a promising alternative to reference-based genomics with the potential to address the full genetic repertoire of a species. Here, we evaluate 260 genome sequences of A. fumigatus including 62 newly sequenced strains, using a combination of population genomics, phylogenomics, and pan-genomics. Our results offer a high-resolution assessment of population structure and recombination frequency, phylogenetically structured gene presence-absence variation, evidence for metabolic specificity, and the distribution of putative antifungal resistance genes. Although A. fumigatus disperses primarily via asexual conidia, we identified extraordinarily high levels of recombination with the lowest linkage disequilibrium decay value reported for any fungal species to date. We provide evidence for 3 primary populations of A. fumigatus, with recombination occurring only rarely between populations and often within them. These 3 populations are structured by both gene variation and distinct patterns of gene presence-absence with unique suites of accessory genes present exclusively in each clade. Accessory genes displayed functional enrichment for nitrogen and carbohydrate metabolism suggesting that populations may be stratified by environmental niche specialization. Similarly, the distribution of antifungal resistance genes and resistance alleles were often structured by phylogeny. Altogether, the pan-genome of A. fumigatus represents one of the largest fungal pan-genomes reported to date including many genes unrepresented in the Af293 reference genome. These results highlight the inadequacy of relying on a single-reference genome-based approach for evaluating intraspecific variation and the power of combined genomic approaches to elucidate population structure, genetic diversity, and putative ecological drivers of clinically relevant fungi.

Investigating and modeling the effect of light intensity on Rhodopseudomonas palustris growth.

Photosynthetic bacteria can be useful biotechnological tools-they produce a variety of valuable products, including high purity hydrogen, and can simultaneously treat recalcitrant wastewaters. However, while photobioreactors have been designed and modeled for photosynthetic algae and cyanobacteria, there has been less work on understanding the effect of light in photosynthetic bacterial fermentations. To design photobioreactors, and processes using these organisms, robust models of light penetration, utilization, and conversion are needed. This stydy uses experimental data from a tubular photobioreactor designed to focus in on light intensity effects, to model the effect of light intensity on the growth of Rhodopseudomonas palustris, a model photosynthetic bacterium. The work demonstrates that growth is controlled by light intensity, and that this organism does experience photolimitation below 200 W/m2 and photoinhibition above 600 W/m2 . This has implications for outdoor applications, as there will be low growth during the periods of limited light, and growth may be inhibited during the light intensive hours of mid-day. Further, the work presents a model for light penetration in cylindrical photobioreactors, which tends to be the most common geometry. The model developed showed good fit to the experimental data for each light intensity investigated, with high R2 values and NRMSE values all below 20%. The work extends the modeling tools for these organisms, and will allow for better photobioreactor design, and the integration of modeling tools in designing processes which use photosynthetic bacteria.

Host Lung Environment Limits Aspergillus fumigatus Germination through an SskA-Dependent Signaling Response.

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential, but the mechanism(s) regulating A. fumigatus germination in the airways is unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine-lung-based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named LH-EVOL for lung homogenate evolved). We identified a potential loss-of-function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 ΔsskA and CEA10. In support of the whole-genome variant analyses, sskA, sakA, or mpkC loss-of-function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions, the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA mitogen-activated protein kinase (MAPK) pathway and drives enhanced disease initiation and inflammation in the lungs. IMPORTANCE Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus that has increased germination potential and inflammation in the lungs. Using this serially passaged strain, we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.

Aspergillus fumigatus In-Host HOG Pathway Mutation for Cystic Fibrosis Lung Microenvironment Persistence.

The prevalence of Aspergillus fumigatus colonization in individuals with cystic fibrosis (CF) and subsequent fungal persistence in the lung is increasingly recognized. However, there is no consensus for clinical management of A. fumigatus in CF individuals, due largely to uncertainty surrounding A. fumigatus CF pathogenesis and virulence mechanisms. To address this gap in knowledge, a longitudinal series of A. fumigatus isolates from an individual with CF were collected over 4.5 years. Isolate genotypes were defined with whole-genome sequencing that revealed both transitory and persistent A. fumigatus in the lung. Persistent lineage isolates grew most readily in a low-oxygen culture environment, and conidia were more sensitive to oxidative stress-inducing conditions than those from nonpersistent isolates. Closely related persistent isolates harbored a unique allele of the high-osmolarity glycerol (HOG) pathway mitogen-activated protein kinase kinase, Pbs2 (pbs2C2). Data suggest this novel pbs2C2 allele arose in vivo and is necessary for the fungal response to osmotic stress in a low-oxygen environment through hyperactivation of the HOG (SakA) signaling pathway. Hyperactivation of the HOG pathway through pbs2C2 comes at the cost of decreased conidial stress resistance in the presence of atmospheric oxygen levels. These novel findings shed light on pathoadaptive mechanisms of A. fumigatus in CF, lay the foundation for identifying persistent A. fumigatus isolates that may require antifungal therapy, and highlight considerations for successful culture of persistent Aspergillus CF isolates. IMPORTANCE Aspergillus fumigatus infection causes a spectrum of clinical manifestations. For individuals with cystic fibrosis (CF), allergic bronchopulmonary aspergillosis (ABPA) is an established complication, but there is a growing appreciation for A. fumigatus airway persistence in CF disease progression. There currently is little consensus for clinical management of A. fumigatus long-term culture positivity in CF. A better understanding of A. fumigatus pathogenesis mechanisms in CF is expected to yield insights into when antifungal therapies are warranted. Here, a 4.5-year longitudinal collection of A. fumigatus isolates from a patient with CF identified a persistent lineage that harbors a unique allele of the Pbs2 mitogen-activated protein kinase kinase (MAPKK) necessary for unique CF-relevant stress phenotypes. Importantly for A. fumigatus CF patient diagnostics, this allele provides increased fitness under CF lung-like conditions at a cost of reduced in vitro growth under standard laboratory conditions. These data illustrate a molecular mechanism for A. fumigatus CF lung persistence with implications for diagnostics and antifungal therapy.

Aspergillus fumigatus Strain-Specific Conidia Lung Persistence Causes an Allergic Broncho-Pulmonary Aspergillosis-Like Disease Phenotype.

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic broncho-pulmonary aspergillosis (ABPA) is a disease diagnosed primarily in cystic fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310, which was isolated from the expectorated sputum of an ABPA patient, by whole-genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide, and menadione, in vitro In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease.IMPORTANCE Allergic broncho-pulmonary aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can be used not only to better understand disease pathogenesis of ABPA and potential antifungal treatments but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question of when to utilize antifungal therapies in patients with ABPA and fungal allergic-type diseases.