Filamentous Fungal Carbon Catabolite Repression Supports Metabolic Plasticity and Stress Responses Essential for Disease Progression

Aspergillus fumigatus is responsible for a disproportionate number of invasive mycosis cases relative to other common filamentous fungi. While many fungal factors critical for infection establishment are known, genes essential for disease persistence and progression are ill defined. We propose that fungal factors that promote navigation of the rapidly changing nutrient and structural landscape characteristic of disease progression represent untapped clinically relevant therapeutic targets. To this end, we find that A. fumigatus requires a carbon catabolite repression (CCR) mediated genetic network to support in vivo fungal fitness and disease progression. While CCR as mediated by the transcriptional repressor CreA is not required for pulmonary infection establishment, loss of CCR inhibits fungal metabolic plasticity and the ability to thrive in the dynamic infection microenvironment. Our results suggest a model whereby CCR in an environmental filamentous fungus is dispensable for initiation of pulmonary infection but essential for infection maintenance and disease progression. Conceptually, we argue these data provide a foundation for additional studies on fungal factors required to support fungal fitness and disease progression and term such genes and factors, DPFs (disease progression factors)

Host-Derived Leukotriene B4 Is Critical for Resistance against Invasive Pulmonary Aspergillosis

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how immune competent hosts maintain control of fungal infections while constantly being exposed to fungi is rapidly emerging. It is known that timely neutrophil recruitment to and activation in the lungs is critical to the host defense against development of invasive pulmonary aspergillosis, but the inflammatory sequelae necessary remains to be fully defined. Here, we show that 5-Lipoxygenase (5-LO) and Leukotriene B4 (LTB4) are critical for leukocyte recruitment and resistance to pulmonary A. fumigatus challenge in a fungal-strain-dependent manner. 5-LO activity was needed in radiosensitive cells for an optimal anti-fungal response and in vivo LTB4 production was at least partially dependent on myeloid-derived hypoxia inducible factor-1α. Overall, this study reveals a role for host-derived leukotriene synthesis in innate immunity to A. fumigatus

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

Filamentous fungal carbon catabolite repression supports metabolic plasticity and stress responses essential for disease progression

Aspergillus fumigatus is responsible for a disproportionate number of invasive mycosis cases relative to other common filamentous fungi. While many fungal factors critical for infection establishment are known, genes essential for disease persistence and progression are ill defined. We propose that fungal factors that promote navigation of the rapidly changing nutrient and structural landscape characteristic of disease progression represent untapped clinically relevant therapeutic targets. To this end, we find that A. fumigatus requires a carbon catabolite repression (CCR) mediated genetic network to support in vivo fungal fitness and disease progression. While CCR as mediated by the transcriptional repressor CreA is not required for pulmonary infection establishment, loss of CCR inhibits fungal metabolic plasticity and the ability to thrive in the dynamic infection microenvironment. Our results suggest a model whereby CCR in an environmental filamentous fungus is dispensable for initiation of pulmonary infection but essential for infection maintenance and disease progression. Conceptually, we argue these data provide a foundation for additional studies on fungal factors required to support fungal fitness and disease progression and term such genes and factors, DPFs (disease progression factors)

CreA regulates CCR in <i>A</i>. <i>fumigatus</i>.

<p>A) Growth of wild type, Δ<i>creA</i>, and <i>creA</i>^<i>R</i> on 1% glucose minimal media with or without 0.1% allyl alcohol (AA) incubated for 48 hours. B) Growth on 1% glucose or 1% ethanol minimal media for 72 hours.</p

Model for role of CreA in disease progression of invasive aspergillosis.

<p>Model of disease progression, where upon infection initiation, the presence of oxygen and alternative carbon sources allows for gluconeogenesis and oxidative phosphorylation. However increased fungal growth and influx of host immune cells results in depletion of local oxygen concentration, shifting metabolism towards glycolytic fermentative metabolism. Fungal metabolic adaptation is required for progression to invasive disease, and this requires CreA to mediate disease progression. We propose the concept of disease progression factors (DPFs) as factors required to navigate the dynamic microenvironments that occur during infection and disease progression.</p

CreA maintains fungal bioenergetics homeostasis.

<p>A) Metabolites measured with global metabolomics profiling indicate a perturbation in glucose metabolism and metabolism through the tricarboxylic acid (TCA) cycle. The ratio of each metabolite in Δ<i>creA</i>/WT is given under metabolite name. Metabolites that are boxed in red and green are significantly increased and decreased, respectively, in Δ<i>creA</i>, and black are not significantly changed (p≤0.05 by Welch’s two-sample t-test). mRNA levels of key metabolic enzymes are given as fold-change in Δ<i>creA</i> compared to wild type. B) Expression of <i>acuF</i> as measured by qRT-PCR from cultures grown in GMM overnight, then shifted to fresh glucose media with samples taken at indicated time-points post shift. <i>acuF</i> expression is normalized to <i>actA</i> and <i>tub2</i>. ^ap = 0.0046, ^bp = 0.0006, ^cp = 0.0120, ^dp = 0.001, ^ep = 0.0054, n.s. = not significant by unpaired, two-tailed t-test as compared to WT of respective time-point. Data represents the mean of three biological replicates ± SEM. C) ADP/ATP ratio of cultures grown in GMM overnight then shifted to fresh GMM for 2 hours. Data represents mean of biological triplicates ± SEM; **p<0.0001, n.s. = not significant by unpaired, two-tailed t-test, as compared to WT.</p

CreA is required for disease progression, but not for establishment of infection in a triamcinolone model of IPA.

<p>A) Host survival analysis of fungal strains in a triamcinolone immune suppression model of CD-1 mice inoculated with 2x10⁶ conidia intranasally. n = 20 mice/group from two independent experiments, n = 10 for <i>creA</i>^<i>R</i>, n = 4 mice for mock. *p = 0.0217 compared to wild type and p = 0.0425 compared to <i>creA</i>^<i>R</i>, n.s. = not significant by Log-rank test. B) GMS and H&E staining of histological sections of lung tissue from CD-1 mice as treated in (A) collected 48 hpi. C) Fungal burden of CD-1 mice as treated in (A), from lungs collected 48 hpi, as measured by qRT-PCR of 18 rDNA region; n = 7 mice/group, n = 3 for mock; n.s. by Wilcoxon rank-sum test as compared to WT; error bars represent SEM. D) GMS staining of histological sections of lung tissue collected 8 hpi from CD-1 mice treated with triamcinolone immune suppression and inoculated with 1x10⁷ conidia. E) Growth of WT and Δ<i>creA</i> in lung homogenate media over 24 hours measured by Abs₄₀₅. Data represents mean of six replicates ± SEM.</p

The nutrient landscape of murine lung tissue is complex and dynamic and altered upon treatment with corticosteroids and fungal conidia.

<p>A) Heatmap of relative abundance of total detected metabolites from healthy lungs (- Steroids), lungs treated with corticosteroids (+ Steroids) and corticosteroids with fungal conidia (+ Steroids, + Fungus). Each column represents a single outbred CD-1 mouse. B-E) Scaled input values of (B) glucose, (C) glutatmate, (D) oleate and (E) myristate from metabolomics analysis. Significance is calculated with Welch’s Two Sample t-test between indicated groups. Whiskers represent min to max across six biological replicates (6 independent mice).</p

CreA is required for adaptation to changing oxygen environments.

<p>A) Ratio of hypoxia to normoxia growth of fungal colonies grown in normoxia for 24 hours, then shifted to hypoxia (0.2% O₂, 5% CO₂) for 96 hours. *p<0.05; **p<0.01 by unpaired, two-tailed t-test compared to WT of respective time points. Data represents mean of biological triplicates ± SEM. B) Host survival curve of wild type, Δ<i>creA</i> and <i>creA</i>^<i>R</i> in a chemotherapy model of IPA. Mice were treated with 175mg/kg Cyclophosphamide on day -2 and 40mg/kg Kenalog on day -1 then inoculated with 1x10⁶ conidia in 40uL PBS intranasally. n = 12/group, n = 4 for mock. C) Percent fungal damage by bone marrow derived neutrophils of germlings grown in 1% glucose (GMM) or 1% Tween-80 minimal media. Germlings were grown in normoxia conditions, then shifted to hypoxia (0.2% O₂, 5% CO₂) upon addition of neutrophils for 2 hours. No significant differences were observed between any group by Wilcoxon rank-sum test.</p