Biology subjects

Ross, B. S.

Publications and source records attributed to Ross, B. S..

4 recordsLinked to original sources

bioRxiv2021

Host lung environment limits Aspergillus fumigatus germination through a 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 are 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 Lung Homogenate Evolved (LH-EVOL)). 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 {Delta}sskA 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 MAPK pathway and drives enhanced disease initiation and inflammation in the lungs. IMPORTANCEAspergillus 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 which 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.

microbiology↗

bioRxiv2021

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 compared to non-persistent isolates. Closely related persistent isolates harbor 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 conidia 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 fungal CF isolates. ImportanceAspergillus 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 identified a persistent lineage that harbors a unique allele of the Pbs2 MAPKK necessary for unique CF-relevant stress phenotypes. Importantly for A. fumigatus CF patient diagnostics, this allele provides increased CF lung fitness at a cost of reduced in vitro growth in standard laboratory conditions. These data illustrate a molecular mechanism for A. fumigatus CF lung persistence with implications for diagnostics and antifungal therapy.

microbiology↗

bioRxiv2021

Antifungal activity of Propranolol against Fusarium keratitis isolates from the Mycotic Ulcer Treatment Trial (MUTT) and the United States.

SYNOPSISO_ST_ABSBackgroundC_ST_ABSFusarium keratitis is an infection of the cornea that often results in corneal perforation requiring corneal transplantation even with topical ocular antifungal therapy. The polyene natamycin remains the current antifungal of choice for Fusarium keratitis, but prompt sterilization of the cornea is often not achieved with contemporary therapy. Recently, natamycin synergy with the beta-adrenergic antagonist timolol against Fusarium species was reported. ObjectiveOur objective in this study was to characterize the in vitro antifungal effects of additional beta-adrenergic antagonists alone or in combination with natamycin on Fusarium keratitis isolates from the Mycotic Ulcer Treatment Trial (MUTT) and USA. MethodsMicrobroth dilution assays were used to determine the minimal inhibitory concentration (MIC) of beta-adrenergic antagonists against 18 Fusarium spp. keratitis (10 from MUTT, 8 from USA) and 3 Aspergillus fumigatus isolates. The fractional inhibitory concentration index (FICI) was calculated to assess interactions with natamycin. ResultsMost beta-blockers did not show antifungal activity or synergy with natamycin with the exception of propranolol. A racemic mix of propranolol had fungicidal activity with MIC between 31 and 83 g/mL for the Fusarium isolates. The MIC of the less cardioactive R enantiomer was lower (27-83 g/mL) than the MIC of the S enantiomer (42-104 g/mL). The MICs of both propranolol and natamycin were lower in combination but were not synergistic. The MIC of propranolol was 156 g/mL for the A. fumigatus isolates. ConclusionsPropranolol has intrinsic in vitro fungicidal activity and lowers the MIC of natamycin. Both the R and S enantiomers of propranolol had antifungal activity with the MIC modestly but significantly lower for R-propranolol. These findings have relevance both for the treatment of fungal keratitis and of glaucoma in the setting of fungal keratitis. Further study of propranolols antifungal activity may lead to a novel treatment for fungal keratitis and possibly other fungal infections. Trial RegistrationClinicalTrials.gov Identifier: NCT00997035 (MUTT Trial)

microbiology↗

bioRxiv2020

Strain specific persistence in the murine lung of Aspergillus fumigatus conidia 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 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. IMPORTANCEAllergic 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 not only be used to better understand disease pathogenesis of ABPA and potential anti-fungal 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 when to utilize antifungal therapies in patients with ABPA and fungal allergic type diseases.

microbiology↗