Motsi, N. | Search bioRxiv

bioRxiv2024

Inferring fungal growth rates from optical density data

Quantifying fungal growth underpins our ability to effectively treat severe fungal infections. Current methods quantify fungal growth rates from time-course morphology-specific data, such as hyphal length data. However, automated large-scale collection of such data lies beyond the scope of most clinical microbiology laboratories. In this paper, we propose a mathematical model of fungal growth to estimate morphology-specific growth rates from easy-to-collect, but indirect, optical density (OD600) data of Aspergillus fumigatus growth (filamentous fungus). Our method accounts for OD600 being an indirect measure by explicitly including the relationship between the indirect OD600 measurements and the calibrating true fungal growth in the model. Therefore, the method does not require de novo generation of calibration data. Our model outperformed reference models at fitting to and predicting OD600 growth curves and overcame observed discrepancies between morphology-specific rates inferred from OD600 versus directly measured data in reference models that did not include calibration. Author summaryQuantifying fungal growth is essential for antifungal drug discovery and monitoring antifungal resistance. As fungal growth is complex, with fungal morphology (shape) dynamically changing over time, previous studies have quantified fungal growth by estimating growth rates during specific fungal morphologies (morphology-specific growth rates) or by mathematically modelling fungal growth. However, collecting time-series data that captures the morphological information required for mathematical model fitting or estimating morphology-specific growth rates is prohibitively time consuming for large-scale drug testing in most microbiology laboratories. Alternatively, fungal growth can be quickly, although indirectly, quantified by measuring the optical density (OD) of a broth culture. However, changes in OD are not always reflective of true changes in fungal growth because OD is an indirect measure. This paper proposes a method to model fungal growth and estimate a morphology-specific growth rate from indirect OD600 measurements of the major mould pathogen, Aspergillus fumigatus. We explicitly model the relationship between measured indirect OD600 data and true fungal growth (calibration). The presented work serves as the much-needed foundation for estimating and comparing morphology-specific fungal growth rates in varying antifungal drug concentrations using only OD600 data.

systems biology↗

bioRxiv2022

Epithelial uptake of Aspergillus fumigatus drives efficient fungal clearance in vivo and is aberrant in Chronic Obstructive Pulmonary Disease (COPD)

Hundreds of spores of the common mould Aspergillus fumigatus (Af) are inhaled daily by human beings, representing a constant, often fatal, threat to our respiratory health. The small size of Af spores suggest that interactions with Airway Epithelial Cells (AECs) are frequent and we and others have previously demonstrated that AECs are able to internalise Af spores. We thus hypothesised that Af spore uptake and killing by AECs is important for driving efficient fungal clearance in vivo and that defective spore uptake and killing would represent major risk factors for Aspergillus-related diseases. In order to test this, we utilised single-cell approaches based on Imaging Flow Cytometry (IFC) and live-cell microfluidic imaging to measure spore uptake and outcomes in vitro, in vivo and using primary human AECs. In vitro, viability of immortalised AECs was largely unaffected by Af uptake and AECs were able to significantly curtail the growth of internalised spores. Applying our approach directly to infected mouse lungs we demonstrated, for the first time, that Af spores are internalised and killed by AECs during whole animal infection, whereby only ~3% of internalised spores remained viable after 8 hours of co-incubation with murine AECs. Finally, in vitro analysis of primary human AECs from healthy and at-risk donors revealed significant alterations in the uptake and consequent outcomes in Chronic Obstructive Pulmonary Disease (COPD), whereby gorging COPD-derived AECs were unable to quell intracellular Af as efficiently as healthy primary AECs. We have thus demonstrated that AECs efficiently kill Af spores upon uptake in vivo and that this process is altered in COPD, a well-known risk factor for debilitating fungal lung disease, thereby suggesting that AECs critically contribute to the efficient clearance of inhaled Af spores and that dysregulation of curative AEC responses represents a potent driver of Aspergillus-related diseases.

microbiology↗