Metaproteomic Profiling of Fungal Gut Colonization in Gnotobiotic Mice

Background: Eukaryotic microbes can modulate mammalian host health and disease states, yet the molecular contribution of gut fungi remains nascent. We previously showed that mice exclusively colonised with fungi displayed increased sensitivity to allergic airway inflammation and had fecal metabolite profiles similar to germ-free mice. This marginal effect on the host metabolome suggested that fungi do not primarily use metabolites to modulate the host immune system.Methods: To describe functional changes attributed to fungal colonisation, we performed mass spectrometry-based analyses of feces (Label-Free Quantitative; LFQ) and the small intestine (labeling with Tandem Mass Tag; TMT) of gnotobiotic mice colonised with defined consortia of twelve bacterial species, five fungal species, or both. We also evaluated the effect of microbiome perturbances on the metaproteome by analysing feces from mouse pups treated with an antibiotic or antifungal.Results: We detected 6,675 proteins in the mice feces, of which 3,845 had determined LFQ levels. Analysis of variance showed changes in the different gnotobiotic mouse groups; specifically, 46% of 2,860 bacterial, 15% of 580 fungal, and 76% of 405 mouse quantified proteins displayed differential levels. The antimicrobial treatments resulted in lasting changes in the bacterial and fungal proteomes, suggesting that the antimicrobials impacted the entire community. Fungal colonisation resulted in changes in host proteins functional in innate immunity as well as metabolism, predicting specific roles of gut fungi on host systems during early developmental stages. Several of the detected fungal proteins (3% of 1,492) have been previously reported as part of extracellular vesicles and having immunomodulating properties. Using an isobaric labelling TMT approach for profiling low abundant proteins of the jejunal tissue, we confirmed that the five fungal species differentially impacted the host intestinal proteome compared to the bacterial consortium. The detected changes in mouse jejunal proteins (4% of 1,514) were mainly driven by metabolic proteins. Conclusions: We used quantitative proteomic profiling of gnotobiotic conditions to show how colonisation with selected fungal species impacts the host gut proteome. Our results suggest that an increased abundance of certain gut fungal species in early life may affect the developing intracellular attributes of epithelial and immune cells.

Study of spectrum of fungal isolates in the sputum of newly diagnosed pulmonary tuberculosis patients

Tuberculosis is a state of immunosupression which exposes the patients to further opportunistic pathogens like fungus. Methods: 102 newly diagnosed sputum positive pulmonary tuberculosis cases were enrolled. Significant fungal isolates were seen in 31/102 (30.4%) patients. Aspergillus spp. were isolated in 13/31(41.9%) of the positive fungal cultures while Candida spp. were isolated in 15/31 (48.4%). Low body mass index, duration of symptoms, haemoptysis, severity of radiological features and IgG Aspergillus antibodies were independent risk factors for positive fungal culture. Significant proportion of patients with PTB have fungal colonisation of their airways which can lead to poor clinical outcomes. Few easily ascertained clinical parameters can help the clinician to determine patients who are at a higher risk of fungal colonisation.

New Insights in Microbial Species Predicting Lung Function Decline in CF: Lessons from the MucoFong Project

Several predictive models have been proposed to understand the microbial risk factors associated with cystic fibrosis (CF) progression. Very few have integrated fungal airways colonisation, which is increasingly recognized as a key player regarding CF progression. To assess the association between the percent predicted forced expiratory volume in 1 s (ppFEV1) change and the fungi or bacteria identified in the sputum, 299 CF patients from the “MucoFong” project were included and followed-up with over two years. The relationship between the microorganisms identified in the sputum and ppFEV1 course of patients was longitudinally analysed. An adjusted linear mixed model analysis was performed to evaluate the effect of a transient or chronic bacterial and/or fungal colonisation at inclusion on the ppFEV1 change over a two-year period. Pseudomonas aeruginosa, Achromobacter xylosoxidans, Stenotrophomonas maltophilia, and Candida albicans were associated with a significant ppFEV1 decrease. No significant association was found with other fungal colonisations. In addition, the ppFEV1 outcome in our model was 11.26% lower in patients presenting with a transient colonisation with non-pneumoniae Streptococcus species compared to other patients. These results confirm recently published data and provide new insights into bacterial and fungal colonisation as key factors for the assessment of lung function decline in CF patients.

MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.

Artificial intelligence enables the identification and quantification of arbuscular mycorrhizal fungi in plant roots

Soil fungi establish mutualistic interactions with the roots of most vascular land plants. Arbuscular mycorrhizal (AM) fungi are among the most extensively characterised mycobionts to date. Current approaches to quantifying the extent of root colonisation and the relative abundance of intraradical hyphal structures in mutant roots rely on staining and human scoring involving simple, yet repetitive tasks prone to variations between experimenters. We developed the software AMFinder which allows for automatic computer vision-based identification and quantification of AM fungal colonisation and intraradical hyphal structures on ink-stained root images using convolutional neural networks. AMFinder delivered high-confidence predictions on image datasets of colonised roots of Medicago truncatula, Lotus japonicus, Oryza sativa and Nicotiana benthamiana obtained via flatbed scanning or digital microscopy enabling reproducible and transparent data analysis. A streamlined protocol for sample preparation and imaging allowed us to quantify dynamic increases in colonisation in whole root systems over time. AMFinder adapts to a wide array of experimental conditions. It enables accurate, reproducible analyses of plant root systems and will support better documentation of AM fungal colonisation analyses. AMFinder can be accessed here: https://github.com/SchornacklabSLCU/amfinder.git

Pigeon Pea and Cowpea-Based Cropping Systems Improve Vesicular Arbuscular Mycorrhizal Fungal Colonisation of Subsequent Maize on the Alfisols in Central Malawi

Mycorrhizal associations contribute to the sustainability of crop production systems through their roles in nutrient cycling and other benefits in the soil-plant ecosystems. A two-year study was conducted on the Alfisols of Lilongwe and Dowa districts, Central Malawi, to assess the vesicular-arbuscular mycorrhizal (VAM) fungal colonisation levels in pigeon pea, cowpea, and maize grown in sole cropping, legume-cereal, and legume-legume intercropping systems and in the maize grown in short rotation (year 2) as influenced by the previous cropping systems and N fertilizer application. The gridline intersect method was used to assess the VAM fungal colonisation levels. Results showed that all treatments that included legumes whether grown as sole crop, in legume-cereal or in legume-legume cropping systems in the previous year, had significantly higher (P < 0.05) VAM fungal colonisation of the rotational maize crop roots by a range 39% to 50% and 19% to 47% than those in maize supplied and not supplied with N fertilizer, respectively, in a maize-maize short rotation, at the Lilongwe site. A similar trend was reported for the Dowa site. Furthermore, there were positive correlations between VAM fungal colonisation and the plant P content, dry matter yield, and nodule numbers. Further studies may help to assess the diversity of VAM fungal species in Malawi soils and identify more adaptive ones for inoculation studies.