The Nanoscale Architecture of the Fungal Cell wall

Session

AFM in Life Sciences

Authors

Dr Abimbola Feyisara Olulana (2, 4, 5), Eleanor Briggs (1), Cameron Colclough (2, 1), Dr Sam Amsbury (1), Dr Simon A. Johnston (3), Prof Andrew Fleming (1), Prof Jamie K. Hobbs (2, 4, 5)

Affiliations

1. Department of Animal and Plant Sciences, University of Sheffield
2. Department of Physics and Astronomy, University of Sheffield
3. Faculty of Medicine, Dentistry & Health, University of Sheffield
4. The Florey Institute, University of Sheffield
5. Krebs Institute, University of Sheffield

Keywords

High-resolution AFM, Nanoscale Architecture, Cell Wall, Zymoseptoria tritici, Cryptococcus neoformans.

Abstract text

The fungal cell wall is an essential structure for maintaining cellular integrity and viability [1]. The cell wall plays an important role in different biological functions such as controlling cellular permeability and protecting the cell from osmotic and mechanical stress [2,3]. Its disruption can lead to cell death, which makes it a good antifungal target. In contrast to the bacterial cell wall, the fungal cell wall is structured with its main inner core made up of glucan, chitin, and chitosan that then serve as a substrate upon which other components like glycoproteins and other polysaccharides are deposited [4,5]. Even though the molecular composition of the fungal cell wall is extensively studied, the detailed 3D structure of its complex architecture remains elusive. To this end, we used high-resolution atomic force microscope (AFM) to decipher the detailed three-dimensional, molecular organization of the purified extracted cell walls of two non-identical pathogens; Zymoseptoria tritici and Cryptococcus neoformans. The former is a wheat plant pathogen [6] and the latter is a human pathogen that causes severe infection of the central nervous system of immunocompromised individuals[4]. 

Starting with Zymoseptoria, by combining tapping mode and peak force tapping mode AFM, our results show that the longitudinal axis of the rod-shaped cell is predominantly associated with a combination of singular and bundled fibres that are oriented nearly parallel to the short axis of the cell. By doing location-dependent imaging, the AFM images show that the architecture at the pole is characterized with less orientated fibre network with pores smaller than those on the circumference. We further interrogate the inner sections of the wall, in particular, we observed that the inner section of the circumferential section of the wall is a dense layer with fine fibres that are oriented in the same direction as the short axis of the cell. In comparison, the pores in the inner section are smaller than any observed on the external section of the wall. 

To quantify the local orientation across different sections of the cell, we utilized the fibreFinder App [7] —an automated ridge detection app—built in the Matlab workspace, which gives an angular histogram of the local directionality as a function of pixel length and locality. Lastly, in this talk, I will show some of the preliminary results on Cryptococcus neoformans and how it is morphologically distinct compared to the Zymoseptoria tritici. Our findings on these pathogens show an exciting application of high-resolution atomic force microscope in revealing the detailed molecular organization at different locations on the fungal cell wall, which will further aid our understanding of fungi and potentially aid the discovery and design of new anti-fungal drugs.

References

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[2]        S.M. Bowman, S.J. Free, The structure and synthesis of the fungal cell wall, BioEssays. 28 (2006) 799–808. https://doi.org/10.1002/BIES.20441.

[3]        N.A.R. Gow, J.-P. Latge, C.A. Munro, The Fungal Cell Wall: Structure, Biosynthesis, and Function, Microbiol. Spectr. 5 (2017). https://doi.org/10.1128/MICROBIOLSPEC.FUNK-0035-2016/ASSET/EF31457D-DED9-4F8D-B8F6-E301F26771D1/ASSETS/GRAPHIC/FUNK-0035-2016-FIG7.GIF.

[4]        D.P. Agustinho, L.C. Miller, L.X. Li, T.L. Doering, Peeling the onion: The outer layers of cryptococcus neoformans, Mem. Inst. Oswaldo Cruz. 113 (2018) 1–8. https://doi.org/10.1590/0074-02760180040.

[5]        R. Garcia-Rubio, H.C. de Oliveira, J. Rivera, N. Trevijano-Contador, The Fungal Cell Wall: Candida, Cryptococcus, and Aspergillus Species, Front. Microbiol. 10 (2020) 2993. https://doi.org/10.3389/FMICB.2019.02993/BIBTEX.

[6]        S. Kilaru, M. Schuster, W. Ma, G. Steinberg, Fluorescent markers of various organelles in the wheat pathogen Zymoseptoria tritici, Fungal Genet. Biol. 105 (2017) 16–27. https://doi.org/10.1016/J.FGB.2017.05.001.

[7]        Oliver Meacock, Laia Pasquina-Lemonche, Abimbola Feyisara Olulana, William M. Durham, Simon J. Foster, Jamie .K. Hobbs; An Automated Fibre Orientation Analysis--FibreFinder, Manuscript-in-preparation.