High-resolution AFM reveals the nanoscale architecture of MRSA cell wall

Session

Stream 4 (AFM): Quantitative SPM for Biology, Biomedicine, and Bioinspired Technologies

Authors

Abimbola Feyisara Adedeji Olulana (1, 3), Bohdan Bilyk (1, 2), Laia Pasquina-Lemonche (1, 3, 4), Katarzyna Wacnik (1, 2), Xinyue Chen (1, 3), Simon .J. Foster (1, 2, 4), Jamie .K. Hobbs (1, 3, 4)

Affiliations

1. Krebs Institute, University of Sheffield
2. Department of Molecular Biology and Biotechnology
3. Department of Physics and Astronomy, University of Sheffield
4. The Florey Institute, University of Sheffield

Keywords

High-resolution AFM, peptidoglycan, MRSA, 3D architecture.

Abstract text

Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive bacteria that is genetically-distinct from the antibiotic-sensitive Staphylococcus aureus. Also, MRSA is part of WHO priority group of Superdrug that could lead to 10 million death in the year 2050¹. So far, the studies performed on resistance in MRSA have focussed on the genetic-mutation and evolution associated with MRSA but little has been done as touching understanding the physics that underpins resistance in MRSA².   Here are some of the questions that we seek to address; 1) what are the imprints of resistance on the cell wall architecture? 2) Can we distinguish the antimicrobial strains based on the material properties of their associated cell wall? 3) What is the link between the architectural differences and the inherent macroscopic resistance expressed by MRSA? In addition, 4) when the native penicillin-binding proteins are turned off via methicillin treatment, what are the architectural changes observed?

Our goal is to address these questions by using high-resolution atomic force microscopy (AFM) to decipher the associated cell wall, treated and not treated with an antibiotic. We utilized Tapping mode and PeakForce Tapping mode to examine the thickness of the purified sacculi and the 3D molecular architecture associated with the internal and the external surface of MRSA sacculi (extracted cell wall), without and with treatment with antibiotic. In a complementary fashion, we used wide-field fluorescence microscopy to characterize the cell size and cell cycle associated with MRSA cells and other derivatives of S.aureus, with the latter consisting different genetic modifications but within the same genetic background. 

We find that MRSA is associated with thicker cell wall (by approximately 35%) and reduced cell size (by approximately 30%) when compared to other S.aureus derivatives with no and low-level resistance. For the external surface, AFM reveals different subdomain of porous-rich mesh network, with changing depth, and an extra layer of mesh matrix in the Z-direction. In addition, the nascent peptidoglycan structure is characterized by a concentric rings-like structure in the absence of methicillin treatment, but this is replaced with dense but random structure when such cells are treated with methicillin. By studying the impact of antibiotics on cells with mutations in different cell wall synthesis proteins, we are able to gain new insights into which enzymes are responsible for which architectural features in the bacterial cell wall. This work brings us closer to methods for determining the molecular phenotype associated with particular genes, as well as to understanding how MRSA evades antibiotic-induced cell death.

References

References

1. de Kraker MEA, Stewardson AJ, Harbarth S. Will 10 Million People Die a Year due to Antimicrobial Resistance by 2050? PLoS Med. 2016;13(11):1002184. 
2. Panchal V V., Griffiths C, Mosaei H, et al. Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine-tuning of gene expression. Peschel A, ed. PLOS Pathog. 2020; 16(7):e1008672. 
3. Pasquina-Lemonche L, Burns J, Turner RD, et al. The architecture of the Gram-positive bacterial cell wall. Nature. 2020;582(7811):294-297