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Structural sudies of the bacterial lipid rafts.

Periodic Reporting for period 1 - RaftsStruc (Structural sudies of the bacterial lipid rafts.)

Okres sprawozdawczy: 2018-11-01 do 2020-10-31

RaftsStruc MSCA IF project was focused on the structural characterization of the lipids rafts proteins by state-of-the-art technique: cryo-electron microscopy. Special focus was put on the components of the lipid rafts involved in S.aureus pathogenesis: penicillin binding protein 2a and its interacting partners, among them Flotilin- the scaffold protein of the Functional Membrane Microdomains (FMM).
Infection with methicillin-resistant S. aureus (MRSA) is a global health problem and a cause of high morbidity and mortality (Chambers EH et al. Postgrad. Med.2001). This is due to the great resistance of this bacterium to β-lactam antibiotics, due to the presence of an additional penicillin-binding protein (Pbp2a) (Chambers EH et al. Postgrad. Med.2001). Like other PBP proteins, Pbp2a catalyses the formation of peptide crosslinks between glycan chains of the cell wall. β-lactams act as substrate analogues. When bound to PBP, they inhibit their function; as a result the cell wall is weakened and the cell eventually dies (Chambers EH et al. Postgrad. Med.2001). Pbp2a has very low affinity for β-lactams; it therefore remains active and allows cell growth in the presence of the antibiotics. Pbp2a is a membrane protein and in S. aureus is part of the protein cargo of the FMM and interacts with flotillin A (FloA). The scaffold protein FloA was recently shown to influence Pbp2a oligomerization and/or protein-protein interactions, within lipid rafts, thus affects its function (Garcia-Fernandez et al. Cell, 2017). Yet the mechanism and structural character of this interaction is unknown.
The GENERAL OBJECTIVE of this proposal:
1. Obtain a structure by cryo-electron microscopy (cryo-EM) of in vitro-assembled lipid rafts
In order to obtain a structure of the in vitro assembled lipid rafts it is important to understand the architecture of the lipid rafts components. We focused on the structure of the lipid rafts protein components also to understand their function: Pbp2a, FloA, SA1401. To achieve that we overexpressed and purified each protein for structural studies also we studied protein-protein interactions and complex formation conditions.
2. Structural study of the interaction between the raft scaffold proteins FloA and Pbp2a (a S. aureus protein responsible for methicillin-resistant S. aureus (MRSA)) by cryo-EM and single-particle 3D reconstruction
This objective as a part of objective 2) also was based on overexpression and purification of the full length proteins using detergent based methods and affinity purification. Purified proteins were mixed and analysed for complex formation by Size Exclusion Chromatography and glycerol gradients with and without crosslinking. The samples were analysed by negative staining EM. Unfortunately the stability and quality of the sample was not good enough for further structural studies. However a novel interesting discovery was made: dimerisation of the Pbp2a and its implication on the antibiotic resistance. And we proceeded with the cryo-EM analysis of the dimeric Pbp2a.
Main projects results and possibilities to exploit them:
• Establishing heterogenous overexpresion conditions for three S.aureus USA300 proteins, parts of the FMMs: FloA, Pbp2a and SA1401Establishing purification strategy for three membrane proteins using detergents: FloA, Pbp2a and SA1401
• Extensive in vitro interaction studies and complex formation between Pbp2a-FloA-this will allow for further biochemical, biophysical and functional analysis of the proteins, possible complees formation and structural analysis.
• Biochemcal characterization of the newly discovered Pbp2a dimer by Size Exclusion Chromatography, Glycerol gradient, SEC-MALS, crosslinking-Mass-Spec, Bacterial Two hybrid, site-directed mutagenesis, this obtained results represent a novelty in the field of the antibiotic resistance, so all the studies were performed based on the truncated, soluable and monomeric form of the Pbp2a. The dimer formation may implicate different mechanism of antibiotic resistance and function of the protein in cell wall synthesis.
• Structural study of the uncharacterized S. aureus protein SA1401 by cryo-EM, 8 Å resolution structure was obtained allowing for fitting of the predicted atomic model, as a result a pseudo atomic model was obtained showing the mechanism of membrane insertion and remodeling. These results are entirely novel line of research revealing structure and function of uncharacterized protein from MRSA S. aureus that is involved in the formation of Functional Membrane Microdomains and pathogenesis.
• Membrane localization of the FMMs by tagging SA1401 by CLEM and immunolabeling. – CLEM has never been used for the protein localization in organisms as small as bacterial cells this innovative approach can be applied to other bacterial cells types and further developed for cryo-CLEM conditions.
During the development of the MSCA IF project I was studying interaction between two rafts components, flotilin and Pbp2a. They are both small membrane proteins that interact only in their native lipid environment. We were able to over-express and purify to homogeneity both of them.
Strikingly, while working with the purified full length Pbp2a we realized that the protein exists in the different oligomeric states. Moreover, the change in the oligomeric state brought decrease in the antibiotic resistance of S.aureus (Garcia-Fernandez E et al. Cell 2017). Currently we are finalizing structural and functional characterization of the full lenght Pbp2a dimer by cryo-EM combined with biochemical analysis. Our findings illustrate that in vivo Pbp2a forms a dimer and its disruption affects antibiotic resistance which was proven by point mutagenesis in mecA gene of S.aureus USA300 MRSA strain (manuscript in preparation).
In S.aureus MRSA, the sa1401 gene codes a gene for unknown function and is cotranscribed in an operon with a flotillin/prohibitin (PHB) homolog responsible for the formation of Functional Membrane Microdomains (FMM) in bacteria. So far there is no structural nor functional information about the protein. We overexpressed and purified a full length SA1401 from E.coli membranes. We performed cryo-EM analysis that allows us to obtain 8Å resolution structure of SA1401 dimer embedded in the detergent shell mimicking the lipid bilayer. The obtained pseudo-atomic model shows that the N-terminal amphiphatic helix (aa 1-20) serves as membrane anchor and upon insertion provokes membrane remodelling. To localize the SA1401 as a part of FMMs and regions of possible membrane remodelling, we performed CLEM analysis using S.aureus USA300 thin sections expressing SA1401 with the GFP tag and immunolabelling using monoclaonal anti-SA1401 antibodies.
During the fellowship period, I was able to successfully develop and implement the project on structural analysis bacterial lipid rafts and open a structural line of research in the host lab. This experience allowed for my further specialization in the field of EM, a fast-growing, essential technique for structural biology. New acquired skills from the field of microbiology and cell biology of S. aureus in combination with the structural biology techniques defined my future path and encouraged me to further investigate the structure and function of bacterial lipid rafts and proteins involved in the pathogenesis of S.aureus.
Dimerization analysis of the Pbp2a