Molecular modelling and simulation of the N-Glycosylated efflux machinery in Campylobacter jejuni

Abstract

Antimicrobial resistance is a looming global health crisis, with a large proportion of antibiotics becoming increasingly ineffective as bacteria adapt to evade current therapies. The Gram negative bacterium Campylobacter jejuni is one such microbe with broadening resistance profiles. This bacterium is considered the leading cause of gastroenteritis in the developed world, infecting an estimated 400 million people globally every year. Drug-resistant Campylobacter has been labelled a serious threat by the Centers for Disease Control and Prevention; it is thus of interest to better understand the mechanisms by which this bacterium evades treatment.

Efflux machinery is a mode of resistance widely used by Gram negative bacteria to expel antimicrobials from the cell. C. jejuni harbours several efflux pumps, with CmeABC being considered the predominant machinery for this bacterium. Notably, the proteins comprising this assembly are known to be N -glycosylated. This thesis presents atomistic molecular dynamics simulations and molecular modelling of this post-translationally modified efflux machinery in complex membrane models.

The protein and glycan conformational dynamics of the outer membrane factor, CmeC, are explored, alongside substrate extrusion pathways through this channel. Novel models for lysophospholipids, an important class of lipid in C. jejuni, are developed, tested, and incorporated into a complex, species-specific inner membrane model. The induction of conformational cycling through protonation and substrate binding is investigated for the resistance-nodulation-division protein, CmeB, in this new lipid bilayer system. Protein structure prediction techniques are employed to generate a model for the structure of the hexameric periplasmic adaptor protein CmeA, which is combined with the inner and outer membrane components to yield an envelope-spanning complex. Finally, protein structure prediction, molecular dynamics, and quantum mechanical calculations are applied to the acyltransferase protein, OafB of Salmonella enterica, to investigate the structure and possible mechanism of action of the acyltransferase family 3 proteins.

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Identifiers

ORCID for Syma Khalid: orcid.org/0000-0002-3694-5044

ORCID for Jonathan Essex: orcid.org/0000-0003-2639-2746

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