A Physicochemical and Biophysical Investigation into the Role of Lysyl- Phosphatidylglycerol in the Membrane of Staphylococcus aureus under mild acidic conditions

Background: Staphylococcus aureus readily colonises human epithelia despite the presence of innate defences including mild acidity and cationic antimicrobial peptides (CAMPs). Reduced sensitivity of S. aureus to these defences appears partly due to the increased biosynthesis of lysyl-phosphatidylglycerol (L-PG) in its plasma membrane. The precise mechanisms by which L-PG facilitates tolerance to acidity and CAMPs remain under-investigated, due to the lipid’s lability in mild aqueous conditions. This study examines the role of L-PG in responses to epithelial defences and describes the synthesis and characterisation of a stable L-PG analogue designed for use in biophysical experiments to investigate membrane defences in S. aureus.<br/>Methods: The genetic regulation of L-PG biosynthesis was studied by assessing changes in graXRS, vraFG and mprF expression in response to mild acidity. The effect of acidity on S. aureus membrane lipid composition was quantified by 31P NMR. Monolayers and bilayers formed from S. aureus lipid extracts and synthetic lipid models were employed in investigations into the effects of low pH and CAMPs on membrane structure and physicochemical properties. The experimental techniques included neutron diffraction, small-angle neutron scattering, neutron reflectivity, pressure–area isotherms, zeta potential measurements and 2H-NMR.<br/>Results: Increased L-PG synthesis at pH 5.5, to ~50% total phospholipid, correlated with mprF and graRS expression. The L-PG concentrations at pH 5.5 produced a condensing effect on the bacterial membrane (with both natural and synthetic lipids), making it cationically charged and less permeable to solvent. These membrane changes reduced electrostatic attraction of CAMPs and retarded their ability to partition into the membrane.<br/>Conclusions: L-PG plays an important role in S. aureus tolerance to mild acidity by altering plasma membrane charge and lipid packing properties. These membrane alterations also facilitate tolerance to other epithelial defences such as CAMPs, making L-PG biosynthesis a putative target for antimicrobial therapeutics.<br/>

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2014

Author dc:creator

• Rehal, Reg

Advisors dc:contributor.advisor

• Harvey, Richard Douglas
• Bruce, Kenneth

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/187fad13-3e3f-4664-9ebd-6c85b67b50e4

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/187fad13-3e3f-4664-9ebd-6c85b67b50e4