Impact of Prokaryotic Phospholipid Translocation on Antimicrobial Resistance

Abstract

The increasing rates of multi-resistant bacteria represent a major human health threat. Therefore, development of novel anti-infective strategies and understanding of antimicrobial resistance mechanisms is crucial. The clinically relevant pathogen Staphylococcus aureus uses the ‘Multiple Peptide Resistance Factor’ (MprF) to resist treatment with daptomycin, a calcium-dependent antibiotic of last resort whose mode of action is still not fully understood. To gain resistance, MprF synthetizes the positively charged lipid lysyl-phosphatidylglycerol (Lys-PG) and translocates it from the inner to the outer cytoplasmic leaflet. This process leads to an increase in cell surface charge, and thereby repulsion of cationic antimicrobial peptides (CAMP) and CAMP-like antibiotics. During daptomycin treatment, single nucleotide polymorphisms in the mprF gene frequently take place, which can lead to a further increase in resistance and thereby a failure in treatment of S. aureus infections. Here, we analyze frequently found mutations, mediating a daptomycin-resistant phenotype. According to our data, we conclude that in a genetically defined background T345A may alter the substrate range of the MprF flippase to directly translocate daptomycin, because Lys-PG synthesis, translocation or cell surface charge were not affected while mutated MprF exhibited weakened intramolecular domain interaction. Since MprF is not an essential protein but a critical virulence factor, we developed monoclonal antibodies (mAB) targeting several epitopes of potential extracellular loops of MprF. Here, we describe a mAB which rendered S. aureus susceptible to combinational treatment with antibiotics or antimicrobial peptides and reduces the survival in human polymorphonuclear leukocytes (PMN) treatment. Furthermore, we demonstrate novel mechanistical insights into the translocation process of bacterial phospholipids, which we conclude from the proof of the targeted loop being exposed at both sides of the membrane leaflet. Apart from MprF, bacterial flippase proteins translocating phospholipids between the leaflets of the cytoplasmic membrane have remained largely unknown. Here, we demonstrate the widespread presence of the MprF flippase domain in Bacteria and Archaea. This domain can be found fused to different types of enzymes or encoded as a separate protein. The interaction with many phospholipid-synthetic enzymes and the impact on membrane fluidity and fitness in Escherichia coli led us to name this domain ‘Prokaryotic Phospholipid Translocator’ (PplT) and to propose a critical role in cellular homeostasis. Our findings highlight novel mechanistical insights into the translocation process of bacterial membrane lipids, which may instruct new anti-infective approaches to eradicate or disarm invading pathogens.