Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides

Abstract

Staphylococcus aureus is the second leading cause of bacteremia worldwide and the most important pathogen in endovascular infections (1, 2). S. aureus has a particular propensity for developing multidrug resistance (e.g., methicillin-resistant S. aureus [MRSA] and vancomycin-intermediate S. aureus [VISA]), and serious infections with such strains result in enhanced attributable mortality (3).Since its FDA approval in 2003, daptomycin (DAP) has been utilized in many clinical settings, especially for recalcitrant MRSA infections, such as endocarditis (4-6). However, there have been a number of recently reported cases of clinical S. aureus strains that have evolved in vitro DAP resistance in the context of failing DAP treatment regimens, especially in endovascular syndromes. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation [7][8][9][10][11][12].) One of the major features of DAP-resistant (DAP r ) S. aureus strains is the acquisition of gain-in-function mutations in a relatively restricted cadre of genes, especially in the mprF locus (13-18). The MprF protein is responsible for the synthesis and translocation (flipping) of the unique positively charged phospholipid (PL) lysyl-phosphotidylglycerol (L-PG) from the inner-to-outer cell membrane (CM) leaflet (18)(19)(20). Increases in L-PG synthesis and flipping usually result in augmented positive surface charge; many investigators have speculated that this event leads to a charge-repulsive milieu for cationic molecules, such as host defense peptides (HDPs) and calcium-complexed DAP (13,(19)(20)(21)(22)(23). Over the past several years, a number of laboratories, including ours, have linked the presence of singlenucleotide polymorphisms (SNPs) within the mprF locus to the DAP r phenotype (13,14, 21,(24)(25)(26). These SNPs have occurred throughout the mprF open reading frame (ORF), although there are clearly hot spots within this locus for those SNPs linked to DAP resistance (13,14,19, 23). Investigations of mprF SNPs associated with DAP resistance in S. aureus have principally emerged from studies that used only a few or individual isogenic DAP-susceptible (DAP s ) and DAP r strain pairs. To address this limitation, in the current study, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs using 22 DAP s /DAP r isogenic clinical MRSA strain pairs, (ii) the relation-

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…2). However, SNPs in mprF were the most frequently encountered, and were accompanied by an increase in resistance to daptomycin and other CAMPs (nisin and human CAMPs e.g., [75,79]). Most mprF gain-of-function SNPs are located in the flippase domain, and they generally increase the total amount of Lys-PG in the membrane as well as the amount of Lys-PG translocated to the outer surface of the cytoplasmic membrane (e.g., [77,78]).…”

Phenotypic and genotypic correlates of daptomycin-resistant methicillin-susceptible Staphylococcus aureus clinical isolates

“…However, there have been numerous recent reports of clinical S. aureus strains that have evolved DAP-resistance (DAP-R) in vivo during DAP treatment (Kaatz et al ., 2006; Jones et al ., 2008; Marco et al ., 2008; Murthy et al ., 2008; Bayer et al ., 2014, 2015). Previous studies have linked several characteristic phenotypes with DAP resistance in S. aureus : i) enhanced positive surface charge (Jones et al ., 2008; Yang et al ., 2010); ii) altered cell membrane (CM) phospholipid profiles, especially increased content of positively-charged phospholipids (lysyl-phosphatidylglycerol [L-PG]) (Mishra et al ., 2012b, 2014); iii) CM fluidity/rigidity.…”

“…Previous studies have linked several characteristic phenotypes with DAP resistance in S. aureus : i) enhanced positive surface charge (Jones et al ., 2008; Yang et al ., 2010); ii) altered cell membrane (CM) phospholipid profiles, especially increased content of positively-charged phospholipids (lysyl-phosphatidylglycerol [L-PG]) (Mishra et al ., 2012b, 2014); iii) CM fluidity/rigidity. These phenotypes were often accompanied by a single or multiple genotypic changes such as upregulation of mprF and/or dlt-ABCD transcription (Yang et al ., 2009a, 2009b; Bayer et al ., 2016) and single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) (Murthy et al ., 2008; Bayer et al ., 2013, 2014, 2015, 2016; Yang et al ., 2013a). The mprF gene product, MprF, is a L-PG synthase which adds positively-charged lysine to the negatively-charged phosphatidylglyderol (PG) molecule within the staphylococcal CM (Peschel et al ., 2001; Staubitz et al ., 2004).…”

“…The mprF gene product, MprF, is a L-PG synthase which adds positively-charged lysine to the negatively-charged phosphatidylglyderol (PG) molecule within the staphylococcal CM (Peschel et al ., 2001; Staubitz et al ., 2004). It has been shown that the mprF SNPs were correlated with excess synthesis of L-PG in S. aureus CM and enhanced surface positive charge (Yang et al ., 2013a; Bayer et al ., 2014, 2015, 2016). More recently, it has been reported that all the mprF SNPs observed in the vast majority of DAP-R S. aureus strains are clustered within either one of the two ‘hot spot’ MprF domains: the central bifunctional domain or the C-terminal L-PG synthase domain (Ernst et al ., 2009; Bayer et al ., 2015).…”

“…It has been shown that the mprF SNPs were correlated with excess synthesis of L-PG in S. aureus CM and enhanced surface positive charge (Yang et al ., 2013a; Bayer et al ., 2014, 2015, 2016). More recently, it has been reported that all the mprF SNPs observed in the vast majority of DAP-R S. aureus strains are clustered within either one of the two ‘hot spot’ MprF domains: the central bifunctional domain or the C-terminal L-PG synthase domain (Ernst et al ., 2009; Bayer et al ., 2015). However, these latter data have mainly emerged from studies of DAP-R MRSA strains.…”