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1 The LA Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA
2 The Department of Medicine, Harbour-UCLA Medical Center, Torrance, CA, USA
3 The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
4 Cellular and Molecular Microbiology, Medical Microbiology and Hygiene, University of Tübingen, Tübingen, Germany
5 Department of Microbiology, California State Polytechnical State University-Pomona, Pomona, CA, USA
6 Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, USA
Correspondence
Arnold S. Bayer
bayer{at}humc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2000; Wu et al., 1994; Yeaman et al., 1997, 1998). The best-characterized PMP is thrombin-induced PMP-1 (tPMP-1), a 
8 kDa peptide released in response to physiological stimuli at damaged or infected endovascular surfaces (Koo et al., 1996a, b, 1999; Xiong et al., 1999, 2002; Yeaman et al., 1998). A large body of evidence suggests that tPMP-1 plays a key role in host defence against endovascular infections, such as infective endocarditis (Dhawan et al., 1997; Fowler et al., 2000; Kupferwasser et al., 2002; Wu et al., 1994; Yeaman, 1997).
Our previous studies have identified the S. aureus cytoplasmic membrane (CM) as an initial target for the microbicidal actions of tPMPs. These peptides cause rapid permeabilization of the CM and the eventual death of susceptible bacteria in vitro (Koo et al., 1997, 2001). Thus, the intrinsic properties of the S. aureus CM may have a significant impact upon the capacity of tPMP-1 to initiate lethality. For example, using isogenic strain pairs, we have recently demonstrated that several engineered mutants that are relatively resistant to the lethal action of tPMP-1 on a tPMP-1-resistant (tPMP-1R) strain have more fluid CMs than their respective tPMP-1-susceptible (tPMP-1S) parental strains (Bayer et al., 2000).
The net charge of the S. aureus cell envelope affects the cells' susceptibility profile towards CAPs (Peschel, 2002). For example, the extent of D-alanylation of cell wall teichoic acids contributes to the relative positive charge of the S. aureus cell surface, and correlates with in vitro resistance to a number of CAPs (Peschel, 2002; Peschel et al., 1999). In addition, several laboratories have documented that the overall S. aureus CM PL composition also contributes to the microbial surface charge, and substantially influences the net antimicrobial activity of CAPs (Koprivnjak et al., 2002; Kristian et al., 2003; Nishi et al., 2004; Peschel, 2002; Peschel et al., 1999; Xiong et al., 2005). CM charge in S. aureus is principally determined by the relative contents of its three major PLs: the two negatively charged PLs, phosphatidylglycerol (PG) and cardiolipin (CL); and the positively charged PL, lysyl-PG (LPG) (Peschel et al., 2001). A related characteristic that may well affect CAP susceptibilities is the distribution of PLs within the CM. It is well known that PLs are asymmetrically distributed in the outer and inner CM leaflets in both prokaryotes and eukaryotes (Pomorski et al., 2004; Tannert et al., 2003). We hypothesized that a PL distribution yielding a relatively more positively charged outer vs inner CM leaflet might be associated with reduced susceptibility to the cationic tPMP-1 through reduced electrostatic affinities. This hypothesis has been verified in the current study through investigation of binding of two non-specific cationic proteins (cytochrome c and poly-L-lysine) as well as for three specific CAPs (tPMP-1, the tPMP peptide mimetic, RP1, and the human neutrophil defensin, hNP-1).
For this investigation, we utilized a well-characterized isogenic S. aureus strain pair, comprising a tPMP-1S parental strain, ISP479C, and its isogenic tPMP-1R counterpart, ISP479R. In addition, we have developed, and report here for the first time, three novel chromatographic and flow cytometric assays to delineate PL asymmetry within the S. aureus CM.
This study was presented in part at the 105th General Meeting of the American Society for Microbiology; Atlanta, GA; June 7th, 2005 (abstract #A-002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Dhawan et al., 1997). These strains are indistinguishable in terms of a number of phenotypic characteristics, including coagulase production and fibrinogen binding (Dhawan et al., 1997). The in vitro susceptibility testing of the isogenic strain pair against tPMP-1 has also been previously reported (Dhawan et al., 1997). These studies revealed that the mean percentage survival of strains ISP479C and ISP479R (103 c.f.u. inoculum) after 2 h exposure to tPMP-1 at 2 µg ml–1 was 20 % and 75 %, respectively (Dhawan et al., 1997). The arbitrary break point for tPMP-1R is 50 % survival (Wu et al., 1994). Complementation of snoD reinstated near parental-level susceptibility (35 % survival; Dhawan et al. 1997). Moreover, the CM fluidity characteristics of this strain set revealed that the tPMP-1R strain, ISP479R, has significantly more fluid CMs than the tPMP-1S parental strain, ISP479C (Bayer et al., 2006). Complementation reverses the fluidity profiles to parental levels. Thus, as previously reported, the polarization indices for the parental strain and complemented variant were 0.32±0.02 and 0.32±0.01, respectively, while that for the tPMP-1R strain, ISP479R, was 0.308±0.01 (P<0.05 vs parent and complemented variant; Dhawan et al., 1997). All the above studies utilized vigorous vortexing to ensure singlet organisms for assays.
Phospholipid (PL) analyses
Extraction, identification and quantification of PLs.
PLs were extracted from S. aureus by standard methods (Dixit & Gupta, 1998). Briefly, cells were grown for 18 h in BHI broth, harvested by centrifugation (3000 g, 4 °C, 15 min), and washed in buffer A (100 mM potassium phosphate, 5 mM EDTA, pH 7.2) followed by buffer B (100 mM potassium phosphate, 600 mM potassium chloride, pH 8.2). Cells (0.6 g wet weight, 1x108 cells ml–1) were suspended in 3 ml buffer B and transferred to a 25 ml glass conical flask and cooled to 4 °C with gentle swirling. The suspension was centrifuged and the pellet washed with buffer C (200 mM potassium acetate, 600 mM potassium chloride, pH 4.5) at 4 °C.
The cell pellet was then extracted using 2 : 1 (v/v) chloroform/methanol, followed by washing with 0.9 % NaCl to remove non-lipid contaminants. The extracted organic layer was evaporated to dryness under nitrogen and stored at –20 °C until analysis. The major PL species, PG, CL and LPG, were separated by two-dimensional thin-layer chromatography (2D-TLC) using Silica 60 F254 HPTLC plates (Merck) and subsequently developed with chloroform/methanol/25 % ammonium hydroxide (65 : 25 : 6, by vol.) in the vertical orientation, and chloroform/acetone/acetic acid/methanol/water (45 : 16 : 9 : 8 : 4, by vol.) in the horizontal orientation as detailed elsewhere (Dogra et al., 1999). LPG (positively charged) was identified by ninhydrin staining (Peschel et al., 2001). PG, CL and other minor PLs were visualized by exposure of the TLC plate to iodine vapour. All PLs were purchased from Avanti Polar Lipids and used as internal standards on the 2D-TLC plates.
For quantitative analysis, isolated PLs were individually recovered from TLC plates, and digested at 180 °C for 3 h with 0.3 ml 70 % perchloric acid. The digested samples were incubated with a colorimetric reagent [10 % ascorbic acid, 2.5 % ammonium molybdate, 5 % perchloric acid (1 : 1 : 8, by vol.)] for 2 h at 37 °C, and PLs quantified spectrophotometrically at 660 nm. The content of each PL species was expressed as a percentage of total PL content. All assays were performed a minimum of five times on separate days.
LPG distribution.
Fluorescamine, a fluorescent probe which specifically labels CM surface-exposed (outer leaflet), positively charged amino-PLs, was used to assay for CM LPG distribution (Balasubramanian & Gupta, 1996; Dixit & Gupta, 1998). Fluorescamine labelling was carried out by a modification of the 2D-TLC protocol above. To the cell pellet, 90 µl fluorescamine solution (0.52 M) in dehydrated dimethylsulfoxide was added drop-wise, and then subjected to 2D-TLC as described above. Fluorescamine labelling of outer-leaflet LPG was detected by using a UV detector (365 nm excitation). Once bound to LPG, fluorescamine alters its mobility characteristics, and its ability to be detected by ninhydrin staining is attenuated (Huijbregts et al., 1998). Inner-leaflet LPG was detected by ninhydrin staining.
After detection of the fluorescamine-labelled LPG, its relative content (normalized with respect to total PL and to total LPG) was quantified by the colorimetric assay above. All assays were performed a minimum of five times on separate days.
Cardiolipin (CL) distribution by NAO labelling.
For measurement of CL asymmetry, the fluorescent dye 10-N-nonyl acridine orange (NAO), was used. Following exposure to S. aureus cells, two NAO molecules bind to the two phosphate groups of the CL molecule, resulting in the shifting of NAO fluorescence from 525 nm (monomeric form of the dye) to 640 nm (dimeric form of the dye) (Gallet et al., 1995, 1997; Petit et al., 1992, 1994). NAO will bind initially to the outer CM leaflet CL. Following saturation of outer leaflet CL, the probe then initiates binding to CL distributed in inner CM leaflet regions until full saturation (Gallet et al., 1997; Petit et al., 1994).
Mid-exponential phase cells were adjusted spectrophotometrically to 106 cells ml–1 in HEPES buffer containing 10 % BHI broth and kept at 4 °C. Increasing amounts of NAO (1–5 µM) were added to the cell suspension, which was then incubated for 20 min at 4 °C. NAO binding to CL was measured by flow cytometry using a FACScalibur [Becton-Dickinson; excitation 488 nm, emission 
620 nm long-pass filter (FL-4 channel)]. For each analysis, 10 000 cells were acquired and plotted against forward light scatter (flow rate, 200 cells s–1). All assays were performed a minimum of two times on separate days. Quantitative culture was done to ensure that the interaction of NAO with the S. aureus CM did not alter viability.
Phosphatidylglycerol (PG) distribution.
Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine PL distribution and transport (Balch et al., 1994; Dekkers et al., 2000, Grant et al., 2001; Hrafnsdottir et al., 1997; McIntyre & Sleight, 1991). To investigate the distribution of PG, C6-NBD-PG was introduced into the CM of S. aureus cells by modification of the methods of Pomorski et al. (1996). At equilibrium, the distribution of the C6-NBD-PG probe closely reflects the overall distribution of the specific endogenous CL (Dekkers et al., 2000; Kol et al., 2004). Extensive pilot studies were performed with flow cytometry to define the optimal assay conditions. We determined that exposing S. aureus cells (2x107 cells) to C6-NBD-CL at 5 nmol per assay for 30 min yielded maximal labelling (representing saturation of both inner and outer CM leaflets). The ethanolic solution of C6-NBD-PG was added (1 % of the final volume) to the desired volume of cell suspension in HEPES buffer containing 10 % BHI broth with vigorous vortexing. Before labelling, the cell suspension was cooled to 4 °C to minimize C6-NBD-PG internalization. Next, the fluorescence of cell suspensions was measured by flow cytometry until saturation of fluorescence intensity occurred. The NBD moiety was excited at 488 nm with an argon laser, and the resulting fluorescence emission was collected through a 525 nm band pass filter (FL-1 channel). To quantify the specific outer leaflet distribution of C6-NBD-PG, NBD fluorescence was quenched by the CM-impermeant molecule sodium dithionite (final concentration 25 mM; Angeletti & Nichols, 1998; Balch et al., 1994; McIntyre & Sleight, 1991; Pomorski et al., 1996). Fluorescence remaining after dithionite treatment reflects the amount of C6-NBD-PG residing in the inner CM leaflet (McIntyre & Sleight, 1991).
Cytochrome c and poly-L-lysine binding.
Cytochrome c is a cationic protein derived from equine heart. It has been previously used to estimate the relative surface charge of the cell envelope of isogenic S. aureus strain pairs (Peschel et al., 1999). Poly-L-lysine is a polycationic molecule which is widely used to study the interaction between cationic peptides and charged bilayer membranes (Rossetti et al., 2004). We performed cytochrome c and FITC-labelled poly-L-lysine binding assays (Sigma) according to a previously described method (Peschel et al., 1999). In brief, S. aureus cells were grown overnight, and then washed twice with MOPS buffer (20 mM, pH 7 for cytochrome c) and HEPES buffer (20 mM, pH 7.25 for poly-L-lysine). The cells were suspended in the corresponding buffer to a final OD578 of 7 for cytochrome c and OD578 of 0.1 for poly-L-lysine. The suspension was incubated with 0.5 mg cytochrome c ml–1 or 10 µg poly-L-lysine ml–1 for 10 min in the corresponding buffer and centrifuged (for liposome binding assays, see below). The amount of cytochrome c remaining in the supernatant (i.e. unbound) was determined spectrophotometrically at 530 nm, and the amount of poly-L-lysine remaining in the supernatant was determined fluorometrically (excitation at 500 nm and emission at 530 nm) without or with bacterial exposure. The quantity of bound molecule was calculated from the difference between these values. The lower the amount of bound cytochrome c/poly-L-lysine, the more positively charged the S. aureus cell envelope (Peschel et al., 1999).
Peptide binding to S. aureus whole cells and model membranes
To differentiate the initial binding of tPMP-1 to the whole S. aureus cell (a combination of cell envelope and CM binding) from binding exclusively to the target CM, we utilized two complementary assays (whole-cell vs liposomal binding). For liposomal binding assays, we utilized purified tPMP-1 (prepared by RP-HPLC, Yeaman et al., 1997) and hNP1 (Peptide International) as a comparator CAP. For whole-cell binding assays, the binding of tPMP-1 to cells could not be done by the radial diffusion assay (see below) due to lack of peptide diffusibility through agar (Xiong et al., 2006). Moreover, the protein-spectrophotometric assay described below for tPMP-1 : liposome interactions could not be utilized for tPMP-1 : whole-cell assays due to interference by secreted exoproteins within whole-cell supernatants. Finally, the amount of RP-HPLC-purified tPMP-1 required for the whole-cell binding assays is prohibitive. Thus, to substantiate our findings, we instead utilized the synthetic tPMP-mimetic peptide RP1, which is an 18-amino acid peptide (N-ALYKK5FKKKL10LKSLK15RLG-C; mass 2163 Da) modelled upon the 
-helical C-terminal microbicidal domain of tPMP-1 (Yeaman et al., 2002; Yount et al., 2004). RP-1 precisely recapitulates the mechanism of action of the native molecule (Xiong et al., 2006). Also, as opposed to tPMP-1, this peptide diffuses readily through nutrient agar. RP-1 was synthesized with a symphony multiplex synthesizer (Rainin, Woburn, MA, USA) and authenticated by mass spectroscopy and amino acid analysis as previous described (Yeaman et al., 2002). Purified RP-1 was lyophilized and resuspended in sterile 0.01 % acetic acid for use in the microbiological assays.
Peptide binding to whole cells.
We performed whole-cell binding assays by a modification of a radial diffusion assay previously described (Takemura et al., 1996; Yeaman et al., 2002). Briefly, an agar diffusion plate was prepared by first seeding 106 c.f.u. ml–1 of Bacillus subtilis (ATCC 6633), a tPMP-1-hypersusceptible indicator strain (Yeaman et al., 1997), into 20 ml 2-(N-morpholino)ethanesulfonic acid (MES) agar supplemented with 1 % molecular-grade agarose and 0.03 % glucose (Sigma). This reaction mixture was then poured into 95x15 mm Petri dishes. After allowing agar solidification, approximate 50 µl wells (evenly spaced) were cut with a sterile instrument. For constructing the RP1 and hNP-1 standard curves, a range of peptide concentrations was added to the wells and allowed to incubate in each well for 3 h at 37 °C, then BHI agar was overlain. After the agar overlay had solidified, plates were incubated for 18 h at 37 °C. Zone sizes (mm) were measured using the SpotDense software within the FluorChem 8900 Imaging System (Alpha Innotech). The assay sensitivity was 1 µg ml–1. For determining peptide binding to whole cells, each strain was grown for 18 h in BHI broth at 37 °C, washed, and resuspended to a final inoculum of 108 c.f.u. ml–1. Both peptides (final concentration of 40 µg ml–1 in 0.01 % acetic acid) were then added to each organism and incubated for 10 min at room temperature, followed by centrifugation at 10 000 r.p.m. Supernatants were removed, lyophilized, and stored at 4 °C until assayed by the radial diffusion assay described above. Supernatant peptide concentrations were determined by achievable zone sizes referable to the above standard curve. Three independent assays were performed on different days. The amount of peptide bound to each S. aureus whole cell preparation was then calculated and displayed as peptide bound (µg ml–1; ±SD).
Peptide binding to model liposomes.
Liposomes containing synthetic LPG (DP-LPG), DPPG and bovine-CL were prepared as previously described (Xiong et al., 2005). We formulated liposomes of the following compositions to reflect a proportional range of the three major PLs in S. aureus whole cells: LPG : PG : CL of 0 : 7 : 1, 2 : 7 : 1, and 3 : 7 : 1. All lipid constituents were dissolved in chloroform : methanol (1 : 1), and dried under a stream of nitrogen followed by desiccation overnight. The lipid film was hydrated with HEPES buffer containing 9 % sucrose and then sonicated for 4 min and used within 2–3 days. Liposome stability was verified by calcein retention as previously described (data not shown) (Xiong et al., 2005). We confirmed that added LPG (the positively charged PL within the liposomal membrane) asymmetrically distributed within liposomes similar to its behaviour in whole cells, using the fluorescamine assay as described above. Thus, at liposomal formulations of LPG : PG : CL of 2 : 7 : 1 and 3 : 7 : 1, 30 % of LPG was distributed to the outer membranes (data not shown).
For cytochrome c and poly-L-lysine binding to model membranes, we followed essentially the same protocols as above for whole cells. We used 0.5 mg ml–1 liposomal concentrations for the binding assays. For the peptide-binding assays, a standard curve correlating each peptide concentration with spectrophotometric signals (595 nm) was established by employing a Coomassie–Bradford kit (Pierce) to determine the concentration of soluble peptide. We then added varying quantities of both peptides to liposomal solutions (0.5 mg ml–1) and incubated for 10 min at room temperature, followed by ultracentrifugation at 30 000 r.p.m. for 40 min to pellet the liposome–peptide aggregates (Satchell et al., 2003). The concentration of soluble (i.e. unbound) peptide in the supernatant was determined spectrophotometrically from the standard curve. The quantity of bound peptide (Peptideb) was calculated from the differences between the initial peptide concentration and that remaining in the supernatant. Total bound peptide (PeptideT) was calculated similarly as the highest concentration of peptide used (Moore et al., 1986).
Statistical analyses.
Data were analysed by the Kruskal–Wallis analysis of variance with corrections made for multiple comparisons where appropriate. Results are reported as means±SD. A P value 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The snoD-repaired variant of strain ISP479R, ISP479R-comp, exhibited parental-level PL content profiles.
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(a), two distinct LPG spots, labelled with either ninhydrin or fluorescamine, were detected. The UV-positive spot represents fluorescamine-labelled LPG (outer CM leaflet LPG), while the unlabelled, inner CM LPG was identified by ninhydrin spraying. As can be seen from Fig. 1(b), LPG was asymmetrically distributed overall (70–80 % in the inner leaflet). Furthermore, the outer-leaflet LPG content was significantly higher in the tPMP-1R strain, ISP479R, as compared to the tPMP-1S counterpart strain, ISP479C (27.3±11.0 % vs 18.6±7.0 %, respectively; P=0.05). Complementation of snoD in strain ISP479R yielded LPG distribution at near-parental levels. Thus, despite overall equivalent total LPG levels in their CMs amongst the strain set, the tPMP-1R construct translocated this positively charged PL to the outer CM leaflet at a significantly higher level than tPMP-1S strains. This suggested an important linkage between LPG asymmetry, net CM positive charge and tPMP-1R phenotypes.
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). These data confirmed that CL was asymmetrically distributed in the S. aureus CM, with >75 % localized to the outer leaflet (Fig. 2b). It is of interest that for the tPMP-1R strain, ISP479R, relatively more CL was localized to the inner CM than seen in the tPMP-1S strain (22 % vs 14 % for ISP479R vs ISP479C; Fig. 2b). However, this difference was not statistically significant.
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shows the distribution of C6-NBD-PG in the S. aureus CM after labelling the cells with C6-NBD-PG and quenching of outer leaflet C6-NBD-PG using sodium dithionite. PG was also shown to be asymmetrically distributed in the CM of both study strains. Thus, 75–85 % of this PL species was localized to the outer CM leaflet, with no significant distribution differences between strains.
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, the tPMP-1S strain, ISP479C, bound significantly higher amounts of the cationic proteins, cytochrome c and poly-L-lysine, than tPMP-1R strain, ISP479R (P<0.00001 for both comparisons).
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). Thus, ISP479C bound approximately 54 % and 61 % of RP-1 and hNP-1 in reaction mixtures, respectively, while ISP479R bound only approximately 27 % and 38 % of those peptides, respectively (P=0.03 for RP-1 and 0.013 for hNP-1 respectively).
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Cytochrome c and poly-L-lysine binding to model membranes
As seen in Fig. 6(a, b), there was an inverse relationship between relative LPG content of model liposomal membranes and binding of both cytochrome c and poly-L-lysine. For cytochrome c, this difference reached statistical significance (P<0.01 comparing the 0 : 7 : 1 vs 3 : 7 : 1 liposomes). For poly-L-lysine, these differences did not quite reach statistical significance (P=0.08).
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DISCUSSION |
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; Peschel, 2002; Yeaman & Yount, 2003). For PMPs, this interaction results in CM permeabilization, without depolarization or demonstrable pore formation (Koo et al., 1997, 1999; Yeaman et al., 1998). Our prior studies have principally focused on tPMP-1 as a prototype PMP for several reasons: (i) it is the most abundant PMP from rabbit platelets, a relevant animal model for endovascular pathogenesis (Yeaman 1997; Yeaman et al., 1997); (ii) it is microbicidal in vitro against important bloodstream pathogens, including S. aureus (Yeaman et al., 1997); (iii) the primary and secondary structures of tPMP-1 have been solved, and its tertiary structure has been modelled (Yount et al., 2004); (iv) bacterial strains with reduced susceptibilities to tPMP-1 exhibit enhanced survival in experimental endovascular infection models and in humans with such infections (Bayer et al., 1998; Dhawan et al., 1997; Fowler et al., 2000; Wu et al., 1994); and (v) tPMP-1, an analogue of human platelet factor-4, represents an archetypal microbicidal chemokine or kinocidin. Thus, this peptide possesses distinct C-terminal microbicidal and N-terminal chemokine domains, with an interposing domain consisting of anti-parallel 
-sheet domains, two of which constitute the 
-core motif common to other cysteine-stabilized kinocidins (e.g. NAP-2; Rantes; PF-4) (Yount et al., 2004).
We have previously studied S. aureus strains with reduced in vitro susceptibility to tPMP-1 which were derived by distinct methods, including serial passage in tPMP-1 (Yeaman et al., 1994); plasmid carriage of genes encoding efflux pumps for cationic compounds (e.g. QacA) (Kupferwasser et al., 1999); transposon mutagenesis of genes encoding enzymes involved in proton gradient pumps (i.e. snoD) (Bayer et al., 2006; Dhawan et al., 1997); small colony variant formation (e.g. hemB or menD mutants; Bates et al., 2003); and expression of genes encoding the synthesis and incorporation of positively charged amino acid incorporation into CM phospholipids (PLs) (e.g. mprF encoding lysinylation of PG) (Peschel et al., 2001). Although quite diverse, most of these tPMP-1R variant strains shared common CM alterations, especially enhanced fluidity and/or reduced transmembrane potential (
). It is of interest that these latter two features are interrelated (i.e. more fluid CMs leak protons, decreasing the net proton-motive force and of the CM; Haines, 2001). As threshold appears to be critical for the CM interactions of many CAPs, including tPMP-1, even modest reductions in can greatly affect such interactions (Koo et al., 1996a, b, 1997; Yeaman et al., 1998; Bayer et al., 2006).
In the context of these observations, the current investigation focused on two themes: characterizing CM PL content and distribution in an isogenic S. aureus strain pair differing in their in vitro susceptibility profiles to tPMP-1; and delineating the effects of these parameters on net CM charge and peptide binding. It is relevant to mention that even small changes in CM composition can have relatively large effects upon CM function and interaction with CM-active peptides (Lohner & Blondelle, 2005; Pokorny & Almeida, 2005). For example, Pokorny & Almeida (2005) recently showed that 
-lysin (a S. aureus-derived cytotoxic -helical peptide) interacted with small disordered model membrane domains, rather than ordered (lipid raft) domains. Importantly, a small change of the molar fraction of selected lipids (e.g. 5–10 %) significantly shifted the CM state of order, directly affecting the relative binding affinities of -lysin.
Several interesting observations emerged from our studies. In terms of overall PL composition, no substantial differences were noted in comparing the tPMP-1S vs tPMP-1R strains (e.g. ISP479C vs ISP479R). However, in investigating the transbilayer PL distribution, all three PLs were found to be asymmetrically distributed within the S. aureus CM in both strains (Angeletti & Nichols, 1998; Balasubramanian & Gupta, 1996; Dixit & Gupta, 1998; Gallet et al., 1995, 1997; McIntyre & Sleight, 1991; Petit et al., 1992, 1994; Pomorski et al., 1996). Asymmetric PL distribution, a well-defined phenotype in eukaryotic CMs, has pivotal biological impacts (e.g. in CM stability, intercellular recognition and signal transduction: Diaz & Schroit, 1996; Hakomori, 2003; Kol et al., 2002; Pomorski et al., 2004; Tannert et al., 2003). By contrast, positively charged aminophospholipids (particularly phosphatidylserine) are preferentially oriented in the inner CM leaflet of eukaryotes under normal physiological conditions (Balasubramanian & Gupta, 1996; Cerbon & Calderon 1995; Dixit & Gupta, 1998). However, during microenvironmental shifts, this latter PL can translocate to the outer CM leaflet, where it plays an essential role in such diverse processes as blood coagulation, ageing, apoptosis, membrane fusion and cell–cell recognition (reviewed by Diaz & Schroit, 1996; Fadok et al., 2000; Lentz, 2003; Pomorski et al., 2004; Tannert et al., 2003).
It is of particular interest that not only was asymmetric PL distribution common to both study strains, but distinct differences were observed in comparing the parental tPMP-1S and tPMP-1R constructs. For LPG (the positively charged PL species found uniquely in S. aureus), the majority of this PL was distributed within the inner CM leaflet. However, the tPMP-1R construct, ISP479R, exhibited 47 % more outer-leaflet LPG than the tPMP-1S parent, ISP479C, as well as the snoD-complemented variant (Table 1
; Fig. 1b). Such asymmetry differences likely enhance the relative net positivity of the CM surface charge of ISP479R, and might potentially contribute to the reduced tPMP-1 susceptibility phenotype of this strain by a charge repulsion mechanism (Peschel, 2002; Peschel et al., 1999, 2001). Data from the cytochrome c and poly-L-lysine binding assays support this notion, as the tPMP-1R construct bound substantially smaller amounts of these cationic molecules than its tPMP-1S parent (Fig. 4). Our peptide-binding studies further clarified this latter concept. Using the tPMP-1 synthetic analogue RP-1, and hNP-1 (both cationic peptides, having net charges of +8 and +4 respectively), we showed that relative binding of these peptides to the whole cell envelope was decreased in ISP479R as compared with ISP479C (Fig. 5). Similarly, using model liposomal membranes constructed to reflect the range of PL profiles of whole S. aureus cells, increasing LPG content led to substantial reductions in binding of cytochrome c, poly-L-lysine, RP-1, tPMP-1 and hNP-1 (Figs 6 and 7). These data provide support for the idea that differences in interaction of tPMP-1 with the target cell surface envelope and CM are major determinants of tPMP-1 susceptibility profile differences between tPMP-1S and tPMP-1R strains. It should also be pointed out that, in addition to differences in envelope and/or CM charge, the intrinsic influences the interaction of cationic molecules such as tPMP-1 with the CM, and their subsequent transmembrane passage (Koo et al., 1996a, b, 1999; Yeaman et al., 1998).
Additionally, an asymmetric distribution for CL was documented, preferentially within the outer CM leaflet for both strains. As above for LPG and PG, strain ISP479R demonstrated a difference in its CL asymmetry vs strain ISP479C, with relatively less CL (10 %) in its outer CM leaflet. Importantly, CL can serve as a proton reservoir and cap for the CM F0-F1 ATPase proton gradient system (Kates et al., 1993). Thus, CL contributes to generation, and reduced amounts of CL (i.e. as in the ISP479R outer CM leaflet) could contribute to the altered phenotype of this latter strain.
Several recent studies have characterized lipid and PL translocases in Escherichia coli and Salmonella typhimurium (Doerrler et al., 2004; Kol et al., 2003, 2004; Polissi & Georgopoulos, 1996; Reyes & Chang, 2005). Among Gram-positives, only Lactococcus lactis has been shown to possess a gene (lmrA) encoding an ABC transporter involved in transport of amphiphilic drugs, as well as fluorescent lipid analogues, across the CM (Margolles et al., 1999). Our current data suggest that S. aureus possesses translocases for each of the major CM PLs. Interestingly, work by A. Peschel and co-workers has recently suggested that one domain of the mprF operon encoding PG lysinylation may be functioning as an LPG translocase (unpublished data). The genetic linkage between the snoD mutation in ISP-479R and the enhanced LPG translocation and increased membrane fluidity in this construct is not known at this time. Since the snoD mutation disrupts a complex I enzyme gene (an NADH-oxidoreductase maximally active under microaerophilic or anaerobic conditions), it is conceivable that these two distinct membrane adaptations are part of a coordinated response to an altered redox state. We are currently investigating this issue.
As ISP479 background strains are partially deficient in the function of the sigB stress-response operon (Horsburgh et al., 2002), current studies are ongoing in our laboratories to examine PL profiles and asymmetry in other tPMP-1S vs tPMP-1R strain pairs of sigB-intact genetic backgrounds. This will enable us to understand the role of sigB in the regulation of overall PL composition and asymmetry. Finally, our laboratories are also investigating potential contributions of CM fatty acid profiles to tPMP-1 susceptibility phenotypes.
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ACKNOWLEDGEMENTS |
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Edited by: W. B. van Leeuwen
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