Inhibition of Intracellular Macromolecular Synthesis in Staphylococcus aureus by Thrombin-Induced Platelet Microbicidal Proteins

Journal of Infectious Diseases, Feb 2002

Thrombin-induced platelet microbicidal proteins (tPMP-1 and tPMP-2) are believed to initiate their staphylocidal effects via cytoplasmic membrane perturbation. The aim of the present study was to investigate the role of subsequent inhibition of macromolecular synthesis in the staphylocidal mechanisms of tPMP-1 and tPMP-2 in an isogenic tPMP-susceptible and -resistant strain pair (ISP479C and ISP479R, respectively). In ISP479C, tPMP-1 and tPMP-2 (2 mg/mL) exerted significant bactericidal effects and significantly reduced DNAand RNAsynthesis (P <.05 vs. con-trol). In contrast, tPMP-1 and tPMP-2 exerted reduced staphylocidal effects and significantly reduced inhibition of DNA and RNA synthesis against ISP479R, as compared with ISP479C (P <.05). However, tPMP-1 and tPMP-2 (2 µg/mL) caused equivalent degrees of inhibition of protein synthesis in both ISP479C and ISP479R. Collectively, these observations are consistent with the hypothesis that inhibition of specific macromolecular synthesis pathways is integral to the overall staphylocidal mechanism(s) of tPMPs.

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Inhibition of Intracellular Macromolecular Synthesis in Staphylococcus aureus by Thrombin-Induced Platelet Microbicidal Proteins

Yan-Qiong Xiong () 1 Arnold S. Bayer 0 1 Michael R. Yeaman 0 1 0 UCLA School of Medicine , Los Angeles, California 1 Department of Medicine, Division of Infectious Diseases, St. John's Cardiovascular Research Center, Harbor-UCLA Research and Education Institute , Torrance Thrombin-induced platelet microbicidal proteins (tPMP-1 and tPMP-2) are believed to initiate their staphylocidal effects via cytoplasmic membrane perturbation. The aim of the present study was to investigate the role of subsequent inhibition of macromolecular synthesis in the staphylocidal mechanisms of tPMP-1 and tPMP-2 in an isogenic tPMP-susceptible and -resistant strain pair (ISP479C and ISP479R, respectively). In ISP479C, tPMP-1 and tPMP-2 (2 mg/mL) exerted significant bactericidal effects and significantly reduced DNA and RNA synthesis (P , .05 vs. control). In contrast, tPMP-1 and tPMP-2 exerted reduced staphylocidal effects and significantly reduced inhibition of DNA and RNA synthesis against ISP479R, as compared with ISP479C (P , .05). However, tPMP-1 and tPMP-2 (2 mg/mL) caused equivalent degrees of inhibition of protein synthesis in both ISP479C and ISP479R. Collectively, these observations are consistent with the hypothesis that inhibition of specific macromolecular synthesis pathways is integral to the overall staphylocidal mechanism(s) of tPMPs. - Evidence from several laboratories strongly supports the concept that mammalian platelets are integral to host defense against infection [1 6]. Conceptually, the antimicrobial functions of mammalian platelets have recently been linked to their release of a group of small, cationic peptides termed platelet microbicidal proteins (PMPs) [1, 5 17]. Thrombin-induced PMPs (tPMPs) are released in vitro from rabbit and human platelets stimulated with thrombin, a molecule generated at sites of endovascular damage in vivo [18 20]. For example, platelet factor 4, connective tissue activating peptide 3, and derivative thrombocidins are members of the group of tPMPs identified from human platelets [2, 5, 9, 10]. The majority of these peptides exert rapid and potent microbicidal effects in vitro against a broad spectrum of microbial pathogens that commonly invade the bloodstream, including Staphylococcus aureus [1, 3, 5, 6, 1317]. Although the interactions of some tPMPs with target microbial membranes and artificial membrane models have been studied in detail [17, 21 23], their overall microbicidal mechanism(s) have not been fully defined. Studies in our laboratories, focusing on tPMP-1, have demonstrated that this peptide targets and disrupts the S. aureus cytoplasmic membrane, which leads to rapid permeabilization without durable depolarization [17, 21, 22]. Although S. aureus membrane disruption due to tPMP-1 typically occurs within minutes, death of susceptible strains is delayed by 30 120 min after initial exposure [17]. This temporal dissociation between initial tPMP-1 induced membrane damage and eventual staphylococcal death suggests that other, likely intracellular target(s) may also be involved in the lethal mechanism(s) of this peptide. In support of this hypothesis, we recently showed that pretreatment of S. aureus with an inhibitor of DNA gyrase subunit B (novobiocin) or inhibitors of 30S or 50S ribosomal subunits (tetracycline or azithromycin, respectively) significantly reduced subsequent S. aureus killing by tPMP-1 [12]. In contrast, pretreatment of S. aureus with cell wall synthesis inhibitors (i.e., penicillin or vancomycin) significantly enhanced the antiS. aureus effects of tPMP-1 [12]. Thus, our current study was carried out to further examine the relationship between the staphylocidal effects of tPMP-1 and tPMP-2 and macromolecular synthesis in isogenic S. aureus strains differing in their intrinsic tPMP susceptibility profiles. Materials and Methods Organisms. A well-characterized, isogenic tPMP-susceptible (tPMPS; ISP479C) and tPMP-resistant (tPMPR; ISP479R) pair of S. aureus strains was used in these studies. S. aureus ISP479C is a derivative of the parental strain, ISP479, spontaneously cured of the plasmid pl258 containing transposon Tn551 (erythromycin susceptible) [24]. ISP479R is a stable transposon mutant of ISP479, isolated by transposon mutagenesis of a single Tn551 chromosomal insertion (erythromycin resistant) [24]. Recent genetic analyses have indicated that the Tn551 insert appears to reside within a locus with high homology to mnhD, which encodes a key Na+/H+ antiporter in S. aureus and is involved in alkaline and osmotic homeostasis, cation antiport, and multidrug export (P. J. McNamara, R. A. Proctor, M. R. Yeaman, and A. S. Bayer, unpublished data). Extensive phenotypic and genotypic analyses have demonstrated these strains to be identical, except for their difference in tPMP-1 susceptibility [24]. Bacillus subtilis strain ATCC 6633, highly susceptible to tPMPs, was used to standardize the bioactivities of tPMP preparations [13]. All organisms were stored at 270 C until being subcultured onto 6.6% sheep blood agar plates. Logarithmic-phase cells were obtained by inoculating several colonies into brain-heart infusion broth (Difco Laboratories) and incubating at 37 C until an OD at 600 nm of 0.6 was achieved. Cells were harvested by centrifugation, washed twice in PBS at pH 7.2, sonicated for 4 s to ensure single organisms, and adjusted to the desired final inocula by use of a spectrophotometer (Milton Roy Co.). Bacterial inocula were routinely confirmed by quantitative cultures on sheep blood agar plates. Antibiotics. Novobiocin, rifampin, and tetracycline were purchased from Sigma, Marion-Merrell Dow, and Aldrich Chemical, respectively. Novobiocin is a prototype DNA synthesis inhibitor (blocks DNA gyrase subunit B); rifampin is a well-established inhibitor of RNA synthesis (blocks bacterial DNA-dependent RNA polymerase); and tetracycline served as a representative inhibitor of bacterial protein synthesis (30S ribosomal subunit). These antibiotics were prepared in appropriate diluents from standard powders, as directed by the manufacturers recommendations. Antibiotic susceptibilities of S. aureus ISP479C or ISP479R. MICs of novobiocin, rifampin, and tetracycline for S. aureus ISP479C or ISP479R were determined in Mueller-Hinton broth (Difco), following National Committee for Clinical Laboratory Standards guidelines [25]. A broth microdilution technique was used, with final S. aureus inocula of either 105 or 107 cfu/mL [25]. These inocula were chosen because 105 cfu/mL is a standard inoculum for antibiotic susceptibility testing and 107 cfu/mL represented the starting inoculum for all macromolecular synthesis assays (see below). The range of antibiotic concentrations tested was 0.003 128 mg/mL for all agents. MICs were defined as the lowest antibiotic concentration yielding no visible growth after 18 h of incubation at 37 C. All MICs were determined at least twice on separate days, and MICs exhibited <1 dilution variance. Radioisotopes. The following isotopes were purchased from Amersham Life Science: [methyl-3H]thymidine, [5-3H]uridine, and L-[4,5-3H]leucine. The specific activities of these isotopes were adjusted by appropriate additions of stock solution, as recommended by the manufacturer. Purification of tPMP-1 and -2. Rabbit tPMP-1 and -2 were purified by reverse-phase high-performance liquid chromatography (RP-HPLC), as described elsewhere [16, 26, 27]. In brief, rabbit platelets were isolated from freshly collected and anticoagulated blood of the New Zealand white rabbit (Irish Farms Products and Services). Platelets were washed twice in Tyrodes salts solution (pH 6.8; Sigma) and were resuspended in Eagle MEM (pH 7.2; Irvine Scientific). tPMP-1 and tPMP-2 rich preparations were produced by platelet stimulation with bovine thrombin (3 U/mL; Sigma) in the presence of 0.2 M CaCl2 and 0.1% (vol/vol) plateletpoor plasma at 37 C for 30 min. The supernatant was then collected by centrifugation (2000 g for 10 min). Previous analyses by SDS-PAGE and RP-HPLC have demonstrated that tPMP-1 and tPMP-2 are the predominant cationic staphylocidal peptides present in such preparations [13, 16]. tPMP-1 and tPMP-2 were then homogeneously purified from platelet supernatants by gel filtration and analytical RP-HPLC [16]. After their isolation, the purities of tPMP-1 and tPMP-2 were confirmed by acid-urea PAGE, SDSPAGE, and analytical RP-HPLC [16]. In addition, RP-HPLC peak integration and modified Lowry protein assays were used to accurately estimate the final tPMP-1 and tPMP-2 concentrations, as described elsewhere [28]. Purified tPMP-1 and -2 were lyophilized, resuspended in sterile 0.01% acetic acid, and stored until use at 4 C. Bioactivity of tPMP-1 and tPMP-2. The microbicidal activities of purified tPMP-1 and tPMP-2 were confirmed, as described elsewhere, with B. subtilis ATCC 6633 [13, 16]. In brief, a B. subtilis inoculum of 104 cfu/mL was added to microtiter wells containing a range of concentrations of purified tPMP-1 or tPMP-2, to achieve final inocula of 103 cfu/mL and final tPMP-1 or tPMP-2 concentrations of 0 2 mg/mL. The mixtures were incubated at 37 C for 30 min, were briefly sonicated to ensure single organisms, and were quantitatively cultured onto 6.6% sheep blood agar plates. After incubation at 37 C for 24 h, the bactericidal activity of tPMP-1 or -2 was confirmed by 100% killing of 103 cfu/mL of B. subtilis within 30 min at 0.5 mg/mL of tPMP-1 or -2 [13, 16]. Staphylocidal assays of tPMP-1 and tPMP-2. The staphylocidal activities of purified tPMP-1 and -2 were evaluated, as described elsewhere [13, 16]. In brief, purified tPMP-1 or -2 was resuspended in sterile 0.01% acetic acid buffer adjusted to pH 7.2. The S. aureus strains were grown to logarithmic phase, washed twice, and resuspended in 200 mL of MEM, to achieve a final concentration of 107 cfu/mL (the final inoculum for macromolecular synthesis assays; see below) and final concentrations of either purified tPMP-1 or purified tPMP-2 of 2 or 4 mg/mL. After 0, 30, 60, 90, and 120 min of incubation at 37 C, aliquots, as well as untreated controls, were obtained from reaction tubes, sonicated, serially diluted, plated on Trypticase soy agar plates (Difco) containing 0.01% sodium polyanetholsulfonate (an anionic compound used to inhibit further cationic tPMPinduced killing [13]), and incubated for 24 h at 37 C. Surviving colonies were counted, and the staphylocidal activities of tPMPs were expressed as the percentage of surviving colony-forming units versus untreated controls. All assays were done independently >2 times in triplicate, and the mean percentage of survival (^SD) was determined for each exposure condition. Measurement of DNA, RNA, and protein synthesis. The effects of tPMP-1 and tPMP-2 on DNA, RNA, and protein synthesis in the pair of S. aureus strains were determined by measuring the respective incorporations of [methyl-3H]thymidine, [5-3H]uridine, or L[4,5-3H]leucine into trichloroacetic acid insoluble material, as described elsewhere [29]. Novobiocin, rifampin, and tetracycline were used as controls for inhibition of DNA, RNA, and protein synthesis, respectively. Radioactive precursors were added to logarithmicphase S. aureus cells (10 mCi/mL of [methyl-3H]thymidine, 5 mCi/ mL of [5-3H]uridine, or 5 mCi/mL of L-[4,5-3H]leucine) in control medium (MEM only) or in medium containing a single antibiotic alone (5MIC) or tPMP-1 or tPMP-2 alone (2 mg/mL). This latter peptide concentration (2 mg/mL) was chosen for determining macromolecular synthesis inhibition because it represents a concentration at which both tPMPS and tPMPR study strains were progressively killed over 2 h of incubation, although to a greater or lesser extent, respectively (see Results). To assess DNA or RNA synthesis, 25-mL samples were removed from each reaction tube at selected incubation times (30, 60, 90, and 120 min), mixed with 50 mL of 2% Triton X-100 (Sigma; final concentration, 0.04%), and diluted with 2.5 mL of cold 10% trichloroacetic acid (TCA). The Triton step was not used in [3H]leucine incorporation assays, because of the nonspecific protein disruption effects of this reagent (product information, Sigma). Moreover, for protein synthesis studies, we obtained sample aliquots at several additional early time points (e.g., 15, 30, and 45 min). After >30 min in 10% cold TCA with 0.04% Triton X-100 (for DNA and RNA synthesis assays) or 3 h in 10% cold TCA only (for protein synthesis assays), samples were filtered through glass fiber filters (GF/A; Whatman LabSales). These extraction procedures yielded similar and adequate degrees of lysis of both S. aureus strains (see below). The reaction or incubation tubes and filters were washed 3 times with 5 mL of cold 5% TCA and twice with 2 mL of 95% ethanol. Filters were dried and were placed in vials containing 10 mL of counting fluid (Amersham Life Science). Radioactivity was measured by liquid scintillation (LS-100; Beckman Instruments). For each macromolecular synthesis assay, >3 separate experiments were done independently on different days. The impact of tPMP exposure on radioisotopic precursor incorporation over time was determined as 1 experimental counts per minute=control counts per minute 100% and was expressed as the mean percentage of inhibition versus control (^SD). To ensure that the DNA, RNA, and protein extraction procedures would be equivalent for both S. aureus strains, we examined the lytic effects of 10% cold TCA, with or without 0.04% Triton X-100, on these strains. Exponential-phase S. aureus cells grown in brain-heart infusion broth were collected by centrifugation (2000 g for 10 min), washed twice with PBS, resuspended in PBS, and adjusted to an OD at 600 nm of 0.500 ( 108 cfu/mL). After this, 107 S. aureus cells were exposed to 10% cold TCA, with or without 0.04% Triton X100. The cells were incubated on ice, and the OD at 580 nm was measured at 0, 15, 30, 45, 60, 120, 180, and 240 min (time period encompassing nucleic acid and protein synthesis assays). Statistical analyses. Differences in the extent of staphylocidal effects and in the inhibition of radioisotope incorporation between control and experimental groups were compared with the unpaired Students t test. These analyses were done with the statistical package within Sigma Plot (version 2.02). P , :05 was considered to represent a statistically significant difference. Results Antibiotic susceptibilities of S. aureus ISP479C and ISP479R. ISP479C and ISP479R were equally susceptible to all antibiotics tested. The MICs of novobiocin, rifampin, and tetracycline for both strains were identical: 0.25, 0.06, and 0.5 mg/mL, respectively, at an inoculum of 105 cfu/mL and 0.5, 0.125, and 2 mg/mL, respectively, at an inoculum of 107 cfu/mL. Staphylocidal activities of tPMP-1 and tPMP-2. At 4 mg/mL, both tPMP-1 and tPMP-2 rapidly killed each S. aureus strain within 30 min of incubation (figure 1). At 2 mg/mL, each peptide significantly reduced the survival of the tPMPS S. aureus strain ISP479C over the 2-h incubation period (P , :05 compared with untreated controls [figure 1A]). However, the rate at which tPMP-2 killed this strain was somewhat slower than that of tPMP-1. For example, at 60 min of incubation, only 40% of ISP479C cells were killed by tPMP-2, compared with 70% of cells killed by tPMP-1 (figure 1A). In contrast, at the 2-h time point, tPMP-1 and tPMP-2 induced killing of this strain were equivalent. Thus, although the overall extent of killing of ISP479C was identical for tPMP-1 and tPMP-2 at 2 h, the staphylocidal kinetics of tPMP-1 appeared to be more rapid than those of tPMP-2. In contrast, the rate and extent of killing of the tPMPR S. aureus strain, ISP479R, were substantially reduced for both tPMP-1 and tPMP-2 at 2 mg/mL (figure 1B), compared with those of strain ISP479C. For example, only 30% 45% of ISP479R cells had been killed by tPMP-1 and tPMP-2 at 2 h of incubation, compared with 80% 90% killing of ISP479C cells (figure 1B). However, these latter differences did not achieve statistical significance. Inhibition of radioisotope precursor incorporation by tPMP1 and tPMP-2. The effects of tPMP-1 and tPMP-2 on incorporation of selected tracer isotopes reflecting DNA, RNA, and protein synthesis in S. aureus strains ISP479C and ISP479R are summarized in figures 2, 3, and 4, respectively. As noted in Materials and Methods, on the basis of the differential killing of the pair of strains by tPMP-1 and -2 at 2 mg/mL, we chose this concentration for use in the macromolecular synthesis assays described below. It was important to confirm that the TCA-based extraction protocols used for measuring isotope incorporation in this study were equally efficient at lysing the 2 S. aureus strains. We observed that 10% cold TCA with 0.04% Triton X-100 (as used for DNA and RNA synthesis inhibition assays) caused an equal lytic effect on both strains ISP479C and ISP479R over a 4-h incubation period. In addition, 10% cold TCA alone (as used for protein synthesis inhibition assays) exhibited a similar lytic effect on both S. aureus strains. DNA synthesis. Addition of tPMP-1 or tPMP-2 (2 mg/mL) to logarithmic-phase S. aureus ISP479C cells resulted in a rapid and extensive (.95%) inhibition of [3H]thymidine incorporation (figure 2A; P , :05 for tPMP-1 or tPMP-2 vs. untreated control cells). In contrast, for strain ISP479R when exposed to the same tPMP-1 or -2 concentration, the extent of inhibition of [3H]thymidine incorporation was substantially less than that observed for strain ISP479C (figure 2B; P , :05 for tPMP-1 or tPMP-2 vs. ISP479C). As expected, novobiocin (5 MIC) caused substantial inhibition of [3H]thymidine incorporation (50%) in both S. aureus strains, compared with untreated controls (P , :05). RNA synthesis. Profiles for tPMP-induced inhibition of [3H]uridine incorporation paralleled those noted above for inhibition of [3H]thymidine incorporation (figure 3). Thus, a significant inhibition of [3H]uridine incorporation (.90%) was observed within 30 min of exposure of the tPMPS strain, ISP479C, to tPMP-1 or tPMP-2 (2 mg/mL; figure 3A; P , :05 vs. untreated control). In contrast, at the same concentration of tPMP-1 or -2, [3H]uridine incorporation in strain ISP479R was inhibited by only 30% 45% over the same time period (figure 3 B; P , :05 for tPMP-1 or -2, ISP479C vs. ISP479R). As anticipated, rifampin (5 MIC) caused 80% and 65% inhibition of [ 3H]uridine incorporation in strains ISP479C and ISP479R, respectively (P , :05 vs. control). Protein synthesis. In contrast to the differential inhibitory effects of tPMP-1 and tPMP-2 observed for [3H]thymidine and [3H]uridine incorporation between the study strains (figure 4), both tPMP-1 and tPMP-2 (2 mg/mL) reduced [3H]leucine incorporation to equivalent extents in these strains over the 4-h sampling period (P , :05 vs. controls for ISP479C [figure 4C] and ISP479R [figure 4D]). Similarly, the impact of tPMP-1 on [3H]leucine incorporation at early time points (i.e., within the first hour of exposure) was also equivalent for both study strains (ISP479C [figure 4A] and ISP479R [figure 4B]). Tetracycline (5 MIC) reduced [3H]leucine incorporation by 70% 80% over the 4-h incubation period for strains ISP479C and ISP479R (P , :05 vs. controls). Temporal relationship between nucleic acid inhibition and staphylocidal activity of tPMPs. Further analyses were done to characterize the temporal relationship between inhibition of DNA and RNA synthesis and killing of S. aureus after exposure to tPMP-1 (figure 5). For strain ISP479C, tPMP-1 caused a significant staphylocidal effect that corresponded to a significant reduction in DNA and RNA synthesis (P , :05 vs. control). In contrast, for strain ISP479R, tPMP-1 exerted reduced staphylocidal effects, paralleling the inhibition of DNA and RNA synthesis that was significantly less than that in strain ISP479C (P , :05). However, for both S. aureus strains, maximal or nearmaximal inhibition of nucleic acid synthesis occurred well before the maximal staphylocidal impact of tPMP-1 (i.e., 30 min vs. 120 min for strain ISP479C and 60 min vs. 120 min for strain ISP479R). Discussion A number of studies from our laboratory have shown that tPMPs appear to interact with the S. aureus cytoplasmic membrane as an initial step in their eventual lethal mechanism. Moreover, alterations in the normal structure and/or function of the cytoplasmic membrane are associated with resistance to the bactericidal properties of these peptides (e.g., perturbations in membrane fluidity or transmembrane electrical potential [DC] [7, 17, 21 23]). However, several lines of evidence have recently demonstrated that membrane permeabilization alone is unlikely to account for the microbicidal mechanisms of tPMPs. Detailed ultrastructural studies of several tPMPS S. aureus strains have shown a clear temporal dissociation between initial membrane permeabilization (occurring within seconds to minutes of exposure to tPMPs) and subsequent cell death (occurring 30 120 min after tPMP exposures [17, 22]). This fact suggests that the inhibition of other, likely intracellular, pathways is involved in execution of the bactericidal mechanisms of tPMPs. Studies of the mechanisms of action of other antimicrobial peptides have also suggested the involvement of intracellular microbial targets [29 32]. For example, Lehrer et al. [29] showed that neutrophil defensins inhibited DNA, RNA, and protein synthesis in Escherichia coli in a time-dependent manner. Moreover, van den Broek et al. [33] demonstrated that protein synthesis inhibitors, such as azithromycin, could interfere with the antistaphylococcal activities of the neutrophil defensin hNP-1. To investigate the potential role of intracellular targets in the staphylocidal mechanisms of tPMPs, we recently examined the extent of tPMP-1 induced S. aureus killing after exposure to agents that block selected intracellular processes [12]. Inhibition of DNA or protein synthesis by conventional antibiotics (novobiocin or tetracycline, respectively) in a tPMPS S. aureus strain before exposure to tPMP-1 effectively prevented the microbicidal actions of this peptide. Moreover, a recent study from our laboratory showed that antibiotic-mediated inhibition of such intracellular processes could block tPMP-induced killing, without completely mitigating membrane permeabilization caused by these peptides [34]. These findings supported our hypothesis that tPMPs may directly and/or indirectly interfere with macromolecular pathways at the level of transcription or translation, after the initial perturbation of the cytoplasmic membrane. These observations provided the foundation for the present study, which examined the impact of tPMP-1 and tPMP-2 on macromolecular synthetic processes in an isogenic and wellcharacterized pair of S. aureus strains differing in tPMP susceptibility phenotypes. The present investigation has yielded several noteworthy findings. We demonstrated that tPMP-1 and tPMP-2 exerted staphylocidal effects against both tPMPS and tPMPR cells in a time- and concentration-dependent manner. As expected, the microbicidal activities of both peptides against tPMPS cells were substantially more rapid in onset and more extensive than those against tPMPR cells. In addition, tPMP-1 appeared to become staphylocidal somewhat more rapidly than tPMP-2. The differential staphylocidal effects of these peptides are unlikely to be attributable to very minor differences in the molar concentrations of tPMP-1 versus tPMP-2 that we used (249 nM vs. 226 nM, each equating to 2 mg/mL). In addition, our previous studies have demonstrated that tPMP-1 and tPMP-2 appear to differ in several ways, although they are both secreted from rabbit platelets in response to thrombin stimulation [16]. For example, these 2 peptides differ in molecular weight (8036 and 8858 for tPMP-1 and tPMP-2, respectively) and amino acid composition [5, 13, 16]. In addition, the microbicidal profile of tPMP-1 against E. coli, Candida albicans, and other S. aureus strains appears to differ from that of tPMP-2 (mirroring the data generated in the present study), particularly at pH 7.2 [16]. Elucidation of the precise basis for the differential microbicidal profiles of these peptides will involve further analyses of their structure-activity relationship, currently in progress. It is important that tPMP-1 and tPMP-2 each significantly inhibited isotope incorporation into macromolecules, which indicates direct and/or indirect inhibition of DNA, RNA, and protein synthesis in each study strain, compared with untreated controls. Moreover, the extent of DNA and RNA inhibition caused by the peptides was substantially greater in tPMPS cells than in tPMPR cells. However, the degree of inhibition of protein synthesis was equivalent for both peptides against each S. aureus strain over the entire study period. This latter observation points to differential inhibition of macromolecules in the mechanism(s) of action of tPMP-1 and -2. Recent studies from our laboratory have characterized the relationship between tPMP-induced membrane permeabilization and lethality in S. aureus, which may provide insights into subsequent intracellular targeting of these peptides [34, 35]. These latter investigations showed that tPMP-induced membrane permeabilization was initiated rapidly after peptide exposure (within minutes) in both strains. Moreover, there was a direct correlation between the overall extent of membrane permeabilization and degree of tPMP-induced killing, with the tPMPR S. aureus strain exhibiting both reduced membrane permeabilization and reduced killing, compared with those of the tPMPS strain. Therefore, it is reasonable to postulate that the differences in tPMP-induced inhibition of DNA and RNA synthesis between these strains may relate to differences in the extent of peptide access to the S. aureus cytoplasm (see figure 6). Such access may be related to the degree of membrane permeabilization induced by tPMP-1 and tPMP-2. For example, it is conceivable that membrane permeabilization may facilitate subsequent tPMP entry into the cell cytoplasm to access intracellular targets, such as DNA and RNA. Of note, several unrelated cationic antimicrobial peptides (including tachyplesin and buforin) have recently been shown to penetrate across target bacterial membranes and to bind to nucleic acids [36, 37]. Thus, although the degrees of tPMP-induced inhibition of DNA and RNA synthesis were substantially less in tPMPR than in tPMPS cells, it is possible that the extent of such inhibition in tPMPR cells may have exceeded a threshold sufficient to impair downstream protein synthesis. Alternatively, tPMPs may induce sufficient membrane permeabilization in both tPMPS and tPMPR cells to yield direct loss of critical cytoplasmic contents, reduced cellular energetics, and/or other global effects, resulting in secondary interference with protein synthesis. Moreover, it is also possible that different intracellular targets have differential inhibition thresholds. For example, inhibition of protein synthesis may require a relatively low intracellular tPMP concentration threshold, yielding equivalent inhibition of protein synthesis in both tPMPS and tPMPR strains. However, inhibition of nucleic acid synthesis could require a higher intracellular tPMP concentration threshold that is achievable to a greater degree in tPMPS than in tPMPR strains, corresponding to the differential tPMP permeabilization of these strains. Each of these possibilities would be consistent with our present observations of equivalent degrees of inhibition of protein synthesis but different degrees of inhibition of DNA and RNA synthesis in tPMPS versus tPMPR cells. Studies to address these hypotheses are under way but are beyond the scope of this report. In summary, the data from the present investigation serve to emphasize the role of direct and/or indirect inhibition of intracellular processes in contributing to the overall staphylocidal mechanisms of tPMPs. A hypothetical model of the proposed mechanisms of action of tPMPs is shown in figure 6. Taken together, these results support our hypothesis that tPMP-induced membrane permeabilization and inhibition of intracellular processes may be independent yet cooperative events in the staphylocidal pathways of tPMP-1 and tPMP-2. Acknowledgment We thank Kimberly Gank for excellent technical support in purifying the tPMP-1 and tPMP-2.


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Yan-Qiong Xiong, Arnold S. Bayer, Michael R. Yeaman. Inhibition of Intracellular Macromolecular Synthesis in Staphylococcus aureus by Thrombin-Induced Platelet Microbicidal Proteins, Journal of Infectious Diseases, 2002, 348-356, DOI: 10.1086/338514