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Antimicrobial Agents and Chemotherapy, May 2006, p. 1656-1663, Vol. 50, No. 5
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.5.1656-1663.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Proteomic Study of Peptide Deformylase Inhibition in Streptococcus pneumoniae and Staphylococcus aureus

Wen Wang, Richard White, and Zhengyu Yuan^*

Vicuron Pharmaceuticals, 34790 Ardentech Court, Fremont, California 94555

Received 14 September 2005/ Returned for modification 30 January 2006/ Accepted 28 February 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptide deformylase (PDF) is an essential enzyme in both gram-negative and gram-positive bacteria. It hydrolyzes formylated N-terminal peptides to generate free N-terminal peptides during the process of protein maturation. Inhibition of this enzyme results in cessation of bacterial growth. We have examined the effect of a potent PDF inhibitor, LBM-415 (also known as VIC-104959), on the proteomes of Staphylococcus aureus and Streptococcus pneumoniae using two-dimensional electrophoresis. Both S. aureus and S. pneumoniae showed accumulation of many N-terminal formylated peptides/proteins upon PDF inhibition. In S. pneumoniae, formylated peptide/protein accumulation was time dependent. Following inhibition, subsequent removal of the inhibitor resulted in deformylation of formylated peptides/proteins; this recovery process was also time dependent. If instead the inhibited cells were maintained in the presence of sub-MIC levels of the PDF inhibitor, the formylated peptides/proteins remained for a much longer time, which correlated with a prolonged postantibiotic effect in vitro. These observations may have broader implications for the application of this class of antibiotics in vivo.

Top Abstract Introduction Materials and Methods Results Discussion References

 
With the emergence of resistance and cross-resistance to existing antibacterial drugs, agents with novel modes of action against pathogenic bacteria are in great demand. Over the past three decades, the only two antibiotic agents with novel mechanisms that have been brought onto the market are linezolid (brand name Zyvox) and daptomycin (brand name Cubicin). Inhibitors of peptide deformylase (PDF) are a new class of antimicrobials that have a distinctive mode of action and represent a unique opportunity in the antibacterial field (8, 10, 22).

PDF catalyzes the hydrolysis of formylated N-terminal peptides (formyl-NH-peptide) to generate free N-terminal peptides (H₂N-peptide) during the process of protein maturation. The enzyme is essential for the growth of both gram-negative and gram-positive bacteria (14, 16, 17). It is believed that all bacterial protein synthesis is initiated with N-terminal formylated methionine (18). Eukaryotic PDF homologs have also been identified, and the mammalian PDF has been proposed as an anticancer target (7, 13, 19). Unlike prokaryotic PDF, vertebrate homologs only exist in mitochondria and their exact functions are not clear. PDF inhibitors have demonstrated good selectivity toward bacterial cells versus mammalian cells, supporting this enzyme as a valid anti-infective target. Elucidation of the detailed mechanism of bacterial growth inhibition resulting from PDF enzyme inhibition not only will help to reveal how this class of antibacterial works, but also may provide insight into how to improve activity.

PDF inhibitor LBM-415 (also known as VIC-104959) was identified as an effective antibacterial agent and entered phase I clinical trials in preparation for its evaluation in the treatment of upper respiratory tract infections (5, 10, 11). Previous studies have shown that LBM-415 has different inhibitory effects on the growth of Staphylococcus aureus and Streptococcus pneumoniae: it is a slow bactericidal agent for S. pneumoniae but static for S. aureus (5). Although LBM-415's target (PDF) is essential in both organisms, formyl methionine transferase (FMT), the enzyme that formylates methionine to permit the initiation of protein synthesis, is essential in S. pneumoniae, but nonessential in S. aureus (14, 15). S. aureus with deletion of the FMT-encoding gene (fmt) is viable, indicating that the mutant can synthesize protein without formylation of its initial methionine. As expected, PDF is no longer an essential enzyme in this mutant. In S. pneumoniae, on the other hand, the genes encoding both FMT and PDF are essential and the FMT-PDF cycle cannot be bypassed. In the present work, growth inhibition by LBM-415 of S. aureus and S. pneumoniae was subjected to proteomic analysis using two-dimensional (2D) electrophoresis. The fate of N-terminal formylated peptides generated upon PDF inhibition in both organisms was studied, and the potential consequence of accumulation of N-terminal formylated peptide is discussed.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials. LBM-415 was prepared at Vicuron, and the detailed synthesis will be published elsewhere. Linezolid was extracted from commercial Zyvox solution using ethyl acetate. Phenylmethylsulfonyl fluoride, dithiothreitol (DTT), iodoacetamide, and lysostaphin were purchased from Sigma (St. Louis, MO). Urea, dimethyl sulfoxide (DMSO), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), immobilized pH gradient (IPG) buffers, and precast sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were obtained from Amersham Biosciences (Piscataway, NJ). Mueller-Hinton broth (MHB) was purchased from Becton Dickinson (Franklin Lakes, NJ). Horse blood was purchased from Quad Five (Ryegate, MT) and lysed by repeated freeze-thaw cycles followed by centrifugation.

Bacterial cultivation and collection. S. pneumoniae ATCC 49619 was grown in MHB supplemented with 5% lysed horse blood (MHB/LHB) in an incubator at 37°C to an optical density at 600 nm (OD₆₀₀) of 0.08 without shaking. Inhibitor (LBM-415 or linezolid) in DMSO was added to the exponentially growing bacterial culture to achieve a final concentration of 50 µg/ml. The bacteria culture was kept under the same conditions, and portions of the culture were collected at different times via centrifugation for 2D analysis.

S. aureus. Strain RN4220 (12) was grown in MHB at 37°C with shaking to an OD₆₀₀ of 0.14 before addition of the inhibitor (LBM-415 or linezolid) to a final concentration of 50 µg/ml. The bacterial culture was kept under the same conditions for 2 more hours and collected by centrifugation.

PAE and PAESME. S. pneumoniae ATCC 49619 was grown as described above. When the culture reached an OD₆₀₀ of 0.07, LBM-415 was added to a final concentration of 5 µg/ml and incubation continued for 3 more hours. The inhibitor-treated culture was then centrifuged, and the cell pellet was washed with an equal volume of phosphate-buffered saline (pH 7.4) and collected again by centrifugation. This cell pellet was resuspended in 5 times the original volume of MHB/LHB with or without 0.125 µg/ml LBM-415 (postantibiotic effect [PAE] or post-antibiotic sub-MIC effect [PAESME]), and the resulting bacterial culture was allowed to regrow. Fractions of the bacterial culture were collected by centrifugation at different time points after the inhibitor removal and prepared for 2D analysis.

Protein sample preparation and 2D electrophoresis analysis. S. pneumoniae cells were suspended in urea lysis buffer (8 M urea, 4% CHAPS, 2% IPG buffer, pH 3 to 10) and incubated for 30 min at room temperature. The crude lysate was centrifuged at 100,000 x g at 10°C for 1 h, and the supernatant was analyzed by 2D electrophoresis. Typically the soluble protein fraction was loaded on IPG strips (pH 4 to 7) with rehydration loading buffer (8 M urea, 2% CHAPS, 0.28% DTT and 0.5% IPG buffer, pH 4 to 7) and focused on IPGphore (Amersham Biosciences) for 12 h. The IPG strips were subsequently equilibrated with DTT followed by iodoacetamide and applied to precast SDS-PAGE gels. The second-dimension electrophoresis was performed by SDS-PAGE with an EttanDALTsix electrophoresis unit, and the gels were stained with silver stain according to the manufacturer's protocols (Amersham Biosciences). The silver-stained image was scanned and analyzed using ImageMaster 2D Elite software (Amersham Biosciences).

S. aureus cells were washed with 25 mM Tris (pH 7.5), and the pellet was resuspended in the same buffer including 2 mM phenylmethylsulfonyl fluoride. Lysostaphin was added to a final concentration of 50 µg/ml, and the mixture was incubated at 37°C for 20 min followed by passage through a French press. The crude lysate was centrifuged at 48,000 x g for 1 h at 10°C, and the soluble fraction was subjected to 2D analysis as described above. For protein identification, the soluble fraction of the lysate was further purified with a cleanup kit (Amersham Biosciences) before loading on the IPG strips; the final SDS-PAGE gels were stained with Coomassie blue. The protein spots of interest were excised from the stained gel and analyzed by Peptide Mass Mapping at the Protein Chemistry Core Facility of Columbia University (New York, NY).

Top Abstract Introduction Materials and Methods Results Discussion References

 
S. aureus protein expression by 2D electrophoresis. Comparison of protein composition was conducted for S. aureus in the presence and absence of LBM-415. Two hours after LBM-415 addition, the bacteria were collected and the soluble proteins were analyzed by 2D electrophoresis. The 2D image of the proteins between pH 4 to 7 showed many new protein spots emerging while the overall protein expression pattern remained unchanged compared to the control, to which no inhibitor was added (Fig. 1). These new protein spots appear to the left (i.e., more acidic) side of the original spots on the 2D gel; many peptides appear as doublets consisting of both newly generated acidic spots and original spots. To further illustrate this, a few examples of the doublets generated in panel B are compared to the corresponding proteins in panel A, as indicated by the numbered frames in Fig. 1. Even after 8 h of LBM-415 exposure, the total protein pattern remained the same, with many doublets persisting but without complete disappearance of the original protein spots (data not shown). In a separate experiment, the protein expression of a FMT mutant of S. aureus (where the formylation/deformylation process is not required for protein synthesis) (14) was examined and showed an identical 2D proteomic image to its parent strain under our experimental conditions (data not shown).

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FIG. 1. 2D electrophoresis images of soluble S. aureus proteins. (A) Bacteria grown in the absence of LBM-415. (B) Bacteria grown in the presence of 50 µg/ml LBM-415 for 2 h. MW, molecular weight.

Identification of selected S. aureus protein "doublets" by peptide mass mapping. In order to identify the newly generated left acidic spots due to LBM-415 addition, two pairs of the proteins were excised from SDS-PAGE gels stained with Coomassie blue and their trypsin digests were analyzed by peptide mass mapping (Protein Chemistry Core Facility, Columbia University). Both proteins highlighted in frame 4 in panel B of Fig. 1 shared similar peptide fragments after trypsin digestion and were indistinguishable from S. aureus alkyl hydroperoxide reductase subunit C. Table 1 shows the peptides detected by mass spectrometry for both right (R) and left (L) spots in frame 4 along with the predicted sequences of this S. aureus protein. S. aureus alkyl hydroperoxide reductase subunit C has a predicted molecular mass of 20,976.8 Da, which is consistent with the estimated position of these proteins on the gel shown in Fig. 1. Similarly, both proteins indicated in frame 5 in panel B of Fig. 1 were indistinguishable from S. aureus 50S ribosomal protein L7/L12. Table 2 shows the peptides detected for both the right and left spots in frame 5 along with their predicted sequences. S. aureus 50S ribosomal protein L7/L12 has a predicted molecular mass of 12,711.7 Da, which is consistent with the estimated position of these proteins in the gels.

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TABLE 1. Peptides detected by MALDI-MSa of tryptic digestions of proteins in Fig. 1, frame 4, and the corresponding predicted peptide sequences of S. aureus alkyl hydroperoxide reductase subunit C

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TABLE 2. Peptides detected by MALDI-MSa of tryptic digestions of proteins from Fig. 1, frame 5, and the corresponding predicted peptide sequences of S. aureus 50S ribosomal protein L7/L12

For comparison, S. aureus was also grown in the presence of the protein synthesis inhibitor linezolid at 50 µg/ml for 2 h. The overall protein expression after linezolid inhibition was almost identical to the pattern observed with bacteria grown in the absence of any inhibitor (data not shown).

S. pneumoniae growth and inhibition. The growth of S. pneumoniae in the presence and absence of 50 µg/ml LBM-415 (MIC = 0.5 µg/ml) and 50 µg/ml linezolid (MIC = 2 µg/ml) was investigated. The growth curves in Fig. 2 demonstrate that the bacteria responded to linezolid more rapidly than to LBM-415. An inhibitory effect is already apparent for linezolid at the first time point of 1 h, whereas only at the second time point (2 h) does LBM-415 cause a marked inhibition.

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FIG. 2. Time courses of antibacterial activity of linezolid and LBM-415 on S. pneumoniae. Inhibitors were added to the exponentially growing S. pneumoniae cultures at OD600s between 0.07 and 0.08, and the final concentration was 50 µg/ml for both compounds. The OD600s of the cultures were recorded at different times after inhibitor addition. The time of inhibitor addition was used as time zero. Samples for 2D analysis were taken at 3 h as indicated in the figure.

S. pneumoniae protein expression by 2D electrophoresis. The protein composition of S. pneumoniae in the presence and absence of LBM-415 was investigated by 2D electrophoresis. Three hours after inhibitor addition, S. pneumoniae was collected and 2D electrophoresis was performed on the soluble protein fraction (Fig. 3). The two gel images have many differences in the protein expression patterns. Compared to the control (Fig. 3A), the PDF inhibitor-treated sample (Fig. 3B) has many new spots, with the concurrent disappearance of many original spots. The new spots appear to the left of the original spots (acidic shift), and proteins frequently appear as doublets. With so many changes, a direct match between the two images became very difficult. The protein pattern displayed by bacteria after 8 h of incubation with the inhibitor was also compared to that of the bacteria grown for the same period of time in the absence of the inhibitor, and the difference was even more marked (data not shown).

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FIG. 3. 2D electrophoresis images of soluble S. pneumoniae proteins. (A) Bacteria grown in the absence of LBM-415. (B) Bacteria grown in the presence of 50 µg/ml LBM-415 for 3 h. MW, molecular weight.

For comparison, 2D electrophoresis of proteins from S. pneumoniae grown in the presence of linezolid (50 µg/ml for 3 h) was performed; the overall protein expression pattern was similar to that of bacteria grown in the absence of any inhibitor (data not shown).

Time dependence of formyl-peptide accumulation. The S. pneumoniae protein samples collected at different time intervals after LBM-415 addition were analyzed by 2D electrophoresis. Figure 4 shows a small field from the 2D gels generated over a time course of 0 to 3.5 h. For some proteins, no change was observed over time; the relative intensity of other proteins clearly demonstrated significant variation. With longer inhibitor exposure time, the intensity of the newly generated acidic proteins increased and the intensity of the original corresponding proteins decreased. Figure 4 also shows that the rate of accumulation for different acidic proteins was not the same.

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FIG. 4. 2D electrophoresis images of soluble proteins from S. pneumoniae that were collected from cultures exposed to 50 µg/ml LBM-415 for 0 (A), 30 (B), 60 (C), 90 (D), 135 (E), and 180 (F) min.

PAE and PAESME of S. pneumoniae. In the same way bacterial growth inhibition was studied, the recovery of bacteria after removal of the PDF inhibitor was investigated by 2D analysis. In the PAE experiment, S. pneumoniae was first incubated with 5 µg/ml LBM-415 for 3 h and then regrown in the absence of LBM-415. The bacteria were sampled at sequential time points, and the proteins of each sample were analyzed using 2D electrophoresis. A small field of the 2D gels is presented in Fig. 5. The intensity of the acidic proteins that accumulated during inhibition gradually decreased after the removal of the inhibitor. Eventually, all acid-shifted proteins disappeared, and the original protein spots regained their intensities 2.5 h after inhibitor removal. The same field of proteins was also examined in the PAESME experiment with bacteria shifted to 0.125 µg/ml LBM-415 (0.25x MIC; Fig. 6). Under this condition, the acid-shifted proteins remained in the bacteria for a much longer period of time. In fact, no decrease in intensity for these proteins was observed during the entire 2.5-h window of the experiment. Neither was recovery observed for the original basic protein spots during this period of time.

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FIG. 5. 2D electrophoresis demonstration of PAE of S. pneumoniae. S. pneumoniae was incubated with 5 µg/ml LBM-415 in growth medium for 3 h. The inhibitor was removed, and the bacteria were allowed to regrow in the absence of the inhibitor and sampled for 2D electrophoresis analysis (A) immediately or (B) 35 min, (C) 80 min, (D) 150 min, and (E) 210 min after medium shift.

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FIG. 6. 2D electrophoresis demonstration of PAESME of S. pneumoniae. S. pneumoniae was incubated with 5 µg/ml LBM-415 in growth medium for 3 h. The inhibitor was removed, and the bacteria were shifted to fresh growth medium containing 0.125 µg/ml LBM-415. The bacteria were sampled at different times for 2D electrophoresis analysis (A) immediately or (B) 80 min or (C) 210 min after medium shift.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the activity and essentiality of bacterial PDF are well understood, it is not clear how inhibition of PDF leads to the growth inhibition of bacteria. One possibility is that PDF inhibition, like that of many other protein synthesis inhibitors, stops the supply of functional proteins, resulting in growth arrest of the bacteria. Alternatively, the accumulation of nonfunctional formylated peptides due to PDF inhibition leads to the growth inhibition of the bacteria. Here we applied 2D electrophoresis to examine the impact of PDF inhibitor LBM-415 on protein expression in bacteria in order to understand how this novel class of antibacterial agents inhibits bacterial growth. Other PDF inhibitors with diverse chemical structures were also investigated in the same experiments; they exhibited similar effects toward the bacterial proteome compared to LBM-415 (not shown).

Inhibition of deformylase function leads to formyl retention at the N-terminal amino group, resulting in a protein with a more acidic isoelectric point (pI). Thus, 2D electrophoresis can be used to resolve the formyl-NH-peptide and NH₂-peptide induced by PDF inhibition (1, 3, 4). When S. aureus was treated with PDF inhibitor LBM-415, the 2D electrophoresis image showed emergence of many new protein spots (Fig. 1). The newly generated spots have similar molecular weights to their original counterproteins but with more acidic pIs. These new spots are attributed to the retention of the formyl group at the N terminus of nascent proteins due to deformylase inhibition. To substantiate our assumption, two pairs of proteins were analyzed using peptide mass mapping, as seen in Tables 1 and 2. The two proteins in each pair were found to share the same protein sequence. This result provides direct support to the assumption that the newly generated acidic spot is the formylated version of the corresponding original peptide.

Aside from the occurrence of the formylated peptides, the overall protein expression pattern in S. aureus remained unchanged (Fig. 1). Even after prolonged exposure (8 h) to LBM-415, all previously deformylated proteins remained visible (data not shown). Unlike S. aureus, the 2D images from S. pneumoniae treated with LBM-415 showed many changes from the control (Fig. 3): the emergence of many new protein spots and the disappearance of many original protein spots. The change in the protein pattern was so profound that a reliable direct match to the control gel was not possible. This S. pneumoniae result is reminiscent of the protein expression observed in Bacillus subtilis treated with PDF inhibitor actinonin, where dramatic change also occurred (3, 4). To explain the different proteomic responses of S. pneumoniae and S. aureus upon exposure to deformylase inhibitor, we speculate that in S. aureus there is a feedback mechanism that turns off protein synthesis and degradation once a substantial level of formylated peptide has accumulated. It is possible that this feedback mechanism does not exist in S. pneumoniae. Even with the accumulation of formylated peptide, the routine protein turnover process continues, eventually leading to the overexpression of nonfunctional proteins and loss of the critical level of functional proteins needed to sustain the viability of the cell. This hypothesis is consistent with the observation that PDF inhibitors are slow bactericidal agents for S. pneumoniae but static for S. aureus (5). In both S. aureus and S. pneumoniae, many proteins did not exhibit formylated version after exposure to LBM-415 and in S. pneumoniae the formylation and deformylation kinetics were different for different proteins. It is not clear that this was due to the differential formylation/deformylation efficiency or a separate mechanism. These issues warrant further detailed studies on the mechanism of formylation/deformylation.

In S. pneumoniae, LBM-415 inhibits bacterial growth with a delay compared to linezolid (Fig. 2). Linezolid is a protein synthesis inhibitor; it binds to rRNA and inhibits translation (20). At the protein level, the 2D images of S. aureus and S. pneumoniae inhibited by linezolid showed very similar patterns compared to their respective controls (data not shown). As an inhibitor of protein translation, linezolid's effect is almost immediate and its proteome is frozen in time. LBM-415, on the other hand, generated broad changes over protein synthesis in both S. aureus and S. pneumoniae (Fig. 1 and Fig. 3). S. pneumoniae sampled at different times after LBM-415 addition revealed that the spot intensity of newly generated formylated peptides was gradually increasing while the intensity of the mature peptides was gradually decreasing. Even given the quantitative limitations of silver staining, the changes of the spot intensity presumably represent the accumulation of formyl-peptides and the depletion of the deformylated peptides (Fig. 4). It is conceivable that the bacterial growth inhibition correlates with the accumulation of the formyl-peptides; this accumulation of nonfunctional formyl-peptide presumably impedes bacterial growth and eventually leads to growth cessation. Alternatively, PDF inhibition results in the depletion of some proteins that require deformylation to be functional. Although Spector and colleagues showed that both formyl-peptide and NH₂-peptide are substrates of ClpXP degradation and the degradation reactions proceed with similar reaction rates (21), it is not clear that all proteins would fold the same way, have the same stability, have the same function, and/or catalyze with the same reaction rate when formylated.

Giglione and colleagues have suggested that the deformylation process is a cotranslational process (8). It was also reported that nascent peptides containing fewer than 40 ± 5 residues retain formyl-methionine at the N terminus, and this moiety is removed when the nascent chain is 40 to 60 amino acids long (molecular mass of 6,000 Da) (2, 9). This hypothesis contradicts the results of this study on S. aureus and S. pneumoniae and the results of others (1, 3, 4). The fact that we could observe the accumulation of formylated proteins with a molecular mass of 20,000 Da (and higher) suggests that the deformylation does not have to occur during the protein synthesis process. Together with the observation that PDF inhibition is a slow process, we conclude that PDF inhibitors do not have a direct effect on translation. The deformylation step of protein synthesis is not necessarily associated with the bacterial ribosome and can occur after release of the full-length protein from the ribosome.

In S. pneumoniae when the PDF inhibitor was removed and the bacteria were allowed to regrow, we observed that the level of the formyl-peptide gradually decreased and the level of the deformylated peptides gradually increased (Fig. 5). Eventually all acidic shifted proteins disappeared after 3.5 h in the absence of PDF inhibitor. The depletion of formyl-peptide and the restoration of the NH₂-peptide were again time-dependent and cumulative processes. LBM-415 was shown to produce a long in vivo PAE against both S. aureus and S. pneumoniae, with the PAE against S. pneumoniae as long as 10 to 13.5 h (W. Craig and D. Andes, Abstr. 14th Eur. Clin. Congr. Microbiol. Infect. Dis., abstr. 921, 2004). A long PAE in vivo may suggest the deformylation process in vivo is rather slow, even after the concentration of PDF inhibitor falls below its MIC. The known immunostimulatory effects of formyl-peptides may also be relevant in this situation, since it was shown that subinhibitory concentrations of the PDF inhibitor actinonin caused the production and release of neutrophil-activating substances by bacteria. These substances are assumed to be formyl-peptides (6).

By measuring CFU, Lopez and coworkers revealed long in vitro PAESMEs in both S. pneumoniae and H. influenzae (S. Lopez, C. Wu, J. Blais, C. Hackbarth, M. Gomez, A. Kubo, R. Jain, A. Sundaram, S. Alvarez, K. Bracken, K. Dean, B. Weidmann, D. Patel, J. Trias, R. White, and Z. Yuan, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 239, 2004). When S. pneumoniae was allowed to regrow in the presence of 0.25x the MIC of LBM-415, the PAE was increased 10-fold, and with exposure to 0.5x the MIC, the PAE was increased 30-fold. In Fig. 6, we did not see any decrease in the amount of formyl-peptides during 3.5 h in the presence of 0.25x the MIC of LBM-415. Even at the end of the experiment, all identified formyl-peptides remained at the same level, in agreement with the prolonged PAESME observed in vitro. It is conceivable that the depletion of the formyl-peptides correlates with the regrowth of the bacteria (PAE) and the persistence of the formyl-peptides correlates with the prolonged PAESME. It appears that once the peptides were formylated and bacterial growth inhibited, only a very low concentration of the PDF inhibitor was necessary to maintain the inhibition both at the protein level and at the growth level. This dramatic PAESME on the deformylation of peptides might have profound implications in terms of clinical applications for this class of antibacterial agents. One treatment with a higher dose of the PDF inhibitor followed by a few trough dosages might be sufficient to inhibit bacterial growth and eventually allow the body to fully respond and eliminate infection in vivo.

In summary, PDF inhibition led to the accumulation of formyl-peptides in both S. aureus and S. pneumoniae. In S. aureus, the overall protein expression pattern remained the same upon PDF inhibition, while in S. pneumoniae more profound changes occurred. The accumulation of the formyl-peptides in S. pneumoniae was time dependent, and the degree of the accumulation varies among different proteins. The formyl-peptides depleted over time during regrowth of S. pneumoniae after the removal of the inhibitor, although high levels of the formylated proteins remained in bacteria in the presence of sub-MIC concentrations of LBM-415. Further studies are warranted to understand the mechanism of PDF inhibition and to enhance the efficacy of this novel class of antibiotics.

 
We thank Peter Margolis for his assistance with manuscript preparation and editorial processing.

Vicuron Pharmaceuticals is a wholly owned subsidiary of Pfizer, Inc.

 
* Corresponding author. Present address: 555 Bryant St., #433, Palo Alto, CA 94301. Phone: (510) 599-4866. E-mail: zyuan{at}yahoo.com.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Apfel, C. M., H. Locher, S. Evers, B. Takács, C. Hubschwerlen, W. Pirson, M. G. P. Page, and W. Keck. 2001. Peptide deformylase as an antibacterial drug target: target validation and resistance development. Antimicrob. Agents Chemother. 45:1058-1064.[Abstract/Free Full Text]
2
2. Ball, L. A., and P. Kaesberg. 1973. Cleavage of the N-terminal formylmethionine residue from a bacteriophage coat protein in vitro. J. Mol. Biol. 79:531-537.[CrossRef][Medline]
3
3. Bandow, J. E., D. Becher, K. Buttner, F. Hochgrafe, C. Freiberg, H. Brotz, and M. Hecker. 2003. The role of peptide deformylase in protein biosynthesis: a proteomic study. Proteomics 3:299-306.[CrossRef][Medline]
4
4. Bandow, J. E., H. Brötz, L. I. O. Leichert, H. Labischinski, and M. Hecker. 2003. Proteomic approach to understanding antibiotic action. Antimicrob. Agents Chemother. 47:948-955.[Abstract/Free Full Text]
5
5. Fritsche, T. R., H. S. Sader, R. Cleeland, and R. N. Jones. 2005. Comparative antimicrobial characterization of LBM415 (NVP PDF-713), a new peptide deformylase inhibitor of clinical importance. Antimicrob. Agents Chemother. 49:1468-1476.[Abstract/Free Full Text]
6
6. Fu, H., C. Dahlgren, and J. Bylund. 2003. Subinhibitory concentrations of the deformylase inhibitor actinonin increase bacterial release of neutrophil-activating peptides: a new approach to antimicrobial chemotherapy. Antimicrob. Agents Chemother. 47:2545-2550.[Abstract/Free Full Text]
7
7. Giglione, C., A. Serero, M. Pierre, B. Boisson, and T. Meinnel. 2000. Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms. EMBO J. 19:5916-5929.[CrossRef][Medline]
8
8. Giglione, C., M. Pierre, and T. Meinnel. 2000. Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents. Mol. Microbiol. 36:1197-1205.[CrossRef][Medline]
9
9. Housman, D., D. Gillespie, and H. F. Lodish. 1972. Removal of formyl-methionine residue from nascent bacteriophage f2 protein. J. Mol. Biol. 65:163-166.[CrossRef][Medline]
10
10. Jain, R., D. Chen, R. J. White, D. V. Patel, and Z. Yuan. 2005. Bacterial peptide deformylase inhibitors: a new class of antibacterial agents. Curr. Med. Chem. 12:763-771.
11
11. Jones, R. N., G. J. Moet, H. S. Sader, and T. R. Fritsche. 2004. Potential utility of a peptide deformylase inhibitor (NVP PDF-713) against oxazolidinone-resistant or streptogramin-resistant Gram-positive organism isolates. J. Antimicrob. Chemother. 53:804-807.[Abstract/Free Full Text]
12
12. Kreiswirth, B. N., S. Lofdahl, M. J. Betley, M. O'Reilly, P. M. Schlievert, M. S. Bergdoll, and R. P. Novick. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305:709.[CrossRef][Medline]
13
13. Lee, M. D., Y. She, M. J. Soskis, C. P. Borella, J. R. Gardner, P. A. Hayes, B. M. Dy, M. L. Heaney, M. R. Philips, W. G. Bornmann, F. M. Sirotnak, and D. A. Scheinberg. 2004. Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics. J. Clin. Investig. 114:1107-1116.[CrossRef][Medline]
14
14. Margolis, P. S., C. J. Hackbarth, D. C. Young, W. Wang, D. Chen, Z. Yuan, R. White, and J. Trias. 2000. Peptide deformylase in Staphylococcus aureus: resistance to inhibition is mediated by mutations in the formyltransferase gene. Antimicrob. Agents Chemother. 44:1825-1831.[Abstract/Free Full Text]
15
15. Margolis, P., C. Hackbarth, S. Lopez, M. Maniar, W. Wang, Z. Yuan, R. White, and J. Trias. 2001. Resistance of Streptococcus pneumoniae to deformylase inhibitors is due to mutations in defB. Antimicrob. Agents Chemother. 45:2432-2435.[Abstract/Free Full Text]
16
16. Mazel, D., S. Pochet, and P. Marliere. 1994. Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation. EMBO J. 13:914-923.[Medline]
17
17. Meinnel, T., and S. Blanquet. 1994. Characterization of the Thermus thermophilus locus encoding peptide deformylase and methionyl-tRNA_f^Met formyltransferase. J. Bacteriol. 176:7387-7390.[Abstract/Free Full Text]
18
18. Meinnel, T., Y. Mechulam, and S. Blanquet. 1993. Methionine as translation start signal: a review of the enzymes of the pathway in Escherichia coli. Biochimie 75:1061-1075.[Medline]
19
19. Nguyen, K. T., X. Hu, C. Colton, R. Chakrabarti, M. X. Zhu, and D. Pei. 2003. Characterization of a human peptide deformylase: implications for antibacterial drug design. Biochemistry 42:9952-9958.[CrossRef][Medline]
20
20. Shinabarger, D. L., K. R. Marotti, R. W. Murray, A. H. Lin, E. P. Melchior, S. M. Swaney, D. S. Dunyak, W. F. Demyan, and J. M. Buysse. 1997. Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions. Antimicrob. Agents Chemother. 41:2132-2136.[Abstract/Free Full Text]
21
21. Spector, S., J. M. Flynn, B. Tidor, T. A. Baker, and R. T. Sauer. 2003. Expression of N-formylated proteins in Escherichia coli. Protein Expr. Purif. 32:317-322.[Medline]
22
22. Yuan, Z., J. Trias, and R. J. White. 2001. Deformylase as a novel antibacterial target. Drug Discov. Today 6:954-961.[CrossRef][Medline]

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Antimicrobial Agents and Chemotherapy, May 2006, p. 1656-1663, Vol. 50, No. 5
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.5.1656-1663.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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