Autolytic Properties of Glycopeptide-Intermediate Staphylococcus aureus Mu50
http://www.100md.com
《抗菌试剂及化学方法》
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, Illinois 61790-4120
ABSTRACT
Whole-cell autolytic activity of prototypical glycopeptide-intermediate Staphylococcus aureus (GISA) Mu50 was reduced versus that of hetero-GISA Mu3 and glycopeptide-susceptible S. aureus, consistent with other GISA strains. In contrast, autolytic activity was relatively high in Mu50 crude cell walls and autolysin extracts against purified cell walls, reflecting the complexities of autolytic activity regulation.
The in vitro or in vivo selection of glycopeptide-intermediate Staphylococcus aureus (GISA) from susceptible S. aureus strains appears to involve an accumulation of mutations which result in an overabundance of cell wall material that sequesters glycopeptide molecules distal from their targets at the cytoplasmic membrane (6, 16). Extensive variability among traits that are associated with the GISA phenotype has slowed progress in dissecting the underlying mechanism of this antimicrobial resistance (12, 17). Altered autolytic activity is one such trait, with Mu50, the first clinically isolated GISA strain, described as expressing increased whole-cell autolytic activity (6). In contrast, all other GISA strains for which this trait has been examined, both clinically isolated and laboratory selected, express reduced autolytic activity to some extent (1, 2, 7, 11, 12, 14, 15, 16, 17, 20). The enhanced autolytic activity that was reported for Mu50 in 1998 has been applied to the formulation of models of the GISA mechanism of resistance and continues to be cited as a characteristic of the GISA phenotype (6, 8, 17). This study, testing the reproducibility of the previous Mu50 autolysis data, was initiated due to the position that Mu50 occupies as the lone GISA strain with enhanced autolysis and the counterintuitive nature of enhanced peptidoglycan degradation in the context of a resistance mechanism that is presumed to be dependent upon a buildup of cell wall material.
Mu50 has a vancomycin (VAN) MIC of 8.0 μg/ml. The Mu50 strain tested throughout this study was obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), as was hetero-GISA Mu3 (VAN MIC of 3.0 μg/ml) (6). Mu50 strains from Fred Tenover of the Centers for Disease Control and Prevention (CDC) and Henry Chambers of the University of California, San Francisco (UCSF) were included in whole-cell autolysis assays to confirm that their autolytic properties were consistent with those of the NARSA lineage, given the phenotypic instability reported for Mu50 and other clinically isolated GISA strains (12). All the Mu50 strains tested were originally provided by K. Hiramatsu. This study also employed two isogenic pairs of laboratory-selected GISA and glycopeptide-susceptible S. aureus (GSSA) parents, GISA COLV10 and parent COL (VAN MICs of 8.0 and 2.0 μg/ml, respectively) and GISA 13136p–m+ V20 and parent 13136p–m+ (VAN MICs of 16.0 and 1.5 μg/ml, respectively) (11).
Results of autolysis assays are presented graphically as decreases in optical density over time for suspensions of cells or cell walls in buffer. Whole-cell autolysis assays were performed exactly as previously described after growth in tryptic soy broth (TSB; BD Difco) or brain heart infusion broth (BHI; BD Difco), with or without one-half of the MIC of VAN (vancomycin HCl; Eli Lilly Co.) included in the assay buffer (6). The results of whole-cell autolysis assays (Fig. 1A) lead to three conclusions. First, the whole-cell autolytic properties of all three lineages of Mu50 were comparable. Second, whole-cell autolysis by Mu50 after growth in TSB was comparable to that after growth in BHI. Finally, GISA Mu50 expressed reduced whole-cell autolytic activity versus that of surrogate progenitor hetero-GISA Mu3, as did GISA 13136p–m+ V20 versus its parent, 13136p–m+. Whole-cell autolytic activity of Mu50 was further reduced by the inclusion of VAN in the assay buffer, as was that of Mu3 and 13136p–m+ (Fig. 1B) as well as 13136p–m+V20, COLV10, and COL (data not presented).
In addition, Mu50 was shown to be deficient in autolytic activity when whole-cell assays were conducted in 0.05 M Tris-HCl (pH 7.2) containing 0.05% Triton X-100, a commonly used method to assess autolytic activity (2, 5, 7, 11, 13, 14, 16) (Fig. 2).
Freeze-thaw extractions were performed on cells grown in TSB with or without one-half of the MIC of VAN to isolate cell wall-associated peptidoglycan hydrolases (autolysins), the activities of which were visualized as clearings on zymograms containing Micrococcus luteus cell walls after electrophoresis of equal amounts of proteins (Fig. 3A and B) (4, 7, 18). Protein concentrations were determined by using the Bio-Rad protein assay kit, which is based on the Bradford dye-binding assay (3). Three major activity bands were evident, corresponding to ATL at 147 kDa (the unprocessed, bifunctional product of the atl gene, which encodes the primary autolytic activities of S. aureus), an ATL processing intermediate at 115 kDa, and at 50 kDa the endo-?-N-acetylglucosaminidase (GL) released after full proteolytic cleavage of ATL (10, 19). Weaker bands corresponding to processing intermediates at 85 kDa, and the other activity released by the complete processing of ATL, an N-acetylmuramyl-L-alanine amidase (AM) at 62 kDa were present (10, 19). After growth without VAN in the medium, band intensities from Mu50 were generally less than those from other strains, including Mu3, indicating reduced amounts and/or activities of these enzymes (Fig. 3A). This was inconsistent with a previous report of Mu50 GL activity greater than those of control strains and Mu3 (6). The inclusion of VAN in the growth medium of the parent and GISA strains generally increased the intensities of bands corresponding to unprocessed ATL at the expense of bands from GL and AM versus the intensities of those observed after growth in antibiotic-free medium (Fig. 3B). Under these conditions, the GL activity of Mu50 was again the lowest of the six strains analyzed.
Crude cell walls (CCWs) retaining autolytic activities were isolated by centrifugation after the mechanical disruption of TSB cultures grown in the presence or absence of one-half of the MIC of VAN (5). Purified cell walls (PCWs) were prepared by boiling CCWs to inactivate autolysins, followed by nuclease and protease treatments and washing (13). Autolytic activity profiles of CCWs are presented in Fig. 4 and reveal that CCWs from GSSA 13136p–m+ showed the highest autolytic activity, and its GISA derivative 13136p–m+V20 showed the lowest. The autolytic activity of Mu50 CCWs was not only greater than that for Mu3 but also was nearly as high as that from 13136p–m+ CCWs. Differences in whole-cell autolytic activity that are not reflected in the activities of CCWs in some cases have been noted previously (5). The growth of cells with VAN in the medium resulted in CCWs with similar autolytic activity profiles to those from antibiotic-free growth, with only modest reductions in activities.
Autolysis activity profiles were generated from PCWs in lysis buffer that served as substrates for active autolysins released from cells of TSB cultures by three freeze-thaw cycles and collected in supernatants following centrifugation (7, 13). Autolysin extracts from GSSA and GISA were challenged to degrade PCWs from various strains. Presented in Fig. 5A to C are autolysis activity profiles of PCWs from four strains that were degraded by Mu50 autolysins. Mu50 autolysins were very highly and equally active against PCWs produced without VAN exposure from GSSA 13136p–m+ and from Mu50 itself and less highly but equally active against PCWs from GISA 13136p–m+V20 and Mu3 (Fig. 5A). The growth of cells with VAN at one-half of the MIC in the growth medium resulted in PCWs with relatively minor alterations in susceptibilities to Mu50 autolysins, although such PCWs from GISA 13136p–m+V20 were more resistant to degradation, while those from GSSA 13136p–m+ were degraded at an increased rate in the first 90 min of the assay (Fig. 5B). After growth without VAN present, the preincubation of PCWs from cells of these four strains with 1,000 μg/ml VAN, followed by washing to remove unbound glycopeptide, resulted in dramatic reductions in susceptibilities to Mu50 autolysins (Fig. 5C) (16). Final optical densities after 4 h of lysis were higher for preincubated PCWs from both Mu50 itself and GSSA 13136p–m+ than any from PCWs produced without VAN exposure (Fig. 5C). PCWs exposed to VAN twice (growth medium and preincubation) yielded autolysis activity profiles resembling those after preincubation only, and all combinations tested of PCWs and autolysins from various strains consistently showed dramatically reduced PCW susceptibilities to autolysins after VAN preincubation relative to VAN exposure in only the growth medium, although the high degree of susceptibility of Mu50 PCWs to Mu50 autolysins was not observed with autolysins extracted from other strains (data not presented).
GISA strains are known to exhibit reduced whole-cell autolytic activities, and this was demonstrated by Mu50 in this study. Whereas the addition of VAN to growth medium yielded only modest reductions in the autolytic activities of subsequently isolated CCWs and PCWs, the saturation of the peptidoglycan with VAN interfered with the actions of autolysins, perhaps by blocking access to the enzymes' substrate, as was evident from PCW preincubation assays in which no free vancomycin was added to the assay buffer or growth medium. This suggests that modulation of the activities of preexisting autolysins is a significant component of the responses of GSSA and GISA to VAN. Whole cells responded to the presence of vancomycin in the growth medium by down-regulating posttranslational processing of the atl gene product. Mu50 autolysins expressed low autolytic activities in zymograms versus other GISA autolysins after the presence or absence of VAN in the growth medium, but the autolytic activities of CCWs from Mu50 indicated high autolytic activity comparable to GSSA 13136p–m+ and greater than those from the other strains with reduced VAN susceptibilities, Mu3 and 13136p–m+ V20.
The reduced whole-cell autolytic activity and peptidoglycan hydrolase activity in zymograms of Mu50 but significant autolytic activity of CCWs and of freeze-thaw extracts against purified cell walls indicate that the regulation of autolysins in this strain is complex. The regulation of S. aureus autolytic activity is generally complex and potentially occurs at a number of differing levels, including cell wall composition, autolysin enzymatic activities, posttranslational processing of autolysins, and autolysin gene transcription (2, 5, 7, 11, 13, 14, 16, 17, 20). In a previous report, reverse transcriptase PCR results from cells grown in the absence of VAN revealed reduced expression of atl in a majority of the GISA strains tested, Mu50 among them, versus GSSA strains. These results could not be explained by differences in atl promoter region sequences, which were reported to be identical among the GISA and GSSA strains in that study (20). Additionally, a clinically isolated GISA strain has been described as exhibiting reduced Triton X-100-stimulated whole-cell autolysis, but with increased activity from some peptidoglycan hydrolase bands on zymograms, versus an isogenic GSSA that was isolated from the same patient. Furthermore, transcriptional profiling revealed increased expression from two autolysin genes by the GISA member of this isogenic pair, with alterations in the teichoic acid component of the cell wall suggested as responsible for the reduced whole-cell autolysis that was observed (9, 17).
In conclusion, the prototypical GISA Mu50 expressed reduced whole-cell autolytic activity in this study, in contrast to an initial report, but consistent with observations for other GISA, and this cannot be attributed to phenotypic changes arising during laboratory propagation or to differences in growth media. The importance of this finding is that reduced autolytic activity is now established as common to the GISA phenotype, although there may be different pathways leading to this phenotype, which until now has had as a unifying element only a thickened cell wall after growth in the presence of vancomycin (10). This information should promote a more focused approach to the deciphering of the GISA mechanism of resistance.
ACKNOWLEDGMENTS
This work was supported by NIH grant AI049964.
Present address: Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Rangsit Center, Prathum Tani, 12121, Thailand.
Present address: BD Diagnostic Systems, Sparks, MD 21152.
REFERENCES
Boyle-Vavra, S., R. B. Carey, and R. S. Daum. 2001. Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate. J. Antimicrob. Chemother. 48:617-625.
Boyle-Vavra, S., M. Challapalli, and R. S. Daum. 2003. Resistance to autolysis in vancomycin-selected Staphylococcus aureus isolates precedes vancomycin-intermediate resistance. Antimicrob. Agents Chemother. 47:2036-2039.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Foster, S. J. 1992. Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis. J. Bacteriol. 174:464-470.
Gustafson, J. E., B. Berger-B?chi, A. Str?ssle, and B. J. Wilkinson. 1992. Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob. Agents Chemother. 36:566-572.
Hanaki, H., K. Kuwahara-Arai, S. Boyle-Vavra, R. S. Daum, H. Labischinski, and K. Hiramatsu. 1998. Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. J. Antimicrob. Chemother. 42:199-209.
Koehl, J. L., A. Muthaiyan, R. K. Jayaswal, K. Ehlert, H. Labischinski, and B. J. Wilkinson. 2004. Cell wall composition and decreased autolytic activity and lysostaphin susceptibility of glycopeptide-intermediate Staphylococcus aureus. Antimicrob. Agents Chemother. 48:3749-3757.
Maki, H., N. McCallum, M. Bischoff, A. Wada, and B. Berger-B?chi. 2004. tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 48:1953-1959.
McAleese, F., S. W. Wu, K. Sieradzki, P. Dunman, E. Murphy, S. Projan, and A. Tomasz. 2006. Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate-S. aureus-type resistance to vancomycin. J. Bacteriol. 188:1120-1133.
Oshida, T., M. Sugai, H. Komatsuzawa, Y. M. Hong, H. Suginaka, and A. Tomasz. 1995. A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc. Natl. Acad. Sci. USA 92:285-289.
Pfeltz, R. F., V. K. Singh, J. L. Schmidt, M. A. Batten, C. S. Baranyk, M. J. Nadakavukaren, R. K. Jayaswal, and B. J. Wilkinson. 2000. Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds. Antimicrob. Agents Chemother. 44:294-303.
Pfeltz, R. F., and B. J. Wilkinson. 2004. The escalating challenge of vancomycin resistance in Staphylococcus aureus. Curr. Drug Targets Infect. Disord. 4:273-294.
Qoronfleh, M. W., and B. J. Wilkinson. 1986. Effects of growth of methicillin-resistant and -susceptible Staphylococcus aureus in the presence of ?-lactams on peptidoglycan structure and susceptibility to lytic enzymes. Antimicrob. Agents Chemother. 29:250-257.
Sakoulas, G., G. M. Eliopoulos, R. C. Moellering, Jr., R. P. Novick, L. Venkataraman, C. Wennersten, P. C. DeGirolami, M. J. Schwaber, and H. S. Gold. 2003. Staphylococcus aureus accessory gene regulator (agr) group II: is there a relationship to the development of intermediate-level glycopeptide resistance? J. Infect. Dis. 187:929-938.
Sieradzki, K., R. B. Roberts, S. W. Haber, and A. Tomasz. 1999. The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection. N. Engl. J. Med. 340:517-523.
Sieradzki, K., and A. Tomasz. 1997. Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus. J. Bacteriol. 179:2557-2566.
Sieradzki, K., and A. Tomasz. 2003. Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus. J. Bacteriol. 185:7103-7110.
Sugai, M., T. Akiyama, H. Komatsuzawa, Y. Miyake, and H. Suginaka. 1990. Characterization of sodium dodecyl sulfate-stable Staphylococcus aureus bacteriolytic enzymes by polyacrylamide gel electrophoresis. J. Bacteriol. 172:6494-6498.
Takahashi, J., H. Komatsuzawa, S. Yamada, T. Nishida, H. Labischinski, T. Fujiwara, M. Ohara, J. Yamagishi, and M. Sugai. 2002. Molecular characterization of an atl null mutant of Staphylococcus aureus. Microbiol. Immunol. 46:601-612.
Wootton, M., P. M. Bennett, A. P. MacGowan, and T. R. Walsh. 2005. Reduced expression of the atl autolysin gene and susceptibility to autolysis in clinical heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) and GISA strains. J. Antimicrob. Chemother. 56:944-947.(Sugunya Utaida, Richard F)
ABSTRACT
Whole-cell autolytic activity of prototypical glycopeptide-intermediate Staphylococcus aureus (GISA) Mu50 was reduced versus that of hetero-GISA Mu3 and glycopeptide-susceptible S. aureus, consistent with other GISA strains. In contrast, autolytic activity was relatively high in Mu50 crude cell walls and autolysin extracts against purified cell walls, reflecting the complexities of autolytic activity regulation.
The in vitro or in vivo selection of glycopeptide-intermediate Staphylococcus aureus (GISA) from susceptible S. aureus strains appears to involve an accumulation of mutations which result in an overabundance of cell wall material that sequesters glycopeptide molecules distal from their targets at the cytoplasmic membrane (6, 16). Extensive variability among traits that are associated with the GISA phenotype has slowed progress in dissecting the underlying mechanism of this antimicrobial resistance (12, 17). Altered autolytic activity is one such trait, with Mu50, the first clinically isolated GISA strain, described as expressing increased whole-cell autolytic activity (6). In contrast, all other GISA strains for which this trait has been examined, both clinically isolated and laboratory selected, express reduced autolytic activity to some extent (1, 2, 7, 11, 12, 14, 15, 16, 17, 20). The enhanced autolytic activity that was reported for Mu50 in 1998 has been applied to the formulation of models of the GISA mechanism of resistance and continues to be cited as a characteristic of the GISA phenotype (6, 8, 17). This study, testing the reproducibility of the previous Mu50 autolysis data, was initiated due to the position that Mu50 occupies as the lone GISA strain with enhanced autolysis and the counterintuitive nature of enhanced peptidoglycan degradation in the context of a resistance mechanism that is presumed to be dependent upon a buildup of cell wall material.
Mu50 has a vancomycin (VAN) MIC of 8.0 μg/ml. The Mu50 strain tested throughout this study was obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), as was hetero-GISA Mu3 (VAN MIC of 3.0 μg/ml) (6). Mu50 strains from Fred Tenover of the Centers for Disease Control and Prevention (CDC) and Henry Chambers of the University of California, San Francisco (UCSF) were included in whole-cell autolysis assays to confirm that their autolytic properties were consistent with those of the NARSA lineage, given the phenotypic instability reported for Mu50 and other clinically isolated GISA strains (12). All the Mu50 strains tested were originally provided by K. Hiramatsu. This study also employed two isogenic pairs of laboratory-selected GISA and glycopeptide-susceptible S. aureus (GSSA) parents, GISA COLV10 and parent COL (VAN MICs of 8.0 and 2.0 μg/ml, respectively) and GISA 13136p–m+ V20 and parent 13136p–m+ (VAN MICs of 16.0 and 1.5 μg/ml, respectively) (11).
Results of autolysis assays are presented graphically as decreases in optical density over time for suspensions of cells or cell walls in buffer. Whole-cell autolysis assays were performed exactly as previously described after growth in tryptic soy broth (TSB; BD Difco) or brain heart infusion broth (BHI; BD Difco), with or without one-half of the MIC of VAN (vancomycin HCl; Eli Lilly Co.) included in the assay buffer (6). The results of whole-cell autolysis assays (Fig. 1A) lead to three conclusions. First, the whole-cell autolytic properties of all three lineages of Mu50 were comparable. Second, whole-cell autolysis by Mu50 after growth in TSB was comparable to that after growth in BHI. Finally, GISA Mu50 expressed reduced whole-cell autolytic activity versus that of surrogate progenitor hetero-GISA Mu3, as did GISA 13136p–m+ V20 versus its parent, 13136p–m+. Whole-cell autolytic activity of Mu50 was further reduced by the inclusion of VAN in the assay buffer, as was that of Mu3 and 13136p–m+ (Fig. 1B) as well as 13136p–m+V20, COLV10, and COL (data not presented).
In addition, Mu50 was shown to be deficient in autolytic activity when whole-cell assays were conducted in 0.05 M Tris-HCl (pH 7.2) containing 0.05% Triton X-100, a commonly used method to assess autolytic activity (2, 5, 7, 11, 13, 14, 16) (Fig. 2).
Freeze-thaw extractions were performed on cells grown in TSB with or without one-half of the MIC of VAN to isolate cell wall-associated peptidoglycan hydrolases (autolysins), the activities of which were visualized as clearings on zymograms containing Micrococcus luteus cell walls after electrophoresis of equal amounts of proteins (Fig. 3A and B) (4, 7, 18). Protein concentrations were determined by using the Bio-Rad protein assay kit, which is based on the Bradford dye-binding assay (3). Three major activity bands were evident, corresponding to ATL at 147 kDa (the unprocessed, bifunctional product of the atl gene, which encodes the primary autolytic activities of S. aureus), an ATL processing intermediate at 115 kDa, and at 50 kDa the endo-?-N-acetylglucosaminidase (GL) released after full proteolytic cleavage of ATL (10, 19). Weaker bands corresponding to processing intermediates at 85 kDa, and the other activity released by the complete processing of ATL, an N-acetylmuramyl-L-alanine amidase (AM) at 62 kDa were present (10, 19). After growth without VAN in the medium, band intensities from Mu50 were generally less than those from other strains, including Mu3, indicating reduced amounts and/or activities of these enzymes (Fig. 3A). This was inconsistent with a previous report of Mu50 GL activity greater than those of control strains and Mu3 (6). The inclusion of VAN in the growth medium of the parent and GISA strains generally increased the intensities of bands corresponding to unprocessed ATL at the expense of bands from GL and AM versus the intensities of those observed after growth in antibiotic-free medium (Fig. 3B). Under these conditions, the GL activity of Mu50 was again the lowest of the six strains analyzed.
Crude cell walls (CCWs) retaining autolytic activities were isolated by centrifugation after the mechanical disruption of TSB cultures grown in the presence or absence of one-half of the MIC of VAN (5). Purified cell walls (PCWs) were prepared by boiling CCWs to inactivate autolysins, followed by nuclease and protease treatments and washing (13). Autolytic activity profiles of CCWs are presented in Fig. 4 and reveal that CCWs from GSSA 13136p–m+ showed the highest autolytic activity, and its GISA derivative 13136p–m+V20 showed the lowest. The autolytic activity of Mu50 CCWs was not only greater than that for Mu3 but also was nearly as high as that from 13136p–m+ CCWs. Differences in whole-cell autolytic activity that are not reflected in the activities of CCWs in some cases have been noted previously (5). The growth of cells with VAN in the medium resulted in CCWs with similar autolytic activity profiles to those from antibiotic-free growth, with only modest reductions in activities.
Autolysis activity profiles were generated from PCWs in lysis buffer that served as substrates for active autolysins released from cells of TSB cultures by three freeze-thaw cycles and collected in supernatants following centrifugation (7, 13). Autolysin extracts from GSSA and GISA were challenged to degrade PCWs from various strains. Presented in Fig. 5A to C are autolysis activity profiles of PCWs from four strains that were degraded by Mu50 autolysins. Mu50 autolysins were very highly and equally active against PCWs produced without VAN exposure from GSSA 13136p–m+ and from Mu50 itself and less highly but equally active against PCWs from GISA 13136p–m+V20 and Mu3 (Fig. 5A). The growth of cells with VAN at one-half of the MIC in the growth medium resulted in PCWs with relatively minor alterations in susceptibilities to Mu50 autolysins, although such PCWs from GISA 13136p–m+V20 were more resistant to degradation, while those from GSSA 13136p–m+ were degraded at an increased rate in the first 90 min of the assay (Fig. 5B). After growth without VAN present, the preincubation of PCWs from cells of these four strains with 1,000 μg/ml VAN, followed by washing to remove unbound glycopeptide, resulted in dramatic reductions in susceptibilities to Mu50 autolysins (Fig. 5C) (16). Final optical densities after 4 h of lysis were higher for preincubated PCWs from both Mu50 itself and GSSA 13136p–m+ than any from PCWs produced without VAN exposure (Fig. 5C). PCWs exposed to VAN twice (growth medium and preincubation) yielded autolysis activity profiles resembling those after preincubation only, and all combinations tested of PCWs and autolysins from various strains consistently showed dramatically reduced PCW susceptibilities to autolysins after VAN preincubation relative to VAN exposure in only the growth medium, although the high degree of susceptibility of Mu50 PCWs to Mu50 autolysins was not observed with autolysins extracted from other strains (data not presented).
GISA strains are known to exhibit reduced whole-cell autolytic activities, and this was demonstrated by Mu50 in this study. Whereas the addition of VAN to growth medium yielded only modest reductions in the autolytic activities of subsequently isolated CCWs and PCWs, the saturation of the peptidoglycan with VAN interfered with the actions of autolysins, perhaps by blocking access to the enzymes' substrate, as was evident from PCW preincubation assays in which no free vancomycin was added to the assay buffer or growth medium. This suggests that modulation of the activities of preexisting autolysins is a significant component of the responses of GSSA and GISA to VAN. Whole cells responded to the presence of vancomycin in the growth medium by down-regulating posttranslational processing of the atl gene product. Mu50 autolysins expressed low autolytic activities in zymograms versus other GISA autolysins after the presence or absence of VAN in the growth medium, but the autolytic activities of CCWs from Mu50 indicated high autolytic activity comparable to GSSA 13136p–m+ and greater than those from the other strains with reduced VAN susceptibilities, Mu3 and 13136p–m+ V20.
The reduced whole-cell autolytic activity and peptidoglycan hydrolase activity in zymograms of Mu50 but significant autolytic activity of CCWs and of freeze-thaw extracts against purified cell walls indicate that the regulation of autolysins in this strain is complex. The regulation of S. aureus autolytic activity is generally complex and potentially occurs at a number of differing levels, including cell wall composition, autolysin enzymatic activities, posttranslational processing of autolysins, and autolysin gene transcription (2, 5, 7, 11, 13, 14, 16, 17, 20). In a previous report, reverse transcriptase PCR results from cells grown in the absence of VAN revealed reduced expression of atl in a majority of the GISA strains tested, Mu50 among them, versus GSSA strains. These results could not be explained by differences in atl promoter region sequences, which were reported to be identical among the GISA and GSSA strains in that study (20). Additionally, a clinically isolated GISA strain has been described as exhibiting reduced Triton X-100-stimulated whole-cell autolysis, but with increased activity from some peptidoglycan hydrolase bands on zymograms, versus an isogenic GSSA that was isolated from the same patient. Furthermore, transcriptional profiling revealed increased expression from two autolysin genes by the GISA member of this isogenic pair, with alterations in the teichoic acid component of the cell wall suggested as responsible for the reduced whole-cell autolysis that was observed (9, 17).
In conclusion, the prototypical GISA Mu50 expressed reduced whole-cell autolytic activity in this study, in contrast to an initial report, but consistent with observations for other GISA, and this cannot be attributed to phenotypic changes arising during laboratory propagation or to differences in growth media. The importance of this finding is that reduced autolytic activity is now established as common to the GISA phenotype, although there may be different pathways leading to this phenotype, which until now has had as a unifying element only a thickened cell wall after growth in the presence of vancomycin (10). This information should promote a more focused approach to the deciphering of the GISA mechanism of resistance.
ACKNOWLEDGMENTS
This work was supported by NIH grant AI049964.
Present address: Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Rangsit Center, Prathum Tani, 12121, Thailand.
Present address: BD Diagnostic Systems, Sparks, MD 21152.
REFERENCES
Boyle-Vavra, S., R. B. Carey, and R. S. Daum. 2001. Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate. J. Antimicrob. Chemother. 48:617-625.
Boyle-Vavra, S., M. Challapalli, and R. S. Daum. 2003. Resistance to autolysis in vancomycin-selected Staphylococcus aureus isolates precedes vancomycin-intermediate resistance. Antimicrob. Agents Chemother. 47:2036-2039.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Foster, S. J. 1992. Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis. J. Bacteriol. 174:464-470.
Gustafson, J. E., B. Berger-B?chi, A. Str?ssle, and B. J. Wilkinson. 1992. Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob. Agents Chemother. 36:566-572.
Hanaki, H., K. Kuwahara-Arai, S. Boyle-Vavra, R. S. Daum, H. Labischinski, and K. Hiramatsu. 1998. Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. J. Antimicrob. Chemother. 42:199-209.
Koehl, J. L., A. Muthaiyan, R. K. Jayaswal, K. Ehlert, H. Labischinski, and B. J. Wilkinson. 2004. Cell wall composition and decreased autolytic activity and lysostaphin susceptibility of glycopeptide-intermediate Staphylococcus aureus. Antimicrob. Agents Chemother. 48:3749-3757.
Maki, H., N. McCallum, M. Bischoff, A. Wada, and B. Berger-B?chi. 2004. tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 48:1953-1959.
McAleese, F., S. W. Wu, K. Sieradzki, P. Dunman, E. Murphy, S. Projan, and A. Tomasz. 2006. Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate-S. aureus-type resistance to vancomycin. J. Bacteriol. 188:1120-1133.
Oshida, T., M. Sugai, H. Komatsuzawa, Y. M. Hong, H. Suginaka, and A. Tomasz. 1995. A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and characterization. Proc. Natl. Acad. Sci. USA 92:285-289.
Pfeltz, R. F., V. K. Singh, J. L. Schmidt, M. A. Batten, C. S. Baranyk, M. J. Nadakavukaren, R. K. Jayaswal, and B. J. Wilkinson. 2000. Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds. Antimicrob. Agents Chemother. 44:294-303.
Pfeltz, R. F., and B. J. Wilkinson. 2004. The escalating challenge of vancomycin resistance in Staphylococcus aureus. Curr. Drug Targets Infect. Disord. 4:273-294.
Qoronfleh, M. W., and B. J. Wilkinson. 1986. Effects of growth of methicillin-resistant and -susceptible Staphylococcus aureus in the presence of ?-lactams on peptidoglycan structure and susceptibility to lytic enzymes. Antimicrob. Agents Chemother. 29:250-257.
Sakoulas, G., G. M. Eliopoulos, R. C. Moellering, Jr., R. P. Novick, L. Venkataraman, C. Wennersten, P. C. DeGirolami, M. J. Schwaber, and H. S. Gold. 2003. Staphylococcus aureus accessory gene regulator (agr) group II: is there a relationship to the development of intermediate-level glycopeptide resistance? J. Infect. Dis. 187:929-938.
Sieradzki, K., R. B. Roberts, S. W. Haber, and A. Tomasz. 1999. The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection. N. Engl. J. Med. 340:517-523.
Sieradzki, K., and A. Tomasz. 1997. Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus. J. Bacteriol. 179:2557-2566.
Sieradzki, K., and A. Tomasz. 2003. Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus. J. Bacteriol. 185:7103-7110.
Sugai, M., T. Akiyama, H. Komatsuzawa, Y. Miyake, and H. Suginaka. 1990. Characterization of sodium dodecyl sulfate-stable Staphylococcus aureus bacteriolytic enzymes by polyacrylamide gel electrophoresis. J. Bacteriol. 172:6494-6498.
Takahashi, J., H. Komatsuzawa, S. Yamada, T. Nishida, H. Labischinski, T. Fujiwara, M. Ohara, J. Yamagishi, and M. Sugai. 2002. Molecular characterization of an atl null mutant of Staphylococcus aureus. Microbiol. Immunol. 46:601-612.
Wootton, M., P. M. Bennett, A. P. MacGowan, and T. R. Walsh. 2005. Reduced expression of the atl autolysin gene and susceptibility to autolysis in clinical heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) and GISA strains. J. Antimicrob. Chemother. 56:944-947.(Sugunya Utaida, Richard F)