1 Department of Developmental Biology, National Institute of Agrobiological Sciences, Oowashi 1-2, Tsukuba, Ibaraki 305-8634; 2 Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan
Received 19 July 2004; returned 16 October 2004; revised 19 January 2005; accepted 22 January 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: Based on preliminary characterization of the ASABF-resistant strain, Mu50, we speculated that the alternative sigma factor sigB may regulate resistance against antimicrobial peptides. To test this hypothesis, the ASABF susceptibility was compared between NKSB (a sigB-knockout derivative of N315) and its sigB-overexpressing derivative. In addition, similar experiments were carried out for N315ex, a deletion mutant of N315 for SCCmec (Staphylococcus cassette chromosome mec) which contains essential genes for ß-lactam resistance.
Results: The sigB-overexpressing NKSB acquired an increased resistance to ASABF- compared with the parent strain. The sigB-induced ASABF-
resistance was also observed in N315ex.
Conclusions: The overexpression of sigB confers resistance to the antimicrobial peptide, ASABF-. SCCmec is not essential for this resistance.
Keywords:
Caenorhabditis elegans
,
Ascaris suum
,
stress response
,
defensin
,
antibiotics
,
CSß motif
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although antimicrobial peptides are novel agents in practical use, bacterial pathogens, including S. aureus, can develop resistance to these peptides as observed with other antibiotics.10 Some mechanisms of resistance have been proposed, e.g. modification in the cell wall1117 or membrane,18,19 efflux pumps,2024 and protease production.13 In addition to each resistance mechanism, some regulatory systems that respond to environmental changes have also been reported to be involved in the control of antimicrobial peptide resistance, e.g. PhoP/PhoQ12,13,25 and PmrA/PmrB15 in Salmonella enterica, and Agr in S. aureus.26
S. aureus can switch its gene expression to adapt to various environments. Such switching is partly regulated by promoter recognition subunits of RNA polymerases designated sigma factors. At least three sigma factors, sigA,27 sigB,28,29 and SA0492,30 have been identified in S. aureus. Each sigma factor recognizes a different promoter sequence and allows the RNA polymerase to initiate site-specific transcription for a specific group of genes. Although the primary sigma factor, sigA, is constitutively maintained, the activity of the alternative sigma factor, sigB, depends on growth-phase and various environmental stresses, suggesting that staphylococcal sigB may regulate some stress responses.3143 The alkaline shock protein gene, asp23, is under the sole control of SigB and its expression is often used as an indicator of SigB activity.36 Recently, Morikawa et al. reported that the molecular concentration of the SigB protein was remarkably higher in the highly methicillin-resistant and moderately vancomycin-resistant strain, Mu50.44 Interestingly, artificial overexpression of sigB in a sigB-knockout strain, NKSB, exhibited a partially similar phenotype to Mu50, i.e. cell wall thickening and high-level resistance to ß-lactam antibiotics.45 These observations suggest that some characteristic phenotypes of Mu50 may be due to overactivation of SigB. Whereas SigB is a global regulator for stress responses, little is known on its role in bacterial resistance against antimicrobial peptides.
Although bacterial resistance against CSß-type antimicrobial peptides has largely not been investigated, MRSA (methicillin-resistant S. aureus) SR1550 has been reported to be remarkably resistant to some synthetic antimicrobial peptides derived from another CS
ß-type antimicrobial peptide, sapecin B, than MSSA (methicillin-susceptible S. aureus) ATCC 6538P.46
MRSA is defined as a ß-lactam-resistant strain carrying an extra penicillin-binding protein, PBP2', encoded by mecA located on SCCmec (Staphylococcus cassette chromosome mec).47
PBP2' exhibits reduced affinity to ß-lactams, and allows cells to continue cell wall synthesis in the presence of ß-lactams.48
In this study, we tested the susceptibility of various S. aureus strains to ASABF- and detected significant divergence of susceptibility. Since some phenotypes of one of the most resistant strains, Mu50, may be due to SigB overactivation as mentioned above, the role of SigB in ASABF-
resistance was explored. In addition, the contribution of SCCmec to antimicrobial resistance was also examined.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
S. aureus strains used in this study are listed in Table 1. S. aureus NKSB, NKSBn, and NKSBv were previously described in detail.45 N315,49 N315ex,50 and Mu5051 were kindly provided by Professor Kei-ichi Hiramatsu and Professor Teruyo Ito (Juntendo University, Tokyo, Japan). Forty clinically isolated MRSA, including MRSA no. 7 and no. 33, were isolated in Mahidol University Hospital, Thailand, and obtained from Dr Wanpen Chaicumpa (Faculty of Allied Health Sciences, Thammasat University Rangsit Center, Prathum Thani, Thailand). COL52 was a gift from Professor Alexander Tomasz (Rockefeller University, New York, USA) via Professor Hitoshi Komatsuzawa (Hiroshima University, Hiroshima, Japan).
|
The construction of sigB overexpression vectors and transformation experiments were carried out as described previously.45 Briefly, the sigB gene region of N315 (for N315ex-n) or Mu50 (for N315ex-m) was cloned into an expression vector, pRIT5H.53 The cloned sigB was constitutively expressed under the control of the protein A promoter.
Northern blot analysis
To estimate the activity of SigB, asp23 transcripts were quantified by northern blot analysis. Total RNA was isolated using an RNeasy RNA isolation kit (Qiagen, Tokyo, Japan). Five micrograms of the total RNA was separated on a 1.2% (w/v) agarose slab gel containing 6.6% formaldehyde and transferred onto a Gene Screen Plus membrane (NEN Life Science Products, Boston, MA, USA). The asp23 DNA fragment was prepared as described previously and used as a probe.44 The results of hybridization were evaluated using a BAS-2500 Bio Imaging Analyzer (Fuji-film, Tokyo, Japan).
Culture conditions and microbicidal assay
For preliminary screening of ASABF-resistant strains, inhibition zone assay was carried out. Twenty-five millilitres of modified Luria-Bertani (LB) agar (1% tryptone, 0.5% yeast extract, 0.1% NaCl, 2% agar) containing 105 cfu/mL logarithmic-phase bacteria was poured into each sterile Petri dish (9 cm in diameter). ASABF solution (0, 1, 10 and 100 mg/L in 10 mM TrisHCl, pH 7.6) was filled in each well (2 mm in diameter) in the modified LB agar plates. The plates were incubated at 30°C for 18 h. The thickness of inhibition zones around the wells was measured and compared among tested strains.
To study further characterization of ASABF susceptibility, the relationship between ASABF dose and bacterial viability was determined. S. aureus was grown at 37°C in Luria-Bertani medium (LB broth) supplemented with or without appropriate antibiotics. NKSB was cultured with tetracycline (10 mg/L).45
NKSBn and NKSBv strains were cultured with tetracycline (10 mg/L) and chloramphenicol (12.5 mg/L).45,53
N315ex-n, -m, and -v were cultured with chloramphenicol (12.5 mg/L).53
The other strains were cultured without antibiotic. The cultures were collected in the logarithmic phase and washed twice with 10 mM TrisHCl, pH 7.6. The washed bacteria were resuspended in 60 µL of 10 mM TrisHCl, pH 7.6, containing a series of purified recombinant ASABF- for a three-fold increase in peptide concentration. The recombinant ASABF-
was prepared as described previously.9
The optical density of the S. aureus suspension was adjusted to 0.02 at 650 nm. After 2 h of incubation at room temperature, the test suspension (10 µL) was diluted 103
times in LB broth. The diluted sample (0.2 mL) was inoculated onto an LB agar plate containing antibiotics required for selection of each tested strain and incubated at 37°C overnight. The numbers of colonies were counted and plotted against ASABF-
concentration. Each experiment consisted of three or four independent trials using newly prepared bacterial cultures and ASABF-solutions.
Electron microscopy
The thickness of the cell wall was measured using electron micrographs. Electron microscopy was carried out as described previously.45
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To date, we have achieved the production of two recombinant ASABF-type antimicrobial peptides, ASABF- (A. suum origin) and ABF-2 (Caenorhabditis elegans origin), using a yeast expression system.6,9
The recombinant ASABF-
was obtained most efficiently as a mixture of two peptides (the natural form and the C-terminal glycine deleted form), and its antimicrobial activity was characterized in detail.9
In the previous study, we have shown that ASABF-
is most effective against S. aureus ATCC 6538P.9
To search for factors which affect ASABF susceptibility, we first tried to detect ASABF-resistant or hypersensitive S. aureus strains. Forty clinical isolates were tested. Preliminary tests by inhibition zone assay suggested that the susceptibility to ASABF-
was significantly diversified (0 to 7 mm thickness of inhibition zone at 100 mg/L ASABF-
) although this peptide was effective against most tested strains (data not shown). For detailed characterization, the viability versus ASABF-
concentration relationship was estimated for the most resistant (MRSA no. 7), and susceptible (MRSA no. 33) strains selected in the preliminary test, as well as three other well-characterized strains, N315, COL and Mu50 (Figure 1). The diversification of susceptibility was confirmed. N315 and MRSA no. 33 were relatively susceptible, and MRSA no. 7, COL and Mu50 were resistant.
|
Among the above resistant strains, we particularly noted the higher resistance of the Mu50 strain. As mentioned in the Introduction, some characteristic phenotypes of Mu50 may be due to overactivation of SigB. We therefore speculated that SigB may also regulate resistance against antimicrobial peptides. To test this hypothesis, the ASABF susceptibility was compared among NKSB, a sigB-overexpressing derivative NKSBn, and a vector control NKSBv at early-log phase. NKSBn was clearly more resistant to ASABF, suggesting that overexpression of sigB confers resistance to ASABF (Figure 2). The overactivation of SigB in NKSBn was confirmed by quantification of a SigB regulated gene expression (asp23) by northern blot analysis (Figure 3).36 The asp23 signals were detected at 0.7 and 1.5 kb as described previously.44 Both of the asp23 transcripts were clearly detected in NKSBn but not in NKSB and NKSBv.
|
|
SCCmec is not involved in sigB overexpression-induced resistance
To test whether SCCmec is essential for SigB-dependent antimicrobial peptide resistance, we compared ASABF- susceptibility of N315 with its SCCmec deletion mutant N315ex. No significant difference was detected [N315 viability (± SEM)=0.93 ± 0.14 at 0.1 mg/L ASABF-
, 0.57 ± 0.16 at 0.3 mg/L, and 0.02 ± 0.01 at 1 mg/L] suggesting that SCCmec does not affect ASABF susceptibility. Next, to examine whether SCCmec is essential for sigB-induced ASABF-
resistance, sigB was overexpressed in N315ex and its ASABF-
susceptibility was tested (Figure 4). The sigB-overexpressing strains of N315ex (N315ex-n and N315ex-m) also showed elevated resistance, as observed in NKSBn, suggesting that SCCmec is not essential for sigB-dependent resistance to ASABF.
|
As mentioned in the Introduction, a most remarkable change caused by the sigB overexpression is high-level ß-lactam resistance, and thickening of the cell wall.44,45 These two phenotypes are closely related because the target of ß-lactams is the synthesis of the cell wall. It is clear that ß-lactam resistance in MRSA is dependent on the product of the mecA gene in the SCCmec region, PBP2'. On the other hand, we excluded the possibility that SCCmec region is required for antimicrobial peptide resistance. Here, the question remains whether the sigB-induced cell-wall thickening is related to antimicrobial peptide resistance. If antimicrobial peptide resistance is derived from the change in the cell wall, we considered that the cell wall could also be changed by sigB overexpression in N315ex also and tested it by electron microscopy. As expected, the overexpression of sigB produced a thickened cell wall in the absence of the SCCmec region [cell wall thickness (± SEM) of N315=25.2 ± 2.3 nm, N315ex=25.5 ± 2.4 nm, N315ex-n=32.4 ± 3.7 nm, and N315ex-m=30.9 ± 3.3 nm]. Therefore, the sigB overexpression induces both antimicrobial peptide resistance and cell-wall thickening through molecular components independent of the SCCmec region. Not only in the artificial sigB-overexpressing strains, such cell wall thickening is also observed in Mu50, i.e. the ASABF-resistant strains (Mu50, NKSBn, N315ex-n and N315ex-m) identified in this study commonly exhibited remarkable cell wall thickening.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The SigB activity in the artificial sigB-overexpressing strain, NKSBn, was estimated to be approximately 1.82.5 times higher than that in N315. Such overactivation was as high as that in Mu50.44 ASABF-susceptibility of N315, which maintains an intrinsic sigB, was not significantly different from that of NKSB, a sigB-knockout derivative of N315. These results indicate that the constitutive overactivation of SigB at the level detected in NKSBn or Mu50 is needed to acquire ASABF resistance.
Cell-wall thickening and ASABF resistance were suggested to be correlated. Since antimicrobial peptides are believed to kill microbes by destroying selective permeability of cytoplasmic membrane, modification of cell wall is a possible factor affecting susceptibility to those peptides, i.e. those peptides must permeate the cell wall to access their target sites. Indeed, some mechanisms of bacterial resistance to antimicrobial peptides have been proposed by cell wall modification.10 In the future, the changes in cell wall brought about by sigB overexpression should be characterized from the standpoint of antimicrobial peptide resistance.
We previously proposed that ASABF-type antimicrobial peptide is a good candidate as a clinically applicable anti-S. aureus agent.9
Recombinant ASABF was effective against all S. aureus strains tested in this study, i.e. 40 clinical isolates, two well-characterized MRSA (COL and Mu50), and a pre-MRSA (N315), at a concentration lower than 1 µM (7.4 mg/L). Although ASABF- susceptibility is diversified among these S. aureus strains, including the resistance induced by SigB overactivation, these results can support ASABF-type antimicrobial peptide as a promising anti-S. aureus agent.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Hancock, R. E. & Patrzykat, A. (2002). Clinical development of cationic antimicrobial peptides: from natural to novel antibiotics. Current Drug Targets. Infectious Disorders 2, 7983.[Medline]
3 . Dimarcq, J. -L., Bulet, P., Hetru, C. et al. (1998). Cysteine-rich antimicrobial peptides in invertebrates. Biopolymers 47, 46577.[CrossRef][ISI][Medline]
4
.
Landon, C., Barbault, F., Legrain, M. et al. (2004). Lead optimization of antifungal peptides with 3D NMR structures analysis. Protein Science 13, 70313.
5
.
Kato, Y. & Komatsu, S. (1996). ASABF, a novel cysteine-rich antibacterial peptide isolated from the nematode Ascaris suum: purification, primary structure, and molecular cloning of cDNA. Journal of Biological Chemistry 271, 304938.
6 . Kato, Y., Aizawa, T., Hoshino, T. et al. (2002). abf-1 and abf-2, ASABF-type antimicrobial peptide genes in Caenorhabditis elegans. Biochemical Journal 361, 22130.[CrossRef][ISI][Medline]
7 . Pillai, A., Ueno, S., Zhang, H. et al. (2003). Induction of ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides by bacterial injection: novel members of ASABF in the nematode Ascaris suum. Biochemical Journal 371, 6638.[CrossRef][ISI][Medline]
8
.
Zhang, H. & Kato, Y. (2004). Common structural properties specifically found in the CSß-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? Developmental and Comparative Immunology 27, 499503.[ISI]
9
.
Zhang, H., Yoshida, S., Aizawa, T. et al. (2000). In vitro antimicrobial properties of recombinant ASABF, an antimicrobial peptide isolated from the nematode Ascaris suum. Antimicrobial Agents and Chemotherapy 44, 27015.
10 . Peschel, A. (2002). How do bacteria resist human antimicrobial peptides? Trends in Microbiology 10, 17986.[CrossRef][ISI][Medline]
11
.
Peschel, A., Otto, M., Jack, R. W. et al. (1999). Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. Journal of Biological Chemistry 274, 840510.
12 . Guo, L., Lim, K. B., Poduje, C. M. et al. (1998). Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell 95, 18998.[CrossRef][ISI][Medline]
13
.
Guina, T., Yi, E. C., Wang, H. et al. (2000). A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to -helical antimicrobial peptides. Journal of Bacteriology 182, 407786.
14 . Belden, W. J. & Miller, S. I. (1994). Further characterization of the PhoP regulon: identification of new PhoP-activated virulence loci. Infection and Immunity 62, 5095101.[Abstract]
15 . Gunn, J. S., Lim, K. B., Krueger, J. et al. (1998). PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Molecular Microbiology 27, 117182.[CrossRef][ISI][Medline]
16
.
Robey, M., O'Connell, W., Cianciotto, N. P. et al. (2001). Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection. Infection and Immunity 69, 427686.
17
.
Gunn, J. S., Ryan, S. S., Van Velkinburgh, J. C. et al. (2000). Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium. Infection and Immunity 68, 613946.
18
.
McCoy, A. J., Liu, H., Falla, T. J. et al. (2001). Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides. Antimicrobial Agents and Chemotherapy 45, 20307.
19
.
Peschel, A., Jack, R. W., Otto, M. et al. (2001). Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine. Journal of Experimental Medicine 193, 106776.
20 . Staubitz, P., Peschel, A., Nieuwenhuizen, W. F. et al. (2001). Structurefunction relationships in the tryptophan-rich, antimicrobial peptide indolicidin. Journal of Peptide Science 7, 55264.[CrossRef][ISI][Medline]
21
.
Shafer, W. M., Qu, X., Waring, A. J. et al. (1998). Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family. Proceedings of the National Academy of Sciences, USA 95, 182933.
22
.
Bayer, A. S., Cheng, D., Yeaman, M. R. et al. (1998). In vitro resistance to thrombin-induced platelet microbicidal protein among clinical bacteremic isolates of Staphylococcus aureus correlates with an endovascular infectious source. Antimicrobial Agents and Chemotherapy 42, 316972.
23
.
Bayer, A. S., Prasad, R., Chandra, J. et al. (2000). In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity. Infection and Immunity 68, 354853.
24
.
Kupferwasser, L. I., Skurray, R. A., Brown, M. H. et al. (1999). Plasmid-mediated resistance to thrombin-induced platelet microbicidal protein in staphylococci: role of the qacA locus. Antimicrobial Agents and Chemotherapy 43, 23959.
25
.
Guo, L., Lim, K. B., Gunn, J. S. et al. (1997). Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ. Science 276, 2503.
26
.
Dunman, P. M., Murphy, E., Haney, S. et al. (2001). Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. Journal of Bacteriology 183, 734153.
27
.
Deora, R. & Misra, T. K. (1996). Characterization of the primary factor of Staphylococcus aureus. Journal of Biological Chemistry 271, 2182834.
28
.
Wu, S., Lencastre, H. & Tomasz, A. (1996). Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing. Journal of Bacteriology 178, 603642.
29 . Kullik, I. I. & Giachino, P. (1997). The alternative sigma factor sigmaB in Staphylococcus aureus: regulation of the sigB operon in response to growth phase and heat shock. Archives of Microbiology 167, 1519.[CrossRef][ISI][Medline]
30
.
Morikawa, K., Inose, Y., Okamura, H. et al. (2003). A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes. Genes to Cells 8, 699712.
31 . Bateman, B. T., Donegan, N. P., Jarry, T. M. et al. (2001). Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation. Infection and Immunity 69, 78517.
32
.
Benson, A. K. & Haldenwang, W. G. (1993). Regulation of B levels and activity in Bacillus subtilis. Journal of Bacteriology 175, 234756.[Abstract]
33
.
Bischoff, M., Entenza, J. M. & Giachino, P. (2001). Influence of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus. Journal of Bacteriology 183, 51719.
34 . Bischoff, M., Roos, M., Putnik, J. et al. (2001). Involvement of multiple genetic loci in Staphylococcus aureus teicoplanin resistance. FEMS Microbiology Letters 194, 7782.[CrossRef][ISI][Medline]
35
.
Cheung, A. L., Chien, Y. -T. & Bayer, A. S. (1999). Hyperproduction of -hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus. Infection and Immunity 67, 13317.
36
.
Giachino, P., Engelmann, S. & Bischoff, M. (2001). B activity depends on RsbU in Staphylococcus aureus. Journal of Bacteriology 183, 184352.
37
.
Karlsson, A. & Arvidson, S. (2002). Variation in extracellular protease production among clinical isolates of Staphylococcus aureus due to different levels of expression of the protease repressor sarA. Infection and Immunity 70, 423946.
38
.
Kullik, I., Giachino, P. & Fuche, T. (1998). Deletion of the alternative sigma factor B in Staphylococcus aureus reveals its function as a global regulator of virulence genes. Journal of Bacteriology 180, 481420.
39
.
Nair, S. P., Bischoff, M., Senn, M. M. et al. (2003). The B regulon influences internalization of Staphylococcus aureus by osteoblasts. Infection and Immunity 71, 416770.
40
.
Price, C. T. D., Singh, V. K., Jayaswal, R. K. et al. (2002). Pine oil cleaner-resistant Staphylococcus aureus: reduced susceptibility to vancomycin and oxacillin and involvement of SigB. Applied and Environmental Microbiology 68, 541721.
41
.
Rachid, S., Ohlsen, K., Wallner, U. et al. (2000). Alternative transcription factor B is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate. Journal of Bacteriology 182, 68246.
42
.
Somerville, G. A., Saïd-Salim, B., Wickman, J. M. et al. (2003). Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for hostpathogen interactions. Infection and Immunity 71, 472432.
43
.
Ziebandt, A. -K., Weber, H., Rudolph, J. et al. (2001). Extracellular proteins of Staphylococcus aureus and the role of SarA and B. Proteomics 1, 48093.[CrossRef][ISI][Medline]
44
.
Morikawa, K., Inose, Y., Higashide, M. et al. (2004). High-level expression of alternative sigma factor, B, is an important determinant to increase ß-lactam resistance in methicillin-resistant Staphylococcus aureus. Microbial Drug Resistance, under revision.
45
.
Morikawa, K., Maruyama, A., Inose, Y. et al. (2001). Overexpression of sigma factor, B, urges Staphylococcus aureus to thicken the cell wall and to resist ß-lactams. Biochemical and Biophysical Research Communications 288, 3859.[CrossRef][ISI][Medline]
46 . Alvarez-Bravo, J., Kurata, S. & Natori, S. (1994). Novel synthetic antimicrobial peptides effective against methicillin-resistant Staphylococcus aureus. Biochemical Journal 302, 5358.[ISI][Medline]
47
.
Katayama, Y., Ito, T. & Hiramatsu, K. (2000). A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 44, 154955.
48 . Hartman, B. J. & Tomasz, A. (1984). Low-affinity penicillin-binding protein associated with ß-lactam resistance in Staphylococcus aureus. Journal of Bacteriology 158, 5136.[ISI][Medline]
49 . Hiramatsu, K., Asada, K., Suzuki, E. et al. (1991). Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). FEBS Letters 298, 1336.[CrossRef][ISI]
50
.
Ito, T., Katayama, Y. & Hiramatsu, K. (1999). Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrobial Agents and Chemotherapy 43, 144958.
51
.
Tanaka, M., Wada, N., Kurosaka, S. M. et al. (1998). In vitro activity of DU-6859a against methicillin-resistant Staphylococcus aureus isolates with reduced susceptibilities to vancomycin. Journal of Antimicrobial Chemotherapy 42, 5523.
52 . Shafer, W. M. & Iandolo, J. J. (1979). Genetics of staphylococcal enterotoxin B in methicillin-resistant isolates of Staphylococcus aureus. Infection and Immunity 25, 90211.[ISI][Medline]
53 . Nilsson, B., Abrahmsen, L. & Uhlen, M. (1985). Immobilization and purification of enzymes with staphylococcal protein A fusion vectors. EMBO Journal 4, 107580.[Abstract]
54 . Kurz, C. L. & Ewbank, J. J. (2003). Caenorhabditis elegans: an emerging genetic model for the study of innate immunity. Nature Reviews. Genetics 4, 38090.[CrossRef][ISI][Medline]
55
.
Sifri, C. D., Begun, J., Ausubel, F. M. et al. (2003). Caenorhabditis elegans as a model host for Staphylococcus aureus pathogenesis. Infection and Immunity 71, 220817.
|