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1.
A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become inducible.  相似文献   

2.
K Graves-Woodward  R F Pratt 《Biochemistry》1999,38(32):10533-10542
Kinetics studies in homogeneous aqueous solution showed that solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus (a bacterial DD-peptidase) was inhibited by the amphiphilic glycolipid antibiotic moenomycin. Inhibition at the peptidase site was determined by competition experiments between moenomycin and the chromophoric beta-lactam nitrocefin. Under conditions of high salt concentration (1 M NaCl), pseudo-first-order rate constants for the reaction of moenomycin with sPBP2a leading to inhibition of acylation by nitrocefin varied with moenomycin concentration in a biphasic fashion. At low moenomycin concentration (<20 microM) little inhibition occurred, but at higher concentrations a linear increase in rate constant with moenomycin concentration was observed, yielding a second-order rate constant of inhibition of 120 s(-)(1) M(-)(1). Since the cmc of moenomycin under these conditions was shown to be ca. 20 microM, the inhibition was concluded to arise from reaction of sPBP2a with a moenomycin micelle. Protein fluorescence studies showed a pseudo-first-order decrease in fluorescence on reaction of the protein with moenomycin. The variation of this rate constant with moenomycin concentration was consistent with reaction of a moenomycin monomer with the protein with a second-order rate constant of 650 s(-)(1) M(-)(1). This monomer reaction did not occur at the DD-peptidase site since its rate was unaffected by prior acylation of the enzyme by benzylpenicillin; nor did it inhibit reaction at that site by beta-lactams. Under low salt conditions (0.175 M NaCl) where reaction could be studied over a greater range of monomer concentrations since the cmc was ca. 120 microM, similar reactions were involved. Under these circumstances, inhibition was concerted with the reaction of moenomycin monomers, although fast premicellar aggregation of moenomycin with the protein also occurred. All moenomycin interactions with sPBP2a were reversible, as revealed by detergent-extraction chromatography. Lower limits to moenomycin off-rates and equilibrium dissociation constants were 7.7 x 10(-)(4) s(-)(1) and 1.2 microM, respectively. Other amphiphiles did not react in exactly the same manner as moenomycin, indicating some degree of specificity in reactions of the latter. sPBP2a did not have detectable affinity for lipid surfaces (Triton X-114 and phosphatidylglycerol vesicles). A general scheme for reaction of moenomycin with sPBP2a is proposed.  相似文献   

3.
The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.  相似文献   

4.
The effects of fosfomycin on penicillin-binding proteins (PBPs) were studied on the methicillin-resistant Staphylococcus aureus strain CIP (Collection de l'Institut Pasteur, Paris, France) 65-25 and on a methicillin-susceptible S. aureus strain CIP 65-6. The combinations of fosfomycin and oxacillin were synergistic, additive or antagonistic, depending on antibiotic concentrations. Fosfomycin induced modifications of the PBP profile of the two strains studied. In particular, it increased the expression of PBP2. This suggested that this protein is inducible; the only PBP not affected by fosfomycin was PBP3.  相似文献   

5.
The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.  相似文献   

6.
7.
Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k2) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (Kd) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.  相似文献   

8.
Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) is important to identify patients colonized with this pathogen and implement infection control precautions. We aimed to improve the combined use of the mecA gene polymerase chain reaction (PCR) and the SA442 PCR to detect MRSA in clinical screening samples. In a true MRSA the mecA copy number will be equal to the SA442 copy number, whereas in samples with a methicillin-sensitive Staphylococcus aureus (MSSA) combined with a methicillin-resistant coagulase-negative Staphylococcus (MRCNS) the copy numbers will usually differ. Here we introduce a PCR system, relative quantification PCR (RQ-PCR), which takes this difference into account. RQ-PCR identifies true MRSA in clinical samples with a specificity that is comparable to the SCCmec-based PCRs.  相似文献   

9.
10.
The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.  相似文献   

11.
The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.  相似文献   

12.
We found that tau, one of the major microtubule-associated proteins, is a good substrate for protein kinase C. The phosphorylation occurred mainly on serine residues and the sites phosphorylated by protein kinase C were largely different from those phosphorylated by cAMP-dependent protein kinase as analyzed by phosphopeptide mapping. The protein kinase C-mediated phosphorylation of tau reduced its abilities to promote tubulin polymerization and to cross-link actin filaments. The reduction in its abilities was in proportion to the number of phosphates incorporated into tau.  相似文献   

13.
Methicillin-resistant Staphylococcus aureus (MRSA) is a global scourge, and treatment options are becoming limited. The MRSA phenotype reverts to that of beta-lactam-sensitive S. aureus when bacteria are grown at pH 5.0 in broth and, more importantly from a medical perspective (protracted, relapsing infections), after phagocytosis by macrophages, where the bacteria thrive in the acidic environment of phagolysosomes. The central factor for the MRSA phenotype is the function of the penicillin-binding protein (PBP) 2a, which maintains transpeptidase activity while being poorly inhibited by beta-lactams because of a closed conformation of its active site. We document herein by binding, acylation/deacylation kinetics, and circular dichroism spectroscopy with purified PBP 2a that at acidic pH (i) beta-lactams interact with PBP 2a more avidly; (ii) the non-covalent pre-acylation complex exhibits a lower dissociation constant and an increased rate of acyl-enzyme formation (first-order rate constant) without change in hydrolytic deacylation rate; and (iii) PBP 2a undergoes a conformational change in the presence of the antibiotic consistent with the opening of the active site from the closed conformation. These observations argue that PBP 2a most likely evolved for its physiological function at pH 7 or higher by adopting a closed conformation, which is not maintained at acidic pH. Although at the organism level the effect of acidic pH on other biological processes in MRSA could not be discounted, our report should provide the impetus for closer examination of the properties of PBP 2a at low pH and thereby identifying novel points of intervention in combating this problematic organism.  相似文献   

14.
A close homologue of mecA, the determinant of broad-spectrum beta-lactam resistance in Staphylococcus aureus was recently identified as a native gene in the animal commensal species Staphylococcus sciuri. Introduction of the mecA homologue from a methicillin-resistant strain of S. sciuri into a susceptible strain of S. aureus caused an increase in drug resistance and allowed continued growth and cell wall synthesis of the bacteria in the presence of high concentrations of antibiotic. We determined the muropeptide composition of the S. sciuri cell wall by using a combination of high-performance liquid chromatography, mass spectrometric analysis, and Edman degradation. Several major differences between the cell walls of S. aureus and S. sciuri were noted. The pentapeptide branches in S. sciuri were composed of one alanine and four glycine residues in contrast to the pentaglycine units in S. aureus. The S. sciuri wall but not the wall of S. aureus contained tri- and tetrapeptide units, suggesting the presence of dd- and ld-carboxypeptidase activity. Most interestingly, S. aureus carrying the S. sciuri mecA and growing in methicillin-containing medium produced a cell wall typical of S. aureus and not S. sciuri, in spite of the fact that wall synthesis under these conditions had an absolute dependence on the heterologous S. sciuri gene product. The protein product of the S. sciuri mecA can efficiently participate in cell wall biosynthesis and build a cell wall using the cell wall precursors characteristic of the S. aureus host.  相似文献   

15.
Molecular cloning and nucleotide sequence analysis were performed for the identification of the regulator genes of methicillin resistance in the genome of a MRSA strain N315. Two open reading frames (orfs) were identified in the 5'-flanking region of the mecA gene. Predicted amino acid sequences of these orfs showed extensive homology to the co-inducer and the repressor protein of the penicillinase (PCase) production in Staphylococcus aureus as well as in Bacillus licheniformis. These orfs are considered to encode putative co-inducer and repressor proteins specific for the regulation of methicillin resistance in MRSA.  相似文献   

16.
Abstract During growth of Streptomyces niveus wild-type in the novobiocin production medium CDM the resistance of mycelia to novobiocin rises from about 25 μg/ml to over 200 μg/ml. ( S. lividans , a novobiocin-sensitive strain, is resistant to approx. 10 μg/ml novobiocin.) The initial period of low level resistance extends from the time of inoculation of the culture until approx. 70 h when the culture is still in the growth phase. High level resistance is initiated before the start of novobiocin production and rises rapidly to a maximum level beyond the end of the growth phase. The rise in pH of the unbuffered CDM medium which occurs during S. niveus fermentation was shown not to be the cause of the change in novobiocin resistance. However, mycelia-free CDM from S. niveus cultures expressing high level novobiocin resistance was shown to contain a factor which induced high level novobiocin resistance in germinating S. niveus spores. Kinetic studies revealed that the inducer first appears in the culture medium before the switch to high level resistance begins and reaches its highest concentration before resistance reaches its maximum level.  相似文献   

17.
mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌   总被引:9,自引:2,他引:9  
目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌(methicillin resistant staphylococcus aureus,MRSA)。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91.43%。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。  相似文献   

18.
Protein A has been isolated from a methicillin-resistant strain of Staphylococcus aureus, A676, which produces only extracellular protein A. The material is homogeneous after a one-step separation and has a molecular weight of 41000. Its physicochemical and immunochemical properties have been studied.  相似文献   

19.
MRSA CC130 containing the mecA homologue mecA(LGA251) were reported from the UK and from Denmark so far from cattle and humans. Here we report on 11 MRSA CC130 among a sample of 12691 isolates of human origin collected from January 2006 until June 2011. MRSA CC130 grew insufficiently on chromogernic agar plates for detection of MRSA; the agglutination test for presence of PBP2a was negative. We designed primers for specific detection of mecA(LGA251) as well as for concomitant detection of both, mec(LGA251) and mecA. As already described, the isolates exhibited spa-types t843, t1736, and t1773. The ccrA homologue indicated the presence SCCmec(XI). When subjected to further characterization by means of a commercially available microarray the isolates were negative for sak chp, and scn, and as expected positive for hla, untruncated hlb, and hld. They furthermore contained edinB, aur, slpA, slpB, slpE. From genes coding for surface and cell wall associated products the ica-operon, cap8, clfA, clfF, ebpS, fnbA, fnbB, sdrC were detected but not cna. The isolates were negative for enterotoxin genes and tst, as well as for eta, and etb; agr-type was III.  相似文献   

20.
A homolog of the Staphylococcus aureus methicillin resistance gene mecA was recently shown to be ubiquitous in independent isolates of the animal species Staphylococcus sciuri. The mecA gene homolog and regions flanking it were cloned and sequenced from four strains of S. sciuri: strain K1 (ATCC 29062), a representative of S. sciuri subsp. sciuri; two strains (K3 and K8) representing S. sciuri subsp. rodentius; and strain K11, a representative of S. sciuri subsp. carnaticum. Strains K1 and K11 were susceptible to methicillin, while strains K3 and K8 showed heterogeneous resistance. The mecA genes of strains K1 and K11 and one of the two copies of mecA (mecA1) present in strain K3 had virtually identical DNA sequences in the mecA gene and were similar in genetic organization in the flanking regions. In contrast, the single copy of mecA in strain K8 and the second copy of mecA (mecA2) in strain K3 had mecA DNA sequences identical to that of S. aureus mecA, and the mecA region in these two strains was also similar to that of the same region in the S. aureus strain used for comparison. Interestingly, an open reading frame defining an N-terminal truncated polypeptide, NTORF101, with a high degree of homology to a DNA segment in the hypervariable region of methicillin-resistant S. aureus (and also similar to the Escherichia coli gene ugpQ) was also identified downstream of the mecA homolog of strain K11, representing S. sciuri subsp. carnaticum. The ugpQ-like gene is not present in methicillin-susceptible strains of S. aureus. The presence of such a ugpQ-like gene together with the homolog of mecA in strain K11 supports the speculation that these genetic elements may be evolutionary relatives and/or precursors of the genetic determinant of methicillin resistance in S. aureus.  相似文献   

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