Liberation of surface-located penicillinase from Staphylococcus aureus

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Conditions suitable for induced formation and secretion of penicillinase (EC 3.5.2.6) in protoplasts of Staphylococcus aureus were determined. No requirement was found for cells to be exposed to inducer prior to formation of protoplasts. Neither cell wall components nor mesosomes appeared to be necessary for induction or secretion. In medium containing 1.1 M sucrose about half of the formed enzyme was soluble, whereas in medium containing 0.37 M sodium succinate only about 10% of the penicillinase remained protoplast-bound. Low concentrations of polyanions (dextran sulfate and potassium polyvinyl-sulfonate) inhibited the formation of induced penicillinase, as did 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid. None of these compounds inhibited the activity of native penicillinase, and none would be expected to pass through the protoplast membrane. Penicillinase, denatured in 4 M urea, could be renatured by dilution in the presence of benzylpenicillin, and the above three inhibitors interfered strongly with this process. The results are taken as evidence that penicillinase may be secreted through the protoplast membrane in an incompletely folded form.In previous studies of the synthesis and liberation of penicillinase (EC 3.5.2.6) by cells of Staphylococcus aureus (3, 4), we showed that there is a fraction of cell-bound penicillinase, comprising up to 40% of the total, which can be liberated by incubation of the cells for short periods at 37 C in 0.15 M sodium citrate. This fraction of cell-bound exopenicillinase is replenished only under conditions permitting de novo penicillinase synthesis and is the most recently synthesized enzyme (5). No transformation of old remaining cell-bound penicillinase to exopenicillinase takes place. The bacterial cell wall has been implicated in the formation of active exoenzymes by several authors. Spheroplasts of Streptococcus faecalis were unable to produce extracellular proteinase until some cell wall component had first been resynthesized (9), and protoplasts of Bacillus subtilis failed to synthesize extracellular amylase and protease under conditions permitting the unimpaired incorporation of "4C-valine into a trichloroacetic acid-insoluble product (14).Induced enzyme synthesis in protoplasts of bacteria has been shown only in very few cases. It is not too clear from the literature whether true protoplasts of B. cereus have the capacity to produce inducible penicillinase. On the one hand, Sheinin (23) reported that protoplasts of B. cereus 569 could be directly induced to form penicillinase at a rate comparable to that of whole cells, whereas Duerksen (7) was not able to demonstrate induced formation of penicillinase in protoplasts of the same organism unless the cells used to produce these protoplasts had been preinduced. In an attempt to resolve this discrepancy, Wainwright and Martin (25), using spheroplasts of this organism, obtained complex results. The interpretation was further complicated by a high and variable rate of basal synthesis of penicillinase by no...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 91%

Formation and Secretion of Induced Penicillinase in Protoplasts of Staphylococcus aureus

Coles

Gross

1973

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“…ated 3-lactamases have been described previously (22), even the location of the extracellular portion varies according to the medium in which the strain is grown. Coles and Gross have shown with strain PC1 that the proportion of staphylococcal ,-lactamase present in the supernatant of broth medium is much greater when the medium is buffered with phosphate (40% exoenzyme) than when it is buffered with Tris hydrochloride (<2% exoenzyme) (6). Presumably, in Tris the extracellular ,B-lactamase is ionically or hydrophobically bound to cell wall structures (6), whereas the P-lactamase is soluble in phosphate buffer and is displaced from the cell.…”

Section: Discussionmentioning

confidence: 99%

“…Coles and Gross have shown with strain PC1 that the proportion of staphylococcal ,-lactamase present in the supernatant of broth medium is much greater when the medium is buffered with phosphate (40% exoenzyme) than when it is buffered with Tris hydrochloride (<2% exoenzyme) (6). Presumably, in Tris the extracellular ,B-lactamase is ionically or hydrophobically bound to cell wall structures (6), whereas the P-lactamase is soluble in phosphate buffer and is displaced from the cell. The possibility that extracellular staphylococcal P-lactamase normally adheres to cell wall structures and therefore is concentrated close to the bacterium that produced it is teleologically attractive, since it produces a situation similar to that observed with gram-negative bacteria, in which ,-lactamase is restricted to the periplasmic space by its inability to pass through the outer membrane (29,35).…”

Section: Discussionmentioning

confidence: 99%

Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography

Kernodle

Zygmunt

McGraw

et al. 1990

Boronic acids are active-site inhibitors of serine P-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify j-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purffication of Staphylococcus aureus P-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal ,l-lactamase exhibited non-active-site-mediated adsorption to the agarose used as a support for the meta-aminophenylborate ligand, preventing the recovery of j-lactamase from the column. Second, the staphylococcal (-lactamases exhibited low affinity for meta-aminophenylborate with inhibition constants (K's) ranging from 8.0 x 10-3 to 20.0 x 10-3 M. These problems were resolved by modifying the buffers utilized during chromatography and increasing the dimensions of the affinity column, and a two-stage procedure consisting of cation-exchange chromatography followed by affinity chromatography was used to purify each of the four variants of staphylococcal ,I-lactamase. The mean specffic activities of the purified type A, B, C, and D 3-lactamases were 44.6, 12.2, 10.6, and 30.8 ,3-lactamase in pure form from a variety of bacterial species, including Bacillus cereus, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella aerogenes, and Pseudomonas maltophilia (4). The present study investigates the applicability of the chromatographic method described by Cartwright and Waley to the purification of the four recognized variants of staphylococcal 1-lactamase. A modified protocol useful in the preparation of purified P-lactamase from S. aureus is described. MATERIALS AND METHODSOrganisms. S. aureus strains that produce each of the four variants of staphylococcal ,-lactamase were evaluated and included PC1(pl524) and RN11(pI258) (type A P-lactamase), 22260 and ST79M41 (type B P-lactamase), V137 (type C 1-lactamase), and FAR8 and FAR10 (type D P-lactamase).The sources and pedigrees of these strains have been described previously (12-14, 16, 23, 24, 26, 31

“…The proportion of extracellular enzyme depends on the strain studied and on the conditions of growth (2). The majority of penicillin-resistant strains causing nosocomial infections are known to produce large amounts of extracellular 3-lactamase (4).…”

mentioning

confidence: 99%

Importance of extracellular and cell-bound beta-lactamase in mediating resistance of Staphylococcus aureus to mezlocillin

Haller¹

1984

The 3-lactamase of Staphylococcus aureus is excreted into the surrounding medium, although to different extents (1, 3). The proportion of extracellular enzyme depends on the strain studied and on the conditions of growth (2). The majority of penicillin-resistant strains causing nosocomial infections are known to produce large amounts of extracellular 3-lactamase (4). There is some evidence that the cellassociated enzyme molecules are covalently bound to phosphatides (3), but it is not yet clear whether they are precursors of the free enzyme, as has been shown to be the case in Bacillus licheniformis (7).Investigators in this laboratory have an interest in the importance of cell-bound and extracellular 1-lactamase in mediating resistance of S. aureus to mezlocillin because of the potential influence on the outcome of susceptibility tests. When minimal inhibitory concentrations are determined by the broth or the microdilution technique, the excreted 13-lactamase accumulates in a small volume of broth and thus may rapidly inactivate the pool of hydrolyzable antibiotic in the test system. In vivo, on the other hand, a considerable portion of exoenzyme may diffuse away from the microbial cells, depending on the site of infection, and therefore may lose its protective effect. Therefore, the question arose whether the enzyme associated with the cell wall alone constitutes a barrier strong enough to inactivate the intruding antibiotic, thus preventing it from reaching the target sites. This was examined by a new in vitro test model designed to eliminate the effect of exoenzyme. MATERIALS AND METHODSBacteria. The test strains were fresh clinical isolates of S. aureus which were stored in liquid nitrogen until use. Strains Va7871 and Va7906 were selected because of their extracellular 3-lactamase activity. Exponentially growing cultures (35°C) were induced with 1 ,i.g of cefoxitin per ml for 3 h (final cell count, 2 x 108 to 5 x 108 CFU/ml), and the extracellular ,-lactamase was measured after centrifugation by iodometric assay (6). The amounts of penicillin G de-125 stroyed by 1 ml of culture supernatant from strains Va7871 and Va7906 were 1.3 and 1.9 mmol/min, respectively. Minimal activity was found without induction. S. aureus Va8143 proved to be 3-lactamase negative under these conditions, even in the presence of subinhibitory concentrations of cefoxitin.In vitro model for elimination of exoenzyme. Iso-Sensitest broth (Oxoid) was used as the nutrient medium throughout all experiments. Inocula were preincubated at 35°C on a rotary shaker for 5 h to obtain cell material of early stationary growth phase. Approximately 105 cells were spread on a sterile cellulose-nitrate filter (Schleicher & Schull Co.; PH 79/1, 0.1-,um-pore size, 76-mm diameter) and were covered by a second membrane. This membrane sandwich containing the bacteria was then mounted in a sterile ultrafiltration chamber (Amicon 402) and incubated at 35°C. During incubation, a constant flow (280 ml/h) of prefiltered nutrient broth containing the antibiotic c...