Mutanolysin-induced spheroplasts of Streptococcus mutants are true protoplasts

Siegel, Johanna; Hurst, Steven F.; Liberman, E S; Coleman, Sylvia E.; Bleiweis, Arnold S.

doi:10.1128/iai.31.2.808-815.1981

Cited by 83 publications

(26 citation statements)

References 16 publications

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“…However, the present study succeeded in extracting a cell-wall fraction of S. pneumoniae with mutanolysin or high-pH treatment, and this strategy seems to be reliable for the extraction of cell-wall-associated proteins. Mutanolysin produces stable protoplasts of S. pneumoniae, with experiments using ( 3 H)thymidine and ( 14 C)leucine as cytoplasmic pool markers revealing only minimal (10%) leakage during a 1-h incubation period [22]. This is important, since it is known that cell-wall-associated protein preparations are usually contaminated with cytosolic proteins.…”

Section: Discussionmentioning

confidence: 99%

Streptococcus pneumoniae: proteomics of surface proteins for vaccine development

Morsczeck¹,

Prokhorova²,

Sigh³

et al. 2008

Clinical Microbiology and Infection

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Two formulations of pneumococcal vaccines are currently available to prevent invasive disease in adults and children. However, these vaccines will not protect against the majority of Streptococcus pneumoniae serotypes. The use of highly conserved cell-wall-associated proteins in vaccines may circumvent this problem. A proteomics approach was used to identify 270 S. pneumoniae cell-wall-associated proteins, which were then screened in a process that included in-silico, in-vitro and in-vivo validation criteria. Five potential candidates for inclusion in a vaccine were selected, expressed in Escherichia coli, and purified for use in immunisation experiments. These proteins were detected in at least 40 different serotypes of S. pneumoniae, and were expressed in S. pneumoniae isolates causing infection. Two of the five candidate proteins, the putative lipoate protein ligase (Lpl) and the ClpP protease, resulted in a reduced CFU titre and a trend towards reduced mortality in an animal sepsis model for investigating new S. pneumoniae protein vaccines.

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Section: Discussionmentioning

confidence: 99%

Streptococcus pneumoniae: proteomics of surface proteins for vaccine development

Morsczeck¹,

Prokhorova²,

Sigh³

et al. 2008

Clinical Microbiology and Infection

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“…Proteolytic activity has been reported to co-migrate with M-1 on IEF gels, but it can be separated from the muramidase by ion exchange chromatography on CM Sephadex, which was the procedure involved in the purification of the mutanolysin used in this study (14,15,18). Proteolytic activity in the mutanolysin preparations was measured using the Azocoll assay (Calbiochem-Behring Corp., La Jolla, CA), as described by Siegel et al (18). The purified M-1 preparations had no detectable proteolytic activity, while the crude mutanolysin preparation was heavily contaminated.…”

Section: Methodsmentioning

confidence: 99%

Treatment of experimental erosive arthritis in rats by injection of the muralytic enzyme mutanolysin.

Janusz¹,

Chetty²,

Eisenberg³

et al. 1984

The Journal of Experimental Medicine

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A single intravenous injection into rats of 0.4 mg of the muralytic enzyme mutanolysin, given as long as 3 d after an arthropathic dose of peptidoglycan-polysaccharide polymers derived from group A streptococci (PG-APS), resulted in a complete resolution of acute arthritis and the prevention of chronic joint disease. When administration of mutanolysin was delayed until 14 d after the injection of PG-APS, a great reduction in the severity of chronic inflammation was still observed. Quantitation of the amount of PG-APS present in the limbs, spleen, and liver by a solid phase enzyme-linked immunoassay indicated that the tissues of mutanolysin-treated rats contained as much PG-APS as tissues of PBS-treated control rats. In addition, rats treated with mutanolysin immediately after receiving an intraperitoneal injection of PG-APS developed a transient limb edema similar to that seen in rats after the injection of PG-APS digested to a small fragment size in vitro with mutanolysin. We hypothesize that mutanolysin acts in vivo by degrading PG-APS to small fragments that persist but are no longer arthropathic.

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“…M-1 mutanolysin was kindly supplied by Kanae Yokagawa, Dainippon Pharmaceuticals, Ltd., Osaka, Japan. The final purification steps of M-1 preparation were essentially those of Siegel et al (27). Briefly, the preparation was first sonicated for 30 s with a model F sonicator (Heat System Ultrasonics, Inc., Plainview, N.Y.).…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of cell wall peptidoglycan and polysaccharide antigens by protoplasts of type III group B Streptococcus

Yeung¹,

Mattingly²

1983

J Bacteriol

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The formation of a nascent peptidoglycan-group-specific antigen of type III group B Streptococcus at the cell membrane level was demonstrated with an M-1 mutanolysin-prepared protoplast system. Protoplasts of group B streptococci in suitably stabilized medium (20% sucrose) readily incorporated [3H]acetate into cell surface macromolecules. Four major polysaccharides were isolated from the protoplast cultural supernatant fluid: the peptidoglycan group-specific antigen polymer, the group B-specific antigen, and the low-molecular-weight and highmolecular-weight forms of the type III polysaccharide antigen. Biosynthesis of all four polymers was not affected by the action of chloramphenicol, indicating protein synthesis was not required for the production of polysaccharide in this system. However, all but the low-molecular-weight type III antigen were inhibited by the action of bacitracin, suggesting that three of the polymers share a common synthesis-assembly site in the membrane. Attachment of the high-molecularweight antigen to the nascent peptidoglycan-group B antigen complex did not occur in the protoplast system, suggesting that a more complex cell wall matrix may be necessary before linkage of the high-molecular-weight antigen takes place.Due to the recent emergence of group B Streptococcus as an important pathogen in neonatal disease (3, 4), several cell surface components have been examined as possible virulence determinants. Only the type-specific antigens, which are carbohydrate in nature (36) with the exception of the Ibc protein (35, 37), have been shown to have antiphagocytic properties, and antiserum prepared against the type-specific antigen protects against challenge by homologous organisms in animal models (17). Recent studies indicate that the type-specific antigen, as well as the group-specific antigen, are covalently attached to the cell wall peptidoglycan (10, 11). However, the nature of the covalent linkages and the stages involved in the biosynthesis and assembly of these cell wall polymers are not known.Although membrane preparations have been used extensively to study the biosynthesis of peptidoglycan and other cell surface polymers (19,33,34), intact protoplasts are advantageous in that membrane integrity is maintained, permitting studies on the biosynthesis of nascent cell wall components without the complication of an insoluble cell wall. For example, Kessler and Shockman (16), using protoplasts of Streptococcus faecalis 9790, demonstrated that the conversion of the high-molecular-weight (HMW) acylated lipoteichoic acid to the lowmolecular-weight (LMW) deacylated lipotei-choic acid was due to the enzymatic activity located on the outer surface of the membrane. Similarly, Bertram et al. (5), employing protoplasts of Bacillus subtilis and nonpenetrating reagents, demonstrated that the enzymatic activities for teichoic acid synthesis were located exclusively on the outer surface of the membrane. Utilizing protoplasts of S. faecalis 9790, Rosenthal et al. (24) observed the formation of soluble...

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Mutanolysin-induced spheroplasts of Streptococcus mutants are true protoplasts

Cited by 83 publications

References 16 publications

Streptococcus pneumoniae: proteomics of surface proteins for vaccine development

Streptococcus pneumoniae: proteomics of surface proteins for vaccine development

Treatment of experimental erosive arthritis in rats by injection of the muralytic enzyme mutanolysin.

Biosynthesis of cell wall peptidoglycan and polysaccharide antigens by protoplasts of type III group B Streptococcus

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