Protoplast formation and localization of enzymes in Streptococcus mitis

Linder, Lars; Andersson, Carita; Sund, Marie‐Louise; Shockman, Gérald D.

doi:10.1128/iai.40.3.1146-1154.1983

Cited by 22 publications

(6 citation statements)

References 42 publications

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“…Few oral streptococci have been shown to exhibit efficient or extensive autolysis, and no autolysins have been isolated from these organisms (32,33,52). We also were unable to observe extensive autolysis of S. sanguis.…”

Section: Discussioncontrasting

confidence: 58%

Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides

Laible¹,

Germaine²

1985

Infect Immun

173

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The bisis of the bactericidal activity of human lysozyme against Streptococcus sanguis was studied. Experiments were designed to evaluate the role of lysozyme muramidase activity in its bactericidal potency. Inactivation of the muramidase activity of lysozyme was achieved by reduction of essential disulfides with dithiothreitol (DTT) or by incubation with the chitin oligosaccharides chitotriose and chitobiose. Muramidaseinactive lysozyme, prepared by reduction with DTT, was equal in bactericidal potency to native lysozyme. Solutions of native chicken egg white lysozyme and human lysozyme exhibited equal bactericidal potency yet differed ca. fourfold with respect to lytic (muramidase) activity. The above results suggested that the bactericidal activity of lysozyme is not dependent upon muramidase activity. Chitotriose and chitobiose were found to inhibit both lytic and bactericidal activities of lysozyme. The bactericidal activity of mnuramidase-inactive lysozyme (reduction with DTT) was also inhibited by chitotriose and chitobiose. Further investigations demonstrated that chitotriose and chitobiose were also potent inhibitors of the bactericidal activity of the cationic homopolypeptides poly-L-arginine and poly-D-lysine. These latter results suggested that the essential bactericidal property of lysozyme was its extreme cationic nature and that some bacterial endogenous activities, inhibitable by chitotriose and chitobiose, were essential for expression of the bactericidal activity of either native or muramidase-inactive lysozyme or of the cationic homopolypeptides. Experiments with Streptococcus faecalis whole cells, cell walls, and crude autolysin preparations implicated endogenous autolytic muramidases as the bacterial targets of chitotriose and chitobiose. The essentially identical responses of S. sanguis and S. faecalis to chitotriose in bactericidal assays with muramidase-inactive lysozyme and polylysine suggested that muramidase-like enzymes exist in S. sanguis and, furthermore, play an essential role in cationic protein-induced loss of viability of the oral microbe. * Corresponding author. some bacteria to oral surfaces. Lysozyme, with a pI > 10.5, is also a rather extreme example of a cationic protein. Cationic proteins are known bactericidal agents against both gram-positive and gram-negative organisms (15, 17, 38, 41, 49, 50). Two modes of bactericidal action are thus available to lysozyme: a muramidase-dependent mode and a cationicdependent mode. The oral streptococci are typically resistant to direct lysis by either human or chicken lysozyme (3, 25, 40). Lysozyme is, however, bactericidal for the major species of oral streptococci (3, 22, 25, 40; N.

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Section: Discussioncontrasting

confidence: 58%

Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides

Laible¹,

Germaine²

1985

Infect Immun

173

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“…Except when used to provide protoplasts, the bacteria were grown overnight at 37°C in Todd-Hewitt broth in 5 to 10% CO2 (GasPak; BBL Microbiology Systems, Cockeysville, Md. ), harvested by centrifugation (7,500 x g, 20 min, 4C), and washed twice in cold 0.01 M sodium phosphate buffer, pH 7.0, with 0.9% (wt/vol) sodium chloride (PBS). After washing, the bacteria were dispersed and suspended to a final A620 of 2.0 (4 x 109 cells per ml) in PBS precisely as described in previous reports (15,16,18).…”

Section: Methodsmentioning

confidence: 99%

“…While S. sanguis is generally resistant to the muralytic effects of lysozyme, mutanolysin (a muramidase isolated from Streptomyces globisporus) has been used to produce protoplasts of several species of related streptococci, including Streptococcus mutans (26,30), group B streptococci (4,37), and Streptococcus mitis (20). These protoplasts dis-played minimal leakage of cytoplasmic contents and were essentially free of cell wall products.…”

mentioning

confidence: 99%

Platelet-interactive products of Streptococcus sanguis protoplasts

et al. 1990

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To isolate a more native, platelet-interactive macromolecule (class II antigen) of Streptococcus sanguis, cultured protoplasts were used as a source. Protoplasts were optimally prepared from fresh washed cells by digestion with 80 U of mutanolysin per ml for 75 min at 37°C while osmotically stabilized in 26% (wt/vol) raffinose. Osmotically stabilized forms were surrounded by a 9-nm bilaminar membrane, as shown by transmission electron microscopy. Protoplasts were cultured in chemically defined synthetic medium and osmotically stabilized by ammonium chloride. Spent culture media were harvested daily for 7 days. Each day, soluble proteins were isolated from media, preincubated with platelet-rich plasma, and tested for inhibition of platelet aggregation induced by S. sanguis cells. Products released from S. sanguis protoplasts and reactive with an anti-class II antigen immunoaffinity matrix were able to inhibit S. sanguis-induced platelet aggregation. As resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, anti-class II-reactive protoplast products included silver-stained bands of 67, 79, 115, 216, and 248 kDa. The 115-kDa protein fraction was isolated by gel filtration and ion-exchange chromatography. This form of the class II antigen contained N-formylmethionine at its amino terminus. Rhamnose constituted 18.2% of the total residual dry weight and nearly half of its carbohydrate content. Diester phosphorus constituted 1% of this fraction. After trypsinization of the protoplast products from either preparation, a 65-kDa protein fragment was recovered. This protoplast protein fragment and the S. sanguis cell-derived 65-kDa class II antigen, previously implicated in the induction of platelet aggregation, were shown to be functionally and immunologically identical.

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“…Importantly, the total amount of glucose consumed by both strains was equivalent, yet the difference in biomass between the two strains was significant, suggesting that growth alone did not account for all the differences in glucose consumption. The glycolytic end-product of glucose is pyruvate, which, in S. mitis-oralis, is predominantly catabolized by the membrane-associated lactate dehydrogenase [12]. The lactic acid produced by lactate dehydrogenase accumulated in the cultivation media, which caused the pH of the media to decrease over time (Fig.…”

Section: Daptomycin Non-susceptibility Alters Growth Of S Mitismentioning

confidence: 99%

Metabolic changes associated with adaptive resistance to daptomycin in Streptococcus mitis-oralis

et al. 2020

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Background Viridans group streptococci of the Streptococcus mitis-oralis subgroup are important endovascular pathogens. They can rapidly develop high-level and durable non-susceptibility to daptomycin both in vitro and in vivo upon exposure to daptomycin. Two consistent genetic adaptations associated with this phenotype (i.e., mutations in cdsA and pgsA) lead to the depletion of the phospholipids, phosphatidylglycerol and cardiolipin, from the bacterial membrane. Such alterations in phospholipid biosynthesis will modify carbon flow and change the bacterial metabolic status. To determine the metabolic differences between daptomycin-susceptible and non-susceptible bacteria, the physiology and metabolomes of S. mitis-oralis strains 351 (daptomycin-susceptible) and 351-D10 (daptomycin non-susceptible) were analyzed. S. mitis-oralis strain 351-D10 was made daptomycin non-susceptible through serial passage in the presence of daptomycin. Results Daptomycin non-susceptible S. mitis-oralis had significant alterations in glucose catabolism and a re-balancing of the redox status through amino acid biosynthesis relative to daptomycin susceptible S. mitis-oralis. These changes were accompanied by a reduced capacity to generate biomass, creating a fitness cost in exchange for daptomycin non-susceptibility. Conclusions S. mitis-oralis metabolism is altered in daptomycin non-susceptible bacteria relative to the daptomycin susceptible parent strain. As demonstrated in Staphylococcus aureus, inhibiting the metabolic changes that facilitate the transition from a daptomycin susceptible state to a non-susceptible one, inhibits daptomycin non-susceptibility. By preventing these metabolic adaptations in S. mitis-oralis, it should be possible to deter the formation of daptomycin non-susceptibility.

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Protoplast formation and localization of enzymes in Streptococcus mitis

Cited by 22 publications

References 42 publications

Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides

Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides

Platelet-interactive products of Streptococcus sanguis protoplasts

Metabolic changes associated with adaptive resistance to daptomycin in Streptococcus mitis-oralis

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