Conservation of cell wall peptidoglycan by strains of Streptococcus mutans and Streptococcus sanguis

Mychajlonka, Myron; McDowell, T D; Shockman, Gérald D.

doi:10.1128/iai.28.1.65-73.1980

Cited by 15 publications

(6 citation statements)

References 35 publications

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“…Thus, release of the protease to the environment may reflect a natural turnover and shedding of the surface components. However, two other viridans group streptococci, S. mutans and S. sanguis, have been found to not release peptidoglycan fragments into the culture medium during growth (30). This apparent discrepancy from our findings may be explained by the use of TCA precipitation methods by these investigators in their determination of enzymatic degradation of radiolabeled peptidoglycan.…”

Section: Discussioncontrasting

confidence: 99%

An Extracellular Protease ofStreptococcus gordoniiHydrolyzes Type IV Collagen and Collagen Analogues

Juarez

Stinson

1999

Infect Immun

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Streptococcus gordonii is a frequent cause of infective bacterial endocarditis, but its mechanisms of virulence are not well defined. In this study, streptococcal proteases were recovered from spent chemically defined medium (CDM) and fractionated by ammonium sulfate precipitation and by ion-exchange and gel filtration column chromatography. Three proteases were distinguished by their different solubilities in ammonium sulfate and their specificities for synthetic peptides. One of the enzymes cleaved collagen analogs Gly-Pro 4-methoxy-β-naphthylamide, 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), and p-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (pZ-peptide) and was released from the streptococci while complexed to peptidoglycan fragments. Treatment of this protease with mutanolysin reduced its 180- to 200-kDa mass to 98 kDa without loss of enzymatic activity. The purified protease cleaved bovine gelatin, human placental type IV collagen, and the Aα chain of fibrinogen but not albumin, fibronectin, laminin, or myosin. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. Maximum production of the 98-kDa protease occurred during growth of S. gordonii CH1 in CDM containing 0.075% total amino acids at pH 7.0 with minimal aeration. Higher initial concentrations of amino acids prevented the release of the protease without reducing cell-associated enzyme levels, and the addition of an amino acid mixture to an actively secreting culture stopped further enzyme release. The purified protease was stored frozen at −20°C for several months or heated at 50°C for 10 min without loss of activity. These data indicate that S. gordonii produces an extracellular gelatinase/type IV collagenase during growth in medium containing minimal concentrations of free amino acids. Thus, the extracellular enzyme is a potential virulence factor in the amino acid-stringent, thrombotic, valvular lesions of bacterial endocarditis.

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Section: Discussioncontrasting

confidence: 99%

An Extracellular Protease ofStreptococcus gordoniiHydrolyzes Type IV Collagen and Collagen Analogues

Juarez

Stinson

1999

Infect Immun

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“…2). Residual protein indicated by 14C in during growth counts reached a final value of approximately in total trichloro-4% of starting 14C counts after 1 h. In two in T-layer protein additional experiments, counts were 14 and 11% derived from 0. pared with values reported for some other grampositive bacteria (3,16); however, Hungerer and Tipper (14) reported that the peptidoglycan of B. sphaericus 9602, which also contains a T-layer, constituted only 23% of the mass of the cell wall.…”

Section: So -supporting

confidence: 60%

“…The modified Park and Hancock procedure 60 was investigated to determine the amount of c residual contaminating protein and possible hy-40 ;t drolysis of peptidoglycan by hot trichloroacetic acid (3) or pronase (16). Log phase cells labeled with [14C]valine or [3H]lysine were heated in 5% 20 trichloroacetic acid at 100°C for 6 min and subjected to the procedure for measurement of peptidoglycan, using various incubation times in 1 mg of pronase per ml.…”

Section: So -mentioning

confidence: 99%

Expansion of the tetragonally arrayed cell wall protein layer during growth of Bacillus sphaericus.

Howard¹,

Dalton²,

McCoubrey³

1982

Journal of Bacteriology

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The outermost layer of the cell wall of Bacillus sphaericus strain P-1 is a tetragonally arrayed structure (T-layer) which is assembled from a single polypeptide. No turnover of T-layer was detected during growth of cultures. In contrast, the turnover of peptidoglycan was between 20 and 25% per generation. The sites of deposition of new T-layer on the cell surface were identified by the indirect fluorescent antibody technique, which labeled old T-layer, and by the reverse technique, which labeled new T-layer. These experiments demonstrated that the major area of T-layer deposition was a band at the site of an incipient cell division. This band subsequently split and covered the new pole of each progeny cell. Little or no T-layer was inserted into existing poles. In addition, multiple bands of new T-layer, which probably accommodate cell elongation, were inserted along the lateral surface of the cell.

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“…For determining the loss of insoluble peptidoglycan, analyses were carried out in different buffers by methods recently described (8,16). Treatments with HEWL (100 ,ug/ml), protease (0.1 to 10 ,ug/ml), and HEWL plus protease were examined for their effects upon the cell wall peptidoglycan of S. mutans GS5 (5.6 x 108 cells per ml) before and after treatment with sodium thiocyanate (final concentration, 0.1 m).…”

mentioning

confidence: 99%

Bacteriolysis of Streptococcus mutans GS5 by lysozyme, proteases, and sodium thiocyanate

Wilkens¹,

Goodman²,

Mackay³

et al. 1982

Infect Immun

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Streptococcus mutans GS5 was grown in a synthetic medium containing radioactive thymidine to monitor cell lysis by assay of the release of DNA. Bacteriolysis was achieved by sequential treatment of the cells with either hen egg white lysozyme and sodium thiocyanate or a combination of hen egg white lysozyme and a proteolytic enzyme followed by addition of the thiocyanate. In the absence of sodium thiocyanate, a small percentage of the total macromolecular thymidine was released in control reaction mixtures during incubation. This amount of released DNA more than doubled in trypsin-treated cells, but the inclusion of lysozyme in reaction mixtures prevented assay of the DNA. Lysis was found to be optimal in the late log phase of growth and was dependent on the concentrations of both lysozyme and protease. Concentrations of trypsin or chymotrypsin as low as 0.01 Fxg/ml were found to be effective in enhancing the lytic process. The addition of protease to lysozyme-inorganic salt reaction mixtures altered both the pH and ionic strength profiles of cell lysis. At pHs of 5.5 or lower, both the lysozyme-NaSCN and the lysozyme-trypsin-NaSCN systems were inactive in mediating lysis. The loss of insoluble cell wall peptidoglycan by lysozyme treatment was pH independent and did not appear to be affected by the addition of protease. Either diluted whole saliva or neutrophil extracts could replace trypsin to enhance cell lysis further.Detailed studies of the bacteriolysis of a variety of microorganisms exhibiting different degrees of susceptibility to lysozyme and inorganic salts are required for the ultimate understanding of the mechanism of cell lysis. In previous communications from our laboratories, we have examined Streptococcus mutans BHT, a serotype b strain (20), which is very sensitive to the lytic action of hen egg white lysozyme (EC 3.2.1.17) (HEWL) and the sodium salts of thiocyanate, bicarbonate, chloride, and fluoride (9,21,22). In this study, we report on the bacteriolysis of S. mutans GS5, a serotype c strain, which is less susceptible to lysis than strain BHT is. During investigation of the lysis of strain GS5 and other oral microorganisms, it has become apparent that a range of lytic sensitivities does indeed exist. Moreover, in this communication, we report on the enhancement of lysozymeinorganic salt lysis by proteases. Preliminary data are also given which demonstrate that the proteases can be replaced in the assay mixtures by either diluted whole saliva or diluted neutrophil extracts, perhaps suggesting a physiological role for the lysozyme-protease-salt antibacterial lytic system in the oral cavity. MATERIALS AND METHODS Biochemicals. HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], MES [2-(N-morpholino)ethanesulfonic acid], PIPES [piperazine-N,N'bis(2-ethanesulfonic acid)], and CHES [2-(N-cyclohexylamino)ethanesulfonic acid] were obtained from Calbiochem, La Jolla, Calif. Tris, sodium thiocyanate, magnesium sulfate, and trichloroacetic acid were purchased from Fisher Scientific Co., Pittsbur...

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Conservation of cell wall peptidoglycan by strains of Streptococcus mutans and Streptococcus sanguis

Cited by 15 publications

References 35 publications

An Extracellular Protease ofStreptococcus gordoniiHydrolyzes Type IV Collagen and Collagen Analogues

An Extracellular Protease ofStreptococcus gordoniiHydrolyzes Type IV Collagen and Collagen Analogues

Expansion of the tetragonally arrayed cell wall protein layer during growth of Bacillus sphaericus.

Bacteriolysis of Streptococcus mutans GS5 by lysozyme, proteases, and sodium thiocyanate

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