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THE CELL-BOUND ALPHA-AMYLASES OF STREPTOCOCCUS BOVIS.

ABSTRACT: 1. The cell-bound alpha-amylase of Streptococcus bovis has been isolated from other carbohydrases in the cell extract by chromatography on DEAE-cellulose. The enzyme has been compared with the extracellular alpha-amylase produced by this organism. 2. The two amylases had similar action patterns on amylose, the main product being maltotriose with smaller amounts of maltose and a little glucose. 3. The cell-bound amylase hydrolysed maltopentaose and maltohexaose at a similar rate to the hydrolysis of amylose. Maltotetraose was hydrolysed six times more slowly, and maltotriose 280 times more slowly, than amylose. 4. Studies with end-labelled maltodextrins revealed that the cell-bound alpha-amylase preferentially hydrolysed the third linkage from the non-reducing end, liberating maltotriose. The linkage at the reducing end of maltotriose was more easily hydrolysed than the other. 5. Egg-white lysozyme and the extracellular enzymes of Streptomyces albus lysed the cell walls of Streptococcus bovis, releasing amylase into the medium. In the presence of 0.6 m-sucrose 10% of the maximal amylase activity was released by lysozyme. Suspension of the spheroplasts in dilute buffer caused the rupture of the cytoplasmic membrane and the liberation of amylase. 6. A sensitive method for determining the ability of amylases to degrade starch granules is described.

SUBMITTER: WALKER GJ

PROVIDER: S-EPMC1206509 | biostudies-other | 1965 Feb

REPOSITORIES: biostudies-other

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Project description:1. A transglucosylase has been separated from the alpha-amylase of Streptococcus bovis by chromatography of the cell extract on DEAE-cellulose. 2. The transglucosylase can synthesize higher maltodextrins from maltotriose, but maltose, isomaltose and panose do not function as donors. 3. Iodine-staining polysaccharide may be synthesized from maltotriose provided that glucose is removed. Synthesis from maltohexaose results in dextrins of sufficient chain length to stain with iodine, but again maltodextrins of longer chain length are formed when glucose is removed from the system. 4. The transglucosylase degrades amylose in the presence of a suitable acceptor, transferring one or more glucosyl residues from the non-reducing end of the donor to the non-reducing end of the acceptor. With [(14)C]glucose as acceptor the maltodextrins produced were labelled in the reducing glucose unit only. 5. The acceptor activities of 25 sugars have been compared with that of glucose. Maltose has 50%, methyl alpha-glucoside has 15%, isomaltose and panose each has 8% and sucrose has 6% of the accepting efficiency of glucose. Mannose and sorbose also had detectable activity. With the exception of maltose all these sugars produced a different series of dextrins from that obtained with glucose. 6. It was concluded that S. bovis transglucosylase transfers alpha-(1-->4)-glucosidic linkages in the same manner as D-enzyme, but some differences in specificity distinguish the two enzymes. Unlike D-enzyme, S. bovis transglucosylase can transfer glucosyl units, producing appreciable amounts of maltose both during synthesis from maltotriose and during transfer from amylose to glucose. 7. No evidence was found that the transglucosylase was extracellular. The enzyme is cell-bound, and is released by treatment of the cells with lysozyme and by suspension of the spheroplasts in dilute buffer. 8. The transglucosylase may be responsible for the storage of intracellular iodophilic polysaccharide that occurs when the cells are grown in the presence of suitable carbohydrate sources.

Project description:Streptococcus bovis/Streptococcus equinus complex (SBSEC) is a common cause of infective endocarditis (IE). For IE-pathogens, the capacity to activate and aggregate platelets is believed to be an important virulence mechanism. While the interactions between bacteria and platelets have been described in detail for many Gram-positive pathogens, little research has been carried out with SBSEC in this respect. Twenty-six isolates of the four most common species and subspecies of SBSEC identified in bacteremia were collected, and interactions with platelets were investigated in platelet rich plasma (PRP) from three donors. Aggregation was studied using light-transmission aggregometry and platelet activation using flow cytometry detecting surface upregulation of CD62P. Platelets and serum were treated with different inhibitors to determine mechanisms involved in platelet aggregation and activation. Twenty-two of 26 isolates induced aggregation in at least one donor, and four isolates induced aggregation in all three donors. In PRP from donor 1, isolate SL1 induced a rapid aggregation with a median time of 70 s to reach 50% aggregation. Blockade of the platelet Fc-receptor or enzymatic cleavage of IgG abolished platelet activation and aggregation. The capacity for bacteria-induced platelet aggregation was also shown to be transferable between donors through serum. SBSEC mediates platelet aggregation in an IgG and IgG-Fc-receptor dependent manner. Bacterial activation of platelets through this pathway is common for many bacteria causing IE and could be a potential therapeutic target for the prevention and treatment of this infection. IMPORTANCE The capacity of bacteria to activate and aggregate platelets is believed to contribute to the pathogenesis of IE. The Streptococcus bovis/Streptococcus equinus complex (SBSEC) contains known IE-pathogens, but there is limited research on the different subspecies ability to interact with platelets and what signaling pathways are involved. This study reports that 22 of 26 tested isolates of different subspecies within SBSEC can induce aggregation, and that aggregation is host dependent. The Fc-IgG-receptor pathway was shown essential for platelet activation and aggregation. To the best of our knowledge, this is the first study that reports on platelet interactions of SBSEC-isolates other than Streptococcus gallolyticus subspecies gallolyticus as well as the first study to report of mechanisms of platelet interaction of SBSEC-isolates. It adds SBSEC to a group of bacteria that activate and aggregate platelets via the platelet Fc-receptor. This could be a potential therapeutic target for prevention of IE.

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THE CELL-BOUND ALPHA-AMYLASES OF STREPTOCOCCUS BOVIS.

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