Comparison of Three Methods for the Purification of the Delta Haemolysin of Staphylococcus aureus

Smith, Gary Scott; Shaw, William V.

doi:10.1099/00221287-124-2-365

Cited by 4 publications

(4 citation statements)

References 6 publications

(19 reference statements)

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“…However, the picture of their location indicated the possible aggregation of hemolytic proteins. The same observation was made by Smith and Shaw (1981) with -hemolysin from S. aureus. Pure proteins presenting -hemolysin activity were successfully obtained by solubilization of crude freeze-dried preparations in the organic solvents and subsequent extraction (Fig.…”

Section: Resultssupporting

confidence: 68%

Staphylococcus cohnii hemolysins — Isolation, purification and properties

Rózalska

Szewczyk

2008

Folia Microbiol

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A total 355 of Staphylococcus cohnii isolates from hospital environment, patients (newborns), medical staff and from non-hospital environment were tested for hemolytic activity. Ninety-one % of S. cohnii ssp. cohnii and 74.5 % S. cohnii ssp. urealyticus strains exhibited hemolysis synergistic to S. aureus ATCC 25923 strain. Crude preparations of hemolysins of both bacterial subspecies presented delta-hemolysin, but not alpha- and beta-toxin activity. Highly pure hemolysins were obtained by semipreparative SDS-PAGE or by organic solvent extraction from the freeze-dried crude preparations. Native-PAGE and 2D-PAGE showed their high heterogeneity. Molar masses of single hemolysin units estimated by the Tris-Tricine-SDS-PAGE were calculated as 3.47 kDa for S. cohnii ssp. cohnii and 3.53 kDa for S. cohnii ssp. urealyticus.

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Section: Resultssupporting

confidence: 68%

Staphylococcus cohnii hemolysins — Isolation, purification and properties

Rózalska

Szewczyk

2008

Folia Microbiol

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“…␦-Toxin deformylation. ␦-Toxin has been reported to accumulate in S. aureus culture medium in both the formylated (approximately 90%) and deformylated (approximately 10%) forms (7,23). The accumulation of deformylated ␦-toxin in the culture medium unexpectedly stops during the postexponential growth phase (Fig.…”

Section: Discussionmentioning

confidence: 99%

Synthesis and Deformylation of Staphylococcus aureus δ-Toxin Are Linked to Tricarboxylic Acid Cycle Activity

et al. 2003

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In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus ␦-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of ␦-toxin deformylation. We observed that during exponential and early postexponential growth, ␦-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated ␦-toxin accumulated after the early postexponential phase. The transition from producing both species of ␦-toxin to producing only formyl-methionine-containing ␦-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of ␦-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that ␦-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated ␦-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated ␦-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.Staphylococcus aureus uses a complex regulatory network to control the expression of virulence factors. Central to this regulatory network is the accessory gene regulatory (agr) locus (15,19). This locus consists of two divergently transcribed RNAs, encoding a density-dependent autoinducing system and an RNA effector molecule (RNAIII) (16). RNAIII acts primarily at the level of transcription but also has been shown to posttranscriptionally regulate alpha-toxin (hla) production; however, the exact mechanism of regulation is not known (17). In addition to its regulatory role, RNAIII is the mRNA for ␦-toxin (hld) (11). The mature form of ␦-toxin is a heat-stabile, 26-amino-acid peptide that inserts into and disrupts membranes (4). Additionally, it has been implicated in the prevention of biofilm formation and in stimulation of the oxidative burst of human neutrophils (22, 28). However, the precise function of ␦-toxin in S. aureus pathogenesis has yet to be elucidated.Recently, workers in our laboratory reported construction and characterization of an S. aureus aconitase (acnA) mutant (24). During the course of that study, it was observe...

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“…CONS isolates which had been stored at -70°C in skim milk were subcultured in enriched broth (2). After incubation at 37°C for 18 h in a shaking water bath, bacteria were removed from the broth by centrifugation and the supernatant was heated at 100°C for 2 min to inactivate any non-delta hemolysins and then tested for ability to hemolyse washed human type 0 erythrocytes (25) and, in some instances, rabbit and sheep erythrocytes. Hemolysin-containing broths were retested after incubation for 40 min with 1% lecithin solution or 10% calf or human serum.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was concentrated approximately 10-fold by using tangential flow filtration (Minitan; Millipore Corp.) across a filter (cutoff, 30,000 molecular weight). Hemolytic activity was measured by endpoint dilution (1 hemolytic unit [HU50] is defined as the amount of activity required to lyse 50% of the blood cell suspension) (25). Hemolytic activity concentrated in the retentate, which was then acidified to pH 5.3 and combined with chloroform to precipitate toxin (12).…”

Section: Methodsmentioning

confidence: 99%

Delta-like toxin produced by coagulase-negative staphylococci is associated with neonatal necrotizing enterocolitis

Scheifele¹,

Bjornson²,

Dyer³

et al. 1987

Infect Immun

103

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Necrotizing enterocolitis (NEC) is a serious neonatal disorder of uncertain cause, although fecal bacteria have been implicated in some outbreaks. We examined coagulase-negative staphylococci (CONS) as possible etiologic agents. In our unit, CONS colonized the bowels of most infants studied, including 46% of 70 NEC cases (mean concentration, 109 1 CFU/g of stool). Over 90% of tested isolates produced a hemolysin resembling delta toxin of Staphylococcus aureus. Toxin purified from a NEC-associated isolate of Staphylococcus epidermidis resembled reference delta toxin from S. aureus in size, biologic properties, and antigenicity. This delta-like toxin was enteropathic, causing mucosal necrosis and hemorrhage in injected loops of the bowels of infant rats. Adjacent, nonexposed bowel remained normal, as did loops injected with lecithin-neutralized toxin. Using a new enzyme-linked immunosorbent assay (ELISA), we detected delta-like toxin in the stools of 11 of 35 infants colonized with CONS positive for delta-like toxin (Tox+). Positive tests were strongly associated with NEC. Of 18 cases with Tox+ CONS, 10 were positive (56%), whereas only 1 of 17 control infants so colonized was positive (6%, P = 0.002). In NEC patients, the mean fecal toxin concentration was 1,012 ng/g. Toxicity to fibroblasts was demonstrable in filtrates of each of six ELISA-positive samples tested but was absent in all five ELISA-negative samples tested. We conclude that delta-like toxin is elaborated in the bowels of some infants with Tox+ CONS, and its association with NEC suggests that such CONS are enteropathic. In our unit, this mechanism was apparent in 23% of 44 recent cases of endemic NEC.

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Comparison of Three Methods for the Purification of the Delta Haemolysin of Staphylococcus aureus

Cited by 4 publications

References 6 publications

Staphylococcus cohnii hemolysins — Isolation, purification and properties

Staphylococcus cohnii hemolysins — Isolation, purification and properties

Synthesis and Deformylation of Staphylococcus aureus δ-Toxin Are Linked to Tricarboxylic Acid Cycle Activity

Delta-like toxin produced by coagulase-negative staphylococci is associated with neonatal necrotizing enterocolitis

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