Localization of Protein A in the Bacteria

Sjöquist, John; Movitz, Jan; Johansson, Inga‐Britt; Hjelm, Hans

doi:10.1111/j.1432-1033.1972.tb02086.x

Cited by 144 publications

(76 citation statements)

References 22 publications

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“…Muramidase cuts the ␤1-4 glycosidic bond between MurNacGlcNac and disrupts the glycan strands of mature peptidoglycan without affecting its peptide backbone (26). Muramidase solubilizes surface protein as a large spectrum of fragments with linked peptidoglycan, each of which migrates more slowly on SDS-PAGE than the lysostaphin-released counterpart (23,27). This assay was used to assess whether the sortase mutants linked surface protein to the cell wall of S. aureus (Fig.…”

Section: Fig 4 Trpmentioning

confidence: 99%

Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus

Ton‐That¹,

Mazmanian²,

Alksne³

et al. 2002

Journal of Biological Chemistry

151

105

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Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with a LPXTG motif. Sortase cleaves polypeptides between the threonine and the glycine of the LPXTG motif. The carboxyl group of threonine is subsequently amide-linked to the amino group of peptidoglycan cross-bridges. The three-dimensional structure of sortase revealed the close proximity of the catalytic site residue cysteine 184 with histidine 120; however, no structural evidence for a thiolate-imidazolium ion pair could be detected. We report that alanine substitution of either cysteine 184 or histidine 120 abolishes in vivo and in vitro sorting reactions. Further, alanine substitution of tryptophan 194, a residue that is in close proximity of histidine 120, reduces the transpeptidase activity of sortase. These results suggest a model whereby sortase forms a thiolate-imidazolium ion pair for the catalysis of its transpeptidation reaction and that the position of tryptophan 194 assists in the formation of this ion pair.

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Section: Fig 4 Trpmentioning

confidence: 99%

Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus

Ton‐That¹,

Mazmanian²,

Alksne³

et al. 2002

Journal of Biological Chemistry

151

105

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“…The assay is sensitive to interference from aminoglycoside antibiotics, and the extent of interference varies with different PRM reagents (1, 2, 4 -7 ). Thus, the Dade Behring PRM and the Sigma PRM assays are sensitive to aminoglycoside interference, whereas the Cobas Fara and Roche Integra 700 PRM assays are not (4,5 ). Furthermore, the PRM reagents used by Fujita et al (1 ) and Orsonneau et al (3 ) are more sensitive to interference than the reagent used by Watanabe et al (2 ).…”

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confidence: 88%

Detection of Staphylococcus aureus by a Sensitive Immuno-PCR Assay

Huang¹,

Chang

2004

Clinical Chemistry

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Staphylococcus aureus causes a wide variety of diseases in humans, the clinical courses of which range from boils and furuncles to more serious diseases such as septicemia and pneumonia (1 ). Although a significant cause of community-acquired infection, most life-threatening cases of S. aureus disease are hospital-acquired and are associated, in many cases, with indwelling vascular devices or catheters (2 ). The standard method of diagnosing S. aureus is to culture an isolate from a blood agar plate and then use a latex test to identify S. aureus.A specific product of S. aureus is protein A (3 ). Protein A is a cell-wall constituent of S. aureus and is mainly covalently linked to the peptidoglycan structure (4 ); however, ϳ8 -30% of the protein is secreted into the medium during the exponential growth phase (5 ). This property has been used to develop a latex agglutination test and an ELISA to identify and detect S. aureus. We describe here a sensitive immuno-PCR assay to detect S. aureus protein A. After optimization of the reaction conditions and the use of flexible plates with 96 V-bottomed wells, which were compatible with both the ELISA washer and the thermal cycler for PCR, we automated both detection methods.Anti-protein A antisera were created by immunizing rabbits with protein A purified from preparative polyacrylamide gels. The IgG fraction of the antisera was purified by DEAE ion-exchange chromatography (6 ). The protocol for biotinylating the antibody is described elsewhere (7 ). The avidin-biotinylated DNA complex was prepared fresh before use by mixing avidin and the biotinylated DNA at a molar ratio of 1:4. Avidin (2 g/L) was first diluted with bovine serum albumin (BSA) diluent to 2 g/L, and then 10 L was mixed with 2 L of the biotinylated DNA (120 mg/L). The mixture was incubated at room temperature for 10 min and then further diluted with BSA diluent before use.The protocol for immuno-PCR was a modified version of the modified protocol of Chang and Huang (8 ) (Fig. 1A). Briefly, microtiter plates were coated with 50 L/ well of anti-protein A IgG (10 mg/L in phosphate-buffered saline) for 1 h at 37°C, washed five times, and blocked with 75 L of the BSA diluent for 1 h at 37°C. The plates were washed five times, and 50 L of 10-fold serial diluted antigen (protein A; 10 Ϫ9 -10 Ϫ18 g/mL) was added to each well. After incubation for 1 h at 37°C, the plates were washed five times, and 50 L of the biotinylated anti-protein A (1 g/L) was added to each well. The plates were incubated at 37°C for 1 h and then washed 12 times; 50 L of the avidin-biotinylated DNA complex (1:10 000 dilution) was then added to each well. The plates were again incubated at 37°C for 1 h and washed 12 times before the PCR step. The negative control was performed in the same way, except that the BSA diluent was substituted for the antigen solution. The buffer used throughout for plate washing was phosphate-buffered saline containing 0.5 mL/L Tween 20, whereas the BSA diluent was used for the dilution of antigen, biotinylated an...

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“…It is now known that the Fc portion of the IgG molecule is the binding site and pure protein A is able to bind two molecules of IgG per molecule (Forsgren andSjoquist, 1966 : Kronvall andFrommel, 1970). however, the reaction is specific for subclass 1, 2 and 4 of human IgG (Hjelm et al, 1975: Sjoquist et al, 1972 et al, 1950 ;Waller et al, 1953). The traps-effect is similar in nature except that the competing Rh genes are located on the other autosome, traps to the gene being investigated.…”

Section: Introductionmentioning

confidence: 96%

“…Protein A is a protein isolated from the cell wall of Staphylococcus aureus and recently has been shown to be covalently linked to the peptidoglycan part of the wall (Sjoquist et al, 1972).…”

Section: Introductionmentioning

confidence: 99%