Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay (original) (raw)
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Serum and tissue protein binding and cell surface properties of Staphylococcus lugdunensis
Journal of Medical Microbiology, 1993
Eleven strains of Staphylococcus lugdunensis from different clinical sources were investigated for their ability to bind 12'I-labelled collagen (Cn) type I and IV, fibronectin (Fn), vitronectin (Vn), laminin (Lm), fibrinogen (Fg), thrombospondin, plasminogen (glu-and lysform) and human IgG. All the strains bound these proteins, although a higher degree of binding was obtained for Cn types I and IV and IgG with mean values of 36 YO, 32 YO and 26 YO binding. respectively. In tests with proteins immobilised on latex beads in a particle agglutination assay, eight of the 11 strains bound Cn type I and seven bound Fg, whereas no strain bound immobilised IgG. Binding to immobilised Cn-I, Fg, Lm and Vn was abolished when the bacterial cells were treated with proteases or heat, indicating cell-surface receptors with protein characteristics. Cell-surface extracts of S . lugdunensis 2342 were able to totally inhibit binding of the homologous strain and S . aureus Cowan 1 to latex-immobilised proteins Cn-I, Lm, Vn, Fn and Fg. The binding of '"I-labelled Cn IV by S. lugdunensis 2342, was heat sensitive, whereas the binding to S. auieus Cowan 1 was heat resistant. The strains gave negative results in tests for the presence of protein A with a S. aureus protein A gene probe and with sensitised red blood cells. No production of heat-stable nuclease (TNase) could be detected by monoclonal antibodies against TNase or by the polymerase chain reaction with an oligonucleotide sequence from S . aureus TNase as primer. When the cell surface characters of the S. lugdunensis strains were studied, five were found to be hydrophobic and negatively charged. four hydrophilic and positively charged and two hydrophobic with positive net charge.
Surface Proteins of Staphylococcus aureus
Clinical Infectious Diseases, 1988
Staphylococcus aureus is a commensal bacterium that causes infections such as sepsis, endocarditis, and pneumonia. S. aureus can express a variety of virulence factors, including surface proteins. Surface proteins are characterized by presence of a Sec-dependent signal sequence at the amino terminal, and the sorting signal domain. Surface proteins are covalently attached to peptidoglycan and they are commonly known as cell wallanchored (CWA) proteins. CWA proteins have many functions and participate in the pathogenesis of S. aureus. Furthermore, these proteins have been proposed as therapeutic targets for the generation of vaccines. In this chapter, different topics related to CWA proteins of S. aureus are addressed. The molecular structure of CWA proteins and their role as virulence factors of S. aureus are described. Furthermore, the involvement of CWA proteins in the processes of adhesion, invasion of host cells and tissues, evasion of the immune response, and the formation of biofilm is discussed. In addition, the role of CWA proteins in skin infection and the proposal to use them as potential vaccine antigens are described. The information contained in this chapter will help the readers to understand the biology of CWA proteins and to recognize the importance of surface molecules of S. aureus.
Surface proteins of Staphylococcus aureus play an important role in experimental skin infection
APMIS, 2014
Staphylococcus aureus is the most common cause of skin infections that range from mild diseases up to life-threatening conditions. Mechanisms of S. aureus virulence in those infections remain poorly studied. To investigate the impact of S. aureus surface proteins on skin infection, we used mouse models of skin abscess formation and skin necrosis, induced by a subcutaneous injection of bacteria. In the skin abscess model, a sortase-deficient S. aureus strain lacking all of its cell-wall anchored proteins was less virulent than its wild-type strain. Also, strains specifically lacking protein A, fibronecting binding proteins, clumping factor A or surface protein SasF were impaired in their virulence. When a model of dermonecrosis was studied, the S. aureus surface proteins could not be shown to be involved. In summary, surface proteins play an important role in virulence of S. aureus skin abscess infections, but not in formation of skin necrosis.
Analytical Letters, 2005
Different ELISA tests to detect and quantify levels of S. aureus in broth cultures were developed and compared. In all cases the assays were a modification of a "sandwich" format based on the use of common IgG as well as specific antibodies to bind protein A, an antigen localized in the cellular wall of S. aureus and partially extracted by boiling. Initially, human IgG was immobilized on the surface of microtitre plate wells in order to bind, by means of the Fc region, protein A that was present either in standard solutions or broth cultures of S. aureus treated by a boiling step.
Organic & Biomolecular Chemistry, 2013
infection, and were part of a cohort of bacteria taken from UK hospitals. E.coli samples were a combination of clinical isolates and wild type strains (E.coli 1-10), and coagulase negative Staphylococci include strains of S.epidermidis, S. warneri, S.hominus and M. luteus, and were obtained from clinical isolates from Sutton hospital. All bacteria were cultured initially on Brain Heart infusion agar, after which they were subcultured twice onto nutrient agar overnight at 37 °C, under aerobic conditions. Each bacterial isolate possesses individual characteristics, and thus displays a variance in the amount of coagulase produced, and therefore there is a degree of variance in results, lending to a greater amount of error in the results depicted. Preparation of the LGX solution A 100 μM and 50 μM solution of compound 3 was prepared by dissolving it in 2.5 % methanol followed by dilution in 1 x PBS. Solutions of Tris Base (0.05 M) and NaCl (0.1 M) in deionised H 2 O were added to the solution until an optimum pH of 8.5 was achieved. The appropriate amount of human prothrombin in 1 x PBS was then added to produce final prothrombin concentrations of 83.6 nM and 41.8 nM, respectively. The resulting solution was termed "LGX". The concentrations of LGX, subsequently discussed, refer to the concentration of the active constituent compound 3. Testing procedure the efficacy of LGX as a selective and sensitive means of detecting S. aureus In order to detect the presence of staphylocoagulase and thus the efficacy of LGX, varying cell concentrations (10 6 , 10 5 , 10 3 , 10 2 CFU/mL (50 μM LGX) and 10 4 , 10 3 , 10 2 , 10 0 CFU/mL (100 μM LGX) were added to a microtitre plate (Nunclon 96 well plates) in a 1:1 ratio with either 50 μM or 100 μM of prepared LGX solution. This provided a final LGX concentration of 25 μM or 50 μM, respectively. The relative fluorescence was then recorded every fifteen minutes over a six hour time period (λ ex. = 488 nm and λ em. = 525 nm). Positive controls used in each experiment were a control strain of MRSA (NCTC 12493), and the negative control was a clinical isolate of E. coli. A 1:1 ratio of both the 100 μM and 50 μΜ of LGX solution with 1 x PBS was used, and termed "LGX alone". Fluorescence intensity was determined at 15 minute intervals over a 6 hour time period and n=3 samples were assayed in each case. We assayed 15 Strains of MRSA at varying cell concentration (10 6 , 10 5 , 10 3 , 10 2 CFU/mL (50 μM LGX) and 10 4 , 10 3 , 10 2 , 10 0 CFU/mL (100 μM) LGX)) with equal volume of both 50 μM and 100 μM LGX, respectively, to give a final LGX concentration of 25 μM or 50 μM, respectively, in addition to a strain of E-coli, which served as a negative control as described above.
2021
Staphylococcus aureus is one of the important pathogen often termed as superbug. After S. aureus interacting with host innate immune system, it induces the release of proinflammatory cytokines like TNF-α and IL-1β from Immune cells. In the current study, various strains of S. aureus and their cell wall lysates indicated cross reactivity with murine cytokines ELISA antibodies. In cell wall lysates of S. aureus, various proteins are involved in this cross reactivity phenomenon. This cross reactivity was exhibited in the form of unusual exaggerated signals while performing ELISA for quantification of murine cytokines. The proteins of cell wall lysates among various strains of S. aureus were determined using SDS-PAGE. To all of our data, it is revealed that unknown epitopes of S. aureus are involved in producing exaggerated signals of cytokines during their quantification by ELISA. This was generated not only during interaction of various strains of S. aureus with murine bone marrow der...
Journal of Medical Microbiology, 2014
The aim of this study was to compare the performance of serological versus molecular typing methods to detect capsular polysaccharide (CP) and surface-associated polysaccharide antigen 336 phenotypes of Staphylococcus aureus isolates. Molecular typing of CP types 1, 5 and 8 was carried out using PCR, whereas serological typing of CP1, 2, 5, 8 and antigen 336 was carried out by slide agglutination using specific antisera. By genotyping, 14/31 strains were CP8 positive, 12/31 strains were CP5 and the remaining 6/31 isolates were non-typable (NT). One isolate was positive for both CP5 and CP8 by PCR, but was confirmed as CP8 type serologically. Detection of CP2 and type 336 by PCR was not possible because specific primers were either not available or non-specific. Using serotyping, 14/31 strains were CP8 positive, 11/31 CP5 positive and 2/31 positive for antigen 336. The remaining four S. aureus isolates were serologically NT. However, three of four NT and two 336-positive S. aureus is...
Purification and Characterisation of A Plasmin-Sensitive Surface Protein of Staphylococcus aureus
European Journal of Biochemistry, 1996
Certain methicillin-resistant Staphylococcus aureus strains contain a 230-kDa cell-wall protein which is not present on the surface of other staphylococci. The presence of this 230-kDa protein is associated with a negative test result in commercial assays designed to detect fibrinogen-binding proteins andlor protein A on the staphylococcal surface. We have purified and partially characterised the 230-kDa protein from a lysostaphin digest of a non-agglutinating methicillin-resistant S. aureus strain. Partial amino acid sequence data obtained from the purified protein did not reveal any significant similarities to known proteins which indicates that the protein is novel. The 230-kDa protein was very sensitive to proteolysis ; soluble plasmin, or plasmin formed on the bacterial-cell surface, rapidly degraded the 230-kDa protein to a 175-kDa form. The finding that the 230-kDa protein bound to lectins allowed its purification by affinity chromatography on immobilised wheat germ agglutinin. Furthermore, the degradation of the 230-kDa protein was associated with an increased adherence of non-agglutinating methicillin-resistant S. aureus cells to solid-phase fibronectin, fibrinogen or IgG.
Journal of Bacteriology, 2001
The ability to attach to host ligands is a well-established pathogenic factor in invasive Staphylococcus aureus disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (srtA) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene emp (1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR, emp was demonstrated in all 240 S. aureus isolates of a defined clinical strain collection as well as in 6 S. aureus laboratory strains, whereas it is lacking in all 10 S. epidermidis strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50, a significantly decreased adhesion of mEmp50 to immobilized fibronectin and fibrinogen, and restoration of these characteristics upon complementation of mEmp50. Emp expression was also demonstrable upon heterologous complementation of S. carnosus. rEmp expressed in Escherichia coli interacted with fibronectin, fibrinogen, and vitronectin in surface plasmon resonance experiments at a K d of 21 nM, 91 nM, and 122 pM, respectively. In conclusion, the biologic characterization of Emp suggests that it is a member of the group of secreted S. aureus molecules that interact with an extended spectrum of host ligands and thereby contribute to S. aureus pathogenicity.