FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2060023876> ?p ?o ?g. }

• W2060023876 endingPage "6366" @default.
• W2060023876 startingPage "6359" @default.
• W2060023876 abstract "Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein AI and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOFΔFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOFΔFn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOFΔFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFΔFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFΔFn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone. Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein AI and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOFΔFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOFΔFn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOFΔFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFΔFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFΔFn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone. Streptococcus pyogenes (group A streptococcus, GAS) 4The abbreviations used are:GASgroup A streptococcusSOFserum opacity factorOFopacity factorapoAIapolipoprotein AILPSlipopolysaccharideSOFΔFnN-terminal SOF domainFnBDC-terminal fibronectin binding domainWTwild typeHDLhigh density lipoproteinPBSphosphate-buffered salineBSAbovine serum albuminFCSfetal calf serum. is an important human pathogen responsible for a wide variety of skin and mucosal infections ranging from pharyngitis and impetigo to more severe invasive infections, such as necrotizing fasciitis and streptococcal toxic shock-like syndrome (1Tart A.H. Walker M.J. Musser J.M. Trends Microbiol. 2007; 15: 318-325Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 2Walker M.J. McArthur J.D. McKay F. Ranson M. Trends Microbiol. 2005; 13: 308-313Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Cunningham M.W. Clin. Microbiol. Rev. 2000; 13: 470-511Crossref PubMed Scopus (1769) Google Scholar). The serum opacity factor (SOF) is a large protein of ∼110 kDa, which is expressed at the cell surface by approximately half of all clinical isolates (4Goodfellow A.M. Hibble M. Talay S.R. Kreikemeyer B. Currie B.J. Sriprakash K.S. Chhatwal G.S. J. Clin. Microbiol. 2000; 38: 389-392PubMed Google Scholar, 5Beall B. Gherardi G. Lovgren M. Facklam R.R. Forwick B.A. Tyrrell G.J. Microbiology. 2000; 146: 1195-1209Crossref PubMed Scopus (94) Google Scholar). Similar to a number of other surface proteins expressed by S. pyogenes, SOF binds fibronectin via a C-terminal-repeated domain (FnBD) (6Jeng A. Sakota V. Li Z. Datta V. Beall B. Nizet V. J. Bacteriol. 2003; 185: 1208-1217Crossref PubMed Scopus (130) Google Scholar, 7Kreikemeyer B. Martin D.R. Chhatwal G.S. FEMS Microbiol. Lett. 1999; 178: 305-311Crossref PubMed Google Scholar, 8Rakonjac J.V. Robbins J.C. Fischetti V.A. Infect. Immun. 1995; 63: 622-631Crossref PubMed Google Scholar), a function that has been implicated in the adhesion of GAS to epithelial cells (9Oehmcke S. Podbielski A. Kreikemeyer B. Infect. Immun. 2004; 72: 4302-4308Crossref PubMed Scopus (27) Google Scholar). In contrast to the conserved C terminus, the N terminus of SOF (SOFΔFn) is highly variable, exhibiting ∼55% identity between different serotypes of S. pyogenes. The N-terminal domain of SOF was originally thought to cleave apolipoprotein A1 (apoAI) in human serum leading to the precipitation of high density lipoproteins (HDLs) (10Saravani G.A. Martin D.R. FEMS Microbiol. Lett. 1990; 56: 35-39Crossref PubMed Google Scholar, 11Katerov V. Lindgren P.E. Totolian A.A. Schalen C. Curr. Microbiol. 2000; 40: 149-156Crossref PubMed Scopus (19) Google Scholar). However, it has recently been demonstrated that the OF activity of SOF is not enzymatic; rather, the direct binding of apoAI by SOF triggers the release of the HDL lipid cargo of apoAI, initiating the opacity reaction (12Courtney H.S. Zhang Y.M. Frank M.W. Rock C.O. J. Biol. Chem. 2006; 281: 5515-5521Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). This OF domain of SOF promotes GAS invasion of epithelial cells (13Timmer A.M. Kristian S.A. Datta V. Jeng A. Gillen C.M. Walker M.J. Beall B. Nizet V. Mol. Microbiol. 2006; 62: 15-25Crossref PubMed Scopus (43) Google Scholar), but it is not known whether the OF activity itself or a discrete domain within the N terminus of SOF contributes to this phenotype. SOF is a virulence determinant of GAS, with insertional inactivation or allelic replacement of sof reducing mortality in an intraperitoneal and subcutaneous murine infection model (13Timmer A.M. Kristian S.A. Datta V. Jeng A. Gillen C.M. Walker M.J. Beall B. Nizet V. Mol. Microbiol. 2006; 62: 15-25Crossref PubMed Scopus (43) Google Scholar, 14Courtney H.S. Hasty D.L. Li Y. Chiang H.C. Thacker J.L. Dale J.B. Mol. Microbiol. 1999; 32: 89-98Crossref PubMed Scopus (97) Google Scholar). SOF is also a vaccine candidate, parenteral immunization of mice with SOF protects against lethal intraperitoneal challenge (15Courtney H.S. Hasty D.L. Dale J.B. Infect. Immun. 2003; 71: 5097-5103Crossref PubMed Scopus (54) Google Scholar). group A streptococcus serum opacity factor opacity factor apolipoprotein AI lipopolysaccharide N-terminal SOF domain C-terminal fibronectin binding domain wild type high density lipoprotein phosphate-buffered saline bovine serum albumin fetal calf serum. It is not known what physiological effect that the precipitation of HDL would have upon the human host or how the interaction of SOF with apoAI contributes to the pathogenesis of GAS. ApoAI exerts a potent anti-inflammatory effect by preventing contact between infected T-cells and monocytes, thereby inhibiting cytokine production (namely tumor necrosis factor-α (TNF-α) and interleukin-1) in monocytes (16Hyka N. Dayer J.M. Modoux C. Kohno T. Edwards C.K. Roux-Lombard 3rd, P. Burger D. Blood. 2001; 97: 2381-2389Crossref PubMed Scopus (340) Google Scholar). In vitro, both HDL and apoAI exert anti-inflammatory effects against the potent bacterial endotoxins, Gram-negative LPS and Gram-positive lipoteichoic acid, binding strongly to both endotoxins and inhibiting the production of TNF-α (17Levels J.H. Abraham P.R. van Barreveld E.P. Meijers J.C. Deventer van S.J. Infect. Immun. 2003; 71: 3280-3284Crossref PubMed Scopus (84) Google Scholar, 18Grunfeld C. Marshall M. Shigenaga J.K. Moser A.H. Tobias P. Feingold K.R. J. Lipid Res. 1999; 40: 245-252Abstract Full Text Full Text PDF PubMed Google Scholar, 19Emancipator K. Csako G. Elin R.J. Infect. Immun. 1992; 60: 596-601Crossref PubMed Google Scholar). In vivo, transgenic animal studies of the toxicity of LPS have shown that expression of human apoAI transgenes protected mice from a lethal dose of LPS (20Levine D.M. Parker T.S. Donnelly T.M. Walsh A. Rubin A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 12040-12044Crossref PubMed Scopus (429) Google Scholar). ApoAI also possesses specific anti-bacterial and anti-viral properties (21Tada N. Sakamoto T. Kagami A. Mochizuki K. Kurosaka K. Mol. Cell Biochem. 1993; 119: 171-178Crossref PubMed Scopus (37) Google Scholar, 22Srinivas R.V. Venkatachalapathi Y.V. Rui Z. Owens R.J. Gupta K.B. Srinivas S.K. Anantharamaiah G.M. Segrest J.P. Compans R.W. J. Cell Biochem. 1991; 45: 224-237Crossref PubMed Scopus (55) Google Scholar, 23Owens B.J. Anantharamaiah G.M. Kahlon J.B. Srinivas R.V. Compans R.W. Segrest J.P. J. Clin. Investig. 1990; 86: 1142-1150Crossref PubMed Scopus (91) Google Scholar). The administration of active SOF protein as a vaccine when the downstream effects of disrupting HDL and its activity in vivo are unknown would not be recommended, as the most effective protection against GAS infection is delivered when the SOF protein is administered parenterally (15Courtney H.S. Hasty D.L. Dale J.B. Infect. Immun. 2003; 71: 5097-5103Crossref PubMed Scopus (54) Google Scholar), and the localized depletion of HDL may reduce the body's defense against other pathogens or may result in inflammation at the site of immunization. Thus, before SOF could be used as a potential vaccine antigen (alone or as part of a multivalent vaccine formulation) it would be prudent to eliminate the OF activity of the protein. To this end, this study generated mutant forms of recombinant SOF protein with attenuated or eliminated OF activity, for use as vaccine formulations. These mutant SOF proteins have also been used to further delineate OF activity and cell binding activity within the N terminus of SOF. Site-directed Mutagenesis—A pQE30-based vector encoding a fusion protein of residues 33-872 of SOF from a M75 GAS strain (lacking signal sequence and fibronectin binding repeat region, pSOF75ΔFn) (24Kreikemeyer B. Talay S.R. Chhatwal G.S. Mol. Microbiol. 1995; 17: 137-145Crossref PubMed Scopus (114) Google Scholar), was used as the template for all mutagenesis reactions. Site-directed mutagenesis was performed as previously described (25Sanderson-Smith M.L. Walker M.J. Ranson M. J. Biol. Chem. 2006; 281: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Primers for site-directed mutagenesis are given in supplemental Table S1. Two mutagenesis strategies were employed, amino acid residues were substituted with alanine (single residues up to 5 residues) and small deletions were made within rSOF75ΔFn. Deletions within rSOF75ΔFn were generated by using site-directed mutagenesis to introduce two AvrII restriction sites flanking the region to be deleted, followed by digestion with AvrII. The digested fragments were then separated by agarose gel electrophoresis, and the DNA fragment containing the portion of pSOF75ΔFn of interest was extracted from the gel and re-ligated. Expression and Purification of Wild Type and Mutant Forms of rSOF75 and rSOF75ΔFn Protein—Large scale expression and purification of rSOF75 proteins was conducted essentially according to the manufacturer's instructions (Qiagen), and has been previously described (13Timmer A.M. Kristian S.A. Datta V. Jeng A. Gillen C.M. Walker M.J. Beall B. Nizet V. Mol. Microbiol. 2006; 62: 15-25Crossref PubMed Scopus (43) Google Scholar, 26Gillen C.M. Towers R.J. McMillan D.J. Delvecchio A. Sriprakash K.S. Currie B. Kreikemeyer B. Chhatwal G.S. Walker M.J. Microbiology. 2002; 148: 169-178Crossref PubMed Scopus (19) Google Scholar). To ensure correct refolding of proteins was achieved, wild type purified proteins were subsequently tested for opacity factor activity using an agarose overlay method (7Kreikemeyer B. Martin D.R. Chhatwal G.S. FEMS Microbiol. Lett. 1999; 178: 305-311Crossref PubMed Google Scholar). Structural Characterization of Wild Type and Mutant Forms of rSOF75ΔFn Protein—To determine the structural integrity of mutant rSOF75ΔFn proteins in comparison to the wild type, a comparison of the secondary structure of wild type and mutant rSOF75ΔFn proteins was conducted using circular dichroism (CD) spectroscopy. CD spectra were acquired using a Jasco J-810 Spectropolarimeter (Jasco). Experiments were conducted at room temperature with proteins at a concentration of ∼0.2 mg/ml in 10 mm sodium phosphate buffer, pH 7.5 containing 50% trifluoroethanol (27Ionescu R.M. Matthews C.R. Nat. Struct. Biol. 1999; 6: 304-307Crossref PubMed Google Scholar, 28Reymond M.T. Merutka G. Dyson H.J. Wright P.E. Protein Sci. 1997; 6: 706-716Crossref PubMed Scopus (92) Google Scholar). Far UV spectra were recorded from 190-250 nm in a 0.1-cm pathlength cell (Starna) containing 400 μl of protein solution. The data shown represents an average of ten scans, corrected for a buffer baseline. Mean residue ellipticity (MRE; [θ]) was calculated using Equation 1 (29Schmid F.X. Creighton T.E. ed. Protein Structure: A Practical Approach. IRL Press, Oxford University Press, Oxford, UK1989: 251-284Google Scholar). MRE[θ]=θ×100×molecularweightconc(mg/ml)×pathlength×1000×no.ofresidues(Eq. 1) The α-helical content of wild type and mutant forms of rSOF75ΔFn was calculated from the MRE value at 222 nm using Equation 2 as described by Ref. 30Chen Y.H. Yang J.T. Martinez H.M. Biochemistry. 1972; 11: 4120-4131Crossref PubMed Scopus (1913) Google Scholar. %α-Helix=MRE[θ]222-234030300×100(Eq. 2) Opacity Activity Assays—Qualitative opacity factor assays were conducted using the serum agarose overlay method (7Kreikemeyer B. Martin D.R. Chhatwal G.S. FEMS Microbiol. Lett. 1999; 178: 305-311Crossref PubMed Google Scholar). The serum overlay method permits visual confirmation of OF activity, as binding of apoA1 by SOF causes precipitation of apoAI and HDL, which appears as an opaque white band on the solid serum/agarose medium. Data are presented as an inversion of the actual blot with opacity activity appearing as a dark band on a light background. Quantitative opacity factor assays were conducted using purified HDL or human serum using the method of Courtney et al. (12Courtney H.S. Zhang Y.M. Frank M.W. Rock C.O. J. Biol. Chem. 2006; 281: 5515-5521Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), with opacification measured as absorbance at 405 nm. ApoAI Binding Capacity—Wells of a microtiter plate were coated with 20 μg/ml rSOF75 protein or gelatin in 0.01 m sodium bicarbonate for 1 h at 37 °C. Plates were washed with PBS and blocked with gelatin (1 mg/ml in PBS) for 1 h at 37 °C, 100 μl of biotinylated (Pierce) apoAI (Calbiochem) was added and incubated at 37 °C for 1 h. Following washing, 100 μl of avidin-peroxidase (0.5 μg/ml) was added to the wells and incubated at 37 °C for 1 h. The plates were then washed and 3,3′-5,5′-tetramethylbenzidine substrate added. Color development was stopped using 1 m phosphoric acid, and the absorbance at 450 nm was recorded. Assays were performed in triplicate. Interaction of SOF-coated Latex Beads with HEp-2 Cells—Assays of the interaction of SOF-coated latex beads with HEp-2 cells were conducted per previously published methods (13Timmer A.M. Kristian S.A. Datta V. Jeng A. Gillen C.M. Walker M.J. Beall B. Nizet V. Mol. Microbiol. 2006; 62: 15-25Crossref PubMed Scopus (43) Google Scholar, 31Dombek P.E. Cue D. Sedgewick J. Lam H. Ruschkowski S. Finlay B.B. Cleary P.P. Mol. Microbiol. 1999; 31: 859-870Crossref PubMed Scopus (107) Google Scholar, 32Molinari G. Talay S.R. Valentin-Weigand P. Rohde M. Chhatwal G.S. Infect. Immun. 1997; 65: 1357-1363Crossref PubMed Google Scholar). Preliminary assays were conducted in Dulbecco's modified Eagle's medium HEPES supplemented with either 10% FCS or 1% FCS. However, differential binding was observed between these two assay conditions, the rSOF75ΔFn only mediated binding to HEp-2 cells when incubated in DMEM HEPES 1% FCS, and thus these conditions were used for all further latex bead adherence assays. The efficiency of protein loading onto latex beads was measured by FLUOstar fluorescent plate reader (BMG Labtech) using anti-SOF75 rabbit serum and fluorescent labeling with goat anti-rabbit Alexa 488 (green) (Molecular Probes) (data not shown), protein loading efficiency was found to be comparable for all protein domains. To determine the effect of exogenous addition of the rFNBD domain on the adhesion and internalization of rSOFΔFn-coated latex beads, purified rFnBD at 1, 5, or 10 μg/ml was preincubated with the HEp-2 cells for 1 h prior to addition of the coated latex beads, and was maintained throughout the subsequent 4-h incubation with the latex beads. Confocal Microscopy Studies—HEp-2 cells (after incubation with the coated latex beads) were fixed for 30 min on ice in 500 μl/well of prechilled 4% paraformaldehyde in PBS, and then washed twice in PBS. Cells were then blocked by the addition of 200 μl/well of PBS containing 10% fetal calf serum and incubated for 30 min at room temperature. The blocking solution was then removed, and the cells were incubated with either protein-G-purified rabbit polyclonal anti-SOF75 antibodies (26Gillen C.M. Towers R.J. McMillan D.J. Delvecchio A. Sriprakash K.S. Currie B. Kreikemeyer B. Chhatwal G.S. Walker M.J. Microbiology. 2002; 148: 169-178Crossref PubMed Scopus (19) Google Scholar) (30 μg) or rabbit polyclonal anti-FnBD antibodies (1:100 dilution) for 45 min at room temperature. Following washing with PBS, cells were incubated with goat anti-rabbit Alexa 488 diluted 1:400 in PBS containing 10% BSA (Molecular Probes) for 1 h at room temperature and subsequently washed with PBS. Cells were permeabilized with 200 μl/well of 0.1% (v/v) Triton X-100 in PBS for 30 min on ice, washed in PBS, followed by storage at 4 °C overnight. The following day, cells were treated with goat anti-rabbit Alexa 633 diluted 1:400 in PBS containing 10% BSA (Molecular Probes) for 1 h and washed three times in PBS. Cells were then mounted onto a glass slide using Mowiol solution (Calbiochem). Images were recorded using a Leica TCS SP confocal microscope mounted on a Leica DM IRBE inverted microscope with Leica TCS NT software (Version 2.61; Leica Microsystems). Mouse Immunization and Challenge—To determine the protective efficacy of rSOF75ΔFn proteins challenge studies were performed. BALB/c mice (n = 10) were immunized subcutaneously with 25 μg of wild type or mutant forms of rSOF75ΔFn protein in incomplete Freund's adjuvant. Control mice received a subcutaneous injection of PBS. After 2 weeks, the mice were boosted with an intramuscular injection of another 25 μg of each protein in PBS. Control mice received a PBS injection. Two weeks after the booster injections, all mice were challenged by an intraperitoneal injection of ∼1 × 109 CFU of the SOF-positive M49 GAS strain 591 (33Podbielski A. Woischnik M. Leonard B.A.B. Schmidt K.H. Mol. Microbiol. 1999; 31: 1051-1064Crossref PubMed Scopus (158) Google Scholar). The number of surviving mice was recorded daily. Moribund mice were sacrificed and recorded as dead. Serum samples were collected on days 0 and 28, and stored at -20 °C prior to determination of rSOF75ΔFn-specific antibodies. In brief, 96-well Nunc-Immuno MaxiSorp assay plates (Nunc) were coated with 2 μg/ml of wild type rSOF75ΔFn in coating buffer (bicarbonate, pH 9.4). After overnight incubation at 4 °C, plates were blocked with 1% BSA in PBS (pH 7.4) for 1 h at 37 °C. Serial 2-fold dilutions of serum in PBS with 1% BSA were added (100 μl/well), and plates were incubated for 1 h at 37 °C. After four washes, secondary biotinylated antibodies were added followed by 1 h of incubation at 37 °C. After six washes, 50 μl/well of peroxidase-conjugated streptavidin (Pharmingen), diluted 1:1000, was added, and plates were further incubated for 45 min at room temperature. After a final six washes, the substrate ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) in 0.1 m citrate-phosphate buffer containing 0.1% H2O2 was added, and plates were incubated for 30-60 min at room temperature. The absorbance was measured at a wavelength of 405 nm. Statistical Analyses—For apoAI binding experiments, an unpaired Student's t test was used to determine if there was any significant difference in the apoAI binding ability of wild type and mutant SOF proteins. For latex bead experiments, a one way analysis of variance using Bartlett's test for equal variance was used to determine whether there was any significant variation in the median number of beads attached to or taken up by HEp-2 cells, followed by a Tukey's Multiple Comparison Test for individual comparison of adherence and internalization mediated by two different proteins. For immunization and challenge experiments, a Kruskal-Wallis test was used to determine whether there was any significant variation in the median titers of the four groups of antisera. Dunn's Multiple Comparison test was used for individual comparison of two groups of antisera. Difference in survival curves was determined by log rank test. All statistics were performed using GraphPad Prism version 4.02 (Graph-Pad Software Inc., San Diego, CA). SOF is a unique multifunctional protein, capable of binding fibronectin via a C-terminal domain designated FnBD (8Rakonjac J.V. Robbins J.C. Fischetti V.A. Infect. Immun. 1995; 63: 622-631Crossref PubMed Google Scholar, 24Kreikemeyer B. Talay S.R. Chhatwal G.S. Mol. Microbiol. 1995; 17: 137-145Crossref PubMed Scopus (114) Google Scholar) and apoAI via an N-terminal domain (12Courtney H.S. Zhang Y.M. Frank M.W. Rock C.O. J. Biol. Chem. 2006; 281: 5515-5521Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (Fig. 1A). The N-terminal domain of SOF (SOFΔFn) has been shown to mediate adhesion to HEp-2 cells and promote HEp-2 cell invasion by whole GAS cells. To assess the contribution of the OF activity of SOF in the processes of HEp-2 epithelial cell adhesion and invasion, deletion mutagenesis was used to eliminate the OF activity of recombinant rSOF75ΔFn. Deletion was undertaken to remove between 22 and 63 amino acid residues of the rSOF75ΔFn protein (between Pro148 and Lys211, Pro210 and Glu232, Lys231 and Asp286, Val285 and Glu315). Each of the rSOF75ΔFn deletion mutants lacked OF activity (Fig. 1B). The rSOF75ΔFnDEL[P210→ E232] mutant was also found to lack OF activity when incubated in human serum or human HDL (Fig. 2). To delineate specific amino acid residues that contribute to OF activity, site-directed mutagenesis to alanine was undertaken on 52 amino acids of SOF75ΔFn that are 100% conserved in 16 different SOF sequences (supplemental Table S1). A mutant form of rSOF75ΔFn with attenuated OF activity (100-fold reduction in activity) was constructed by simultaneously substituting 5 amino acids with alanine (Asp218, Ser226, Lys228, Met229, Glu232). The rSOF75ΔFn[D218A-S226A-K228A-M229A-E232A] protein has attenuated OF activity when incubated with either human serum or human HDL (Fig. 2). To determine whether the loss or attenuation of OF activity was due to a decrease in the apoAI binding capacity of SOF, the ability of rSOF75ΔFnWT, rSOF75ΔFn[D218A-S226A-K228A-M229A-E232A], and rSOF75ΔFnDEL[P210→ E232] to bind biotinylated apoAI was assayed (Fig. 2D). There was no significant decrease in apoAI binding by the mutant proteins, suggesting that the loss of OF activity occurs via an alternative mechanism.FIGURE 2The OF activity and apoAI binding capacity of wild type and mutant forms of rSOF75ΔFn. A, OF activity over 24 h was determined by adding 1 μg/ml of either rSOF75ΔFnBDWT, rSOF75ΔFn[D218A-S226A-K228A-M229A-E232A], or rSOF75ΔFnDEL[P210→ E232] to human serum and recording the A405 nm at timed intervals. B and C, the opacification of serum (B) or HDL (C) as a function of protein concentration. Human serum or purified human HDL was treated with the indicated concentration of protein for 24 h and the absorbance determined at 405 nm. D, binding of biotinylated apoAI by either rSOF75ΔFnBDWT, rSOF75ΔFn[D218A-S226A-K228A-M229A-E232A], rSOF75ΔFnDEL[P210→ E232], or gelatin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The impact of the mutations to rSOF75ΔFn on protein structure was analyzed using far-UV CD spectroscopy. rSOF75ΔFnWT had a CD emission spectrum typical of proteins containing both α-helices and β-sheets, with a characteristic minimum at ∼220 nm, a second larger minimum at ∼207 nm and a maximum at 190 nm (34Venyaminov S.Y. Yang J.T. Fasman G.D. 1st Ed. Circular Dichroism and the Conformational Analysis of Biomolecules. Plenum Press, New York1996: 69-107Google Scholar). Of the rSOF75ΔFnDEL mutant proteins generated, rSOF75ΔFnDEL[K231→ D286] could not be purified for structural analysis. Far-UV CD spectra obtained for rSOF75ΔFnDEL[P148→ K211], rSOF75ΔFnDEL[P210→ E232], and rSOF75ΔFnDEL[V285→ D315] indicate perturbation to the secondary structure of the proteins. The rSOF75ΔFnWT is predicted to contain 27% α-helicity, with a predicted 24% α-helical content in rSOF75ΔFnDEL[P148→ K211], 21% in rSOF75ΔFnDEL[P210→ E232], and 22% in rSOF75ΔFnDEL[V285→ D315]. A concomitant shift toward a more disordered structure was observed for each deletion mutant, as indicated by a shift in the 207 nm minima to a shorter wavelength of 204 nm for rSOF75ΔFnDEL[P148→ K211] and rSOF75ΔFnDEL[P210→ E232] and 205.5 nm for rSOF75ΔFnDEL[V285→ D315] (Fig. 3) (34Venyaminov S.Y. Yang J.T. Fasman G.D. 1st Ed. Circular Dichroism and the Conformational Analysis of Biomolecules. Plenum Press, New York1996: 69-107Google Scholar, 35Freifelder D. 2nd Ed. Physical Biochemistry. Applications to Biochemistry and Molecular Biology. W. H. Freeman and Company, New York1982Google Scholar). In contrast to the loss of secondary structural elements in the rSOF75ΔFnDEL mutants, the rSOF75ΔFn[D218A-S226A-K228A-M229A-E232A] mutant had increased secondary structure when compared with rSOF75ΔFnWT, with an increase in predicted α-helicity from 27 to 29% (Fig. 3). To assess the direct contribution of the OF activity of SOF in the processes of HEp-2 epithelial cell adhesion and invasion, the ability of latex beads coated with wild type OF-positive, mutant OF-negative, and OF-attenuated forms of the rSOF75ΔFn protein to bind to the human pharyngeal epithelial cell line HEp-2 was assayed. These studies indicate that the SOFΔFn domain mediates attachment to HEp-2 cells, with the latex beads coated with rSOF75ΔFnWT adhering to HEp-2 cells in numbers equivalent to latex beads coated with the full-length rSOF75 (p > 0.05), and significantly more latex beads coated with rSOF75ΔFnWT adhering to HEp-2 cells than latex beads coated with a protein encompassing only the fibronectin binding domain of the SOF protein (rFNBD) (p < 0.01). Furthermore, this HEp-2 adherence mediated by the SOFΔFn domain is not dependent on OF activity, with latex beads coated with rSOF75ΔFn[D218A-S226A-K228A-M229A-E232A] (attenuated OF activity) and rSOF75ΔFnDEL[P148→ K211] (abolished OF activity) mediating adherence at the same level as the rSOF75ΔFnWT protein (p > 0.05) (Fig. 4, A and C). Latex beads coated with rSOF75ΔFnDEL[P210→ E232] and rSOF75ΔFnDEL[V285→ D315] have a significantly reduced capacity to attach to HEp-2 cells when compared with rSOF75ΔFnWT (p < 0.01), however, there were significantly more latex beads coated with rSOF75ΔFnDEL mutants attached to HEp-2 cells than were observed with the BSA control (p < 0.01). It has been previously demonstrated that the SOFΔFn domain of SOF possesses pro-invasive properties when expressed on the surface of non-invasive GAS strains or non-invasive Lactococcus lactis (13Timmer A.M. Kristian S.A. Datta V. Jeng A. Gillen C.M. Walker M.J. Beall B. Nizet V. Mol. Microbiol. 2006; 62: 15-25Crossref PubMed Scopus (43) Google Scholar). However, while the SOFΔFn promotes epithelial cell invasion in these backgrounds, this study has shown that the SOFΔFn protein domain is not sufficient per se to mediate intracellular invasion of HEp-2 cells (Fig. 4, B and C). However, while it is apparent that the N terminus of SOF does not directly mediate epithelial cell invasion, it may be concluded that the higher uptake by epithelial cells of latex beads coated with full-length SOF, in comparison to the FnBD domain alone, is attributable to the N-terminal domain of SOF. A significantly greater proportion of latex beads coated with rSOF75 were found to be intracellular (57.1%) than latex beads coated with FnBD alone (31.8% intracellular) (p < 0.01) (Fig. 4B). While SOF is a protective antigen in murine vaccination studies, the capacity of SOF to opacify serum raises questions about its use in humans. The capacity to knock-out OF activity while retaining the structural and f" @default.
• W2060023876 created "2016-06-24" @default.
• W2060023876 creator A5010455330 @default.
• W2060023876 creator A5013266227 @default.
• W2060023876 creator A5016986473 @default.
• W2060023876 creator A5018911946 @default.
• W2060023876 creator A5034986244 @default.
• W2060023876 creator A5044311719 @default.
• W2060023876 creator A5046541623 @default.
• W2060023876 creator A5047079791 @default.
• W2060023876 creator A5050141430 @default.
• W2060023876 creator A5067852918 @default.
• W2060023876 date "2008-03-01" @default.
• W2060023876 modified "2023-10-15" @default.
• W2060023876 title "Opacity Factor Activity and Epithelial Cell Binding by the Serum Opacity Factor Protein of Streptococcus pyogenes Are Functionally Discrete" @default.
• W2060023876 cites W111924924 @default.
• W2060023876 cites W1497161759 @default.
• W2060023876 cites W1833541376 @default.
• W2060023876 cites W1878018231 @default.
• W2060023876 cites W1959067297 @default.
• W2060023876 cites W1976694791 @default.
• W2060023876 cites W1976800164 @default.
• W2060023876 cites W1987225684 @default.
• W2060023876 cites W1999965330 @default.
• W2060023876 cites W2001273693 @default.
• W2060023876 cites W2015622010 @default.
• W2060023876 cites W2019544594 @default.
• W2060023876 cites W2020361370 @default.
• W2060023876 cites W2020497509 @default.
• W2060023876 cites W2021518309 @default.
• W2060023876 cites W2023901812 @default.
• W2060023876 cites W2025890332 @default.
• W2060023876 cites W2027541156 @default.
• W2060023876 cites W2035089602 @default.
• W2060023876 cites W2040941372 @default.
• W2060023876 cites W2046099835 @default.
• W2060023876 cites W2054041072 @default.
• W2060023876 cites W2055733673 @default.
• W2060023876 cites W2059086719 @default.
• W2060023876 cites W2060486025 @default.
• W2060023876 cites W2062228103 @default.
• W2060023876 cites W2072445985 @default.
• W2060023876 cites W2079695176 @default.
• W2060023876 cites W2084552186 @default.
• W2060023876 cites W2086709746 @default.
• W2060023876 cites W2098823687 @default.
• W2060023876 cites W2104184083 @default.
• W2060023876 cites W2107429021 @default.
• W2060023876 cites W2117422749 @default.
• W2060023876 cites W2120572405 @default.
• W2060023876 cites W2121600157 @default.
• W2060023876 cites W2124108127 @default.
• W2060023876 cites W2125107482 @default.
• W2060023876 cites W2128697027 @default.
• W2060023876 cites W2129194771 @default.
• W2060023876 cites W2130466396 @default.
• W2060023876 cites W2136088720 @default.
• W2060023876 cites W2139026449 @default.
• W2060023876 cites W2139360044 @default.
• W2060023876 cites W2140305366 @default.
• W2060023876 cites W2147203214 @default.
• W2060023876 cites W2151810774 @default.
• W2060023876 cites W2154561757 @default.
• W2060023876 cites W2155035342 @default.
• W2060023876 cites W2157554973 @default.
• W2060023876 cites W2158216804 @default.
• W2060023876 cites W2158610854 @default.
• W2060023876 cites W2158727946 @default.
• W2060023876 cites W2160829992 @default.
• W2060023876 cites W2165408245 @default.
• W2060023876 cites W2165990394 @default.
• W2060023876 cites W2166338009 @default.
• W2060023876 cites W2169893082 @default.
• W2060023876 cites W2170288350 @default.
• W2060023876 cites W2327503467 @default.
• W2060023876 cites W4243413100 @default.
• W2060023876 doi "https://doi.org/10.1074/jbc.m706739200" @default.
• W2060023876 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18180300" @default.
• W2060023876 hasPublicationYear "2008" @default.
• W2060023876 type Work @default.
• W2060023876 sameAs 2060023876 @default.
• W2060023876 citedByCount "26" @default.
• W2060023876 countsByYear W20600238762012 @default.
• W2060023876 countsByYear W20600238762013 @default.
• W2060023876 countsByYear W20600238762014 @default.
• W2060023876 countsByYear W20600238762015 @default.
• W2060023876 countsByYear W20600238762016 @default.
• W2060023876 countsByYear W20600238762017 @default.
• W2060023876 crossrefType "journal-article" @default.
• W2060023876 hasAuthorship W2060023876A5010455330 @default.
• W2060023876 hasAuthorship W2060023876A5013266227 @default.
• W2060023876 hasAuthorship W2060023876A5016986473 @default.
• W2060023876 hasAuthorship W2060023876A5018911946 @default.
• W2060023876 hasAuthorship W2060023876A5034986244 @default.
• W2060023876 hasAuthorship W2060023876A5044311719 @default.
• W2060023876 hasAuthorship W2060023876A5046541623 @default.
• W2060023876 hasAuthorship W2060023876A5047079791 @default.
• W2060023876 hasAuthorship W2060023876A5050141430 @default.

• next