Toxoids of streptococcal pyrogenic exotoxin A are protective in rabbit models of streptococcal toxic shock syndrome.

Streptococcal pyrogenic exotoxins (SPEs) are superantigens that have been implicated in causing streptococcal toxic shock syndrome (STSS). Most notably, SPE serotype A is made by nearly all M-protein serotype 1 and 3 streptococci, the M types most associated with the illness (these strains contain one or more other SPEs, and those proteins are likely also to contribute to disease). We have prepared double-, triple-, and hexa-amino-acid mutants of SPE A by PCR and other mutagenesis procedures. The sites chosen for mutation were solvent-exposed residues thought to be important for T-cell receptor (TCR) or major histocompatibility complex (MHC) class II interaction. These mutants were nonsuperantigenic for human peripheral blood mononuclear cells and rabbit and mouse splenocytes and were nonlethal in two rabbit models of STSS. In addition, these mutants stimulated protective antibody responses. Interestingly, mutants that altered toxin binding to MHC class II were more immunogenic than mutants altering TCR binding. Collectively, these studies indicate that multiple-site mutants of SPE A are toxoids that may have use in protecting against the toxin's effects in STSS.

Development of streptococcal pyrogenic exotoxin C vaccine toxoids that are protective in the rabbit model of toxic shock syndrome.

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.

Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity.

The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.

Characterization of a subunit structure and stability of the recombinant porin from Neisseria gonorrhoeae.

An outer membrane PIA protein from Neisseria gonorrhoeae strain FA19 was expressed in Escherichia coli and refolded in vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50 degrees C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80 degrees C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind with Kd=80 and 130 microM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.

Cross-linking of fibronectin to C-terminal fragments of the fibrinogen alpha-chain by factor XIIIa.

Fibronectin binds specifically to fibrin and is covalently cross-linked to the fibrin alpha chain by activated factor XIII (XIIIa). This reaction is important for wound healing. Here we investigate XIIIa-catalyzed cross-linking of fibronectin and some of its fragments to a recombinant fragment representing the COOH-terminal 30 kDa of the fibrin alpha chain (alpha C30K:His 368-Val 610). Only fibronectin and those fragments containing an intact NH2-terminus were able to form cross-linked complexes. As many as 10 of the 17 lysines in alpha C30K can serve as amine donors in this reaction. Analysis of the rate of XIIIa-catalyzed cross-linking of fibronectin NH2-terminal peptides and fragments with alpha C30K revealed that the presence of the first type I "finger" module accelerates the cross-linking reaction; addition of fingers 2-5 had no further effect.

Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen.

Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.

Interaction of N-terminal fragments of fibronectin with synthetic and recombinant D motifs from its binding protein on Staphylococcus aureus studied using fluorescence anisotropy.

The N-terminal 29-kDa fragment of fibronectin (Fn29K) contains five type I "finger" modules. It binds to heparin, fibrin, and bacteria and is involved in fibronectin (Fn) matrix assembly. Binding to Staphylococcus aureus involves a cell wall-associated protein that contains approximately three repeats of a 38-residue D motif (Signäs, C., Raucci, G., Jönsson, K., Lindgren, P.-E., Anantharamaiah, G.M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). Synthetic peptides representing D1, D2, and D3, when labeled with fluorescein isothiocyanate (FITC), exhibited increases in fluorescence anisotropy upon addition of Fn29K but not other Fn fragments. The response could be reversed by titration with unlabeled peptides to yield inhibition constants that agreed with the dissociation constants obtained by fitting the initial response. Values of Kd ranged between 2 and 12 microM, with D3 having the highest affinity. Specificity of D3 for Fn29K was further illustrated by the fact that its C-terminal half (D3b, Lys801 to Lys821), when immobilized, selectively adsorbed Fn29K from a thermolysin digest of fibronectin. The binding site in Fn was further localized within Fn29K by analyzing smaller proteolytic or recombinant subfragments. Those containing fingers, F3-5 and F4-5, were purified on D3b-Sepharose and bound FITC-D3b with Kd values of 4-6 microM. Subfragments containing pairs of fingers 1-2, 2-3, or single fingers 1, 4, or 5 were inactive. Whole D1-3, expressed in Escherichia coli and labeled with fluorescein, bound 1.9 mol/mol of Fn29K with Kd = 1.5 nM. F4-5 and F2-3 bound with respective Kd values of 0.35 and 4.4 microM. These and other results indicate that binding of the individual D region peptides is mediated through their C-terminal halves, primarily to fingers 4 and 5 of fibronectin. The possible basis of the much higher affinity of D1-3 is discussed.

The NH2-terminal fibrin-binding site of fibronectin is formed by interacting fourth and fifth finger domains. Studies with recombinant finger fragments expressed in Escherichia coli.

The NH2-terminal 29-kDa Fib-1 fragment consisting of the first five finger modules of fibronectin (F1-5) binds reversibly to fibrin and facilitates cross-linking by Factor XIII. To narrow down the fibrin-binding site within this region, we have used recombinant technology to express a number of individual fingers, rF1, rF2, rF3, rF4, and rF5, and their pairs, rF1-2 rF2-3, and rF4-5, as fusion proteins in Escherichia coli. These recombinant fragments were separated from the carrier maltose-binding protein by digestion with human factor Xa or other proteases, and their structural integrity was confirmed by spectroscopic and calorimetric methods. The recombinant F1 and F4-5 exhibited fluorescence-detected melting transitions of the same magnitude and with the same midpoint (Tm) as their natural analogues prepared from Fib-1 by proteolysis. Differential scanning calorimetry experiments further demonstrated that these fragments are properly folded and have compact structures identical to the natural ones. Isolated rF4 melts at a much lower temperature than rF5 or the bimodular fragment rF4-5, indicating the loss of a stabilizing interaction between fingers 4 and 5. Comparison of fluorescence spectra of individual rF4 and rF5 with that of rF4-5 was also consistent with an interaction that affects the environment of Trp residue(s). rF2 also melts at a lower temperature than rF3 or rF2-3, suggesting a stabilizing interaction between the second and third fingers as well. When tested on fibrin-Sepharose, only the bimodular fragment rF4-5 was able to bind. All other fragments, including individual fingers 4 and 5, failed to bind. Thus, fibrin binding is not a common property of all fingers. The results indicate that a recognition site for fibrin is located within fingers 4 and 5. The interaction between these neighboring domains may play an important role in proper orientation of the residues forming this site.

Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen.

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle.