In this study, we have investigated the interactions of a Staphylococcal recombinant fibronectin-binding protein A (rFnbA) with fibronectin, fibrinogen, and fibrin. Using analytical size-exclusion chromatography, we evaluated the stoichiometry of reversible binding of FnbA to fibronectin and demonstrated that, in solution, it can accommodate at least two molecules of fibronectin. Results of ELISA experiments demonstrated that rFnbA binds with equally high affinity to both immobilized fibrinogen and fibrin. When included into a thrombin-induced fibrin polymerization reaction, rFnbA strongly inhibited fibrin assembly in a dose-dependent manner. In this study, we have shown that rFnbA can act as a substrate for coagulation factor XIIIa. Factor XIIIa catalyzes the incorporation of amine donor (dansylacadaverine) and amine acceptor (peptide patterned on the N-terminal sequence of fibronectin) synthetic probes into rFnbA, suggesting that it serves as a bifunctional substrate containing reactive glutamine and lysine residues. We have demonstrated that the reversible complex formed by rFnbA and fibronectin or rFnbA and fibrin is covalently stabilized by the transglutaminase action of factor XIIIa. Incubation of rFnbA in the presence of either of its ligands and factor XIIIa results in the introduction of intermolecular epsilon-(gamma-glutamyl)lysine isopeptide bond(s) and the formation of high molecular mass heteropolymers. These findings suggest a novel mechanism by which pathogenic Staphylococcus aureus may utilize the transglutaminase activity of factor XIIIa for attachment to soluble proteins, cell surfaces, and matrixes.

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The outer membrane protein CD of Moraxella catarrhalis is considered to be a potential vaccine antigen against Moraxella infection. We purified the native CD from isolate O35E, administered it to mice, and detected considerable titers of anti-CD antibodies. Anti-CD sera were cross-reactive towards six different M. catarrhalis isolates and promoted bacterial clearance of O35E in a pulmonary challenge model. To circumvent the difficulty of generating large quantities of CD from M. catarrhalis for vaccine use, the CD gene from O35E was cloned into Escherichia coli, and the recombinant CD, expressed without a signal sequence or fusion tags, represented ϳ70% of the total E. coli proteins. The recombinant CD formed inclusion bodies that were solubilized with 6 M urea and then purified by ion-exchange chromatography, a procedure that produced soluble CD of high purity and yield. Mice immunized with the purified recombinant CD had significant titers of anti-CD antibodies that were cross-reactive towards 24 different M. catarrhalis isolates. Upon challenge, these mice showed enhanced bacterial clearance of both O35E and a heterologous M. catarrhalis isolate, TTA24. In an in vitro assay, antisera to either the native or the recombinant CD inhibited the binding activity of CD to human tracheobronchial mucin in a serum concentration-dependent manner, and the extent of inhibition appeared to correlate with the corresponding anti-CD antibody titer and whole-cell enzyme-linked immunosorbent assay titer. Our results demonstrate that the recombinant CD is a promising vaccine candidate for preventing Moraxella infection.Moraxella catarrhalis is an important human mucosal pathogen of the respiratory tract (20,29,44). It is the third most common cause of bacterial otitis media in infants and young children (3, 40), following Streptococcus pneumoniae and nontypeable Haemophilus influenzae. Among adults, M. catarrhalis is often associated with bronchitis, laryngitis, and other respiratory diseases (1, 5). Patients with chronic obstructive pulmonary disease (COPD) are particularly vulnerable to exacerbations caused by M. catarrhalis (1,6,35). Interest in the development of a Moraxella vaccine is further stimulated by the increasing prevalence of antibiotic resistance among M. catarrhalis strains (2,8,19).The CD outer membrane protein of M. catarrhalis has been identified as a potential vaccine against Moraxella infection (9, 26) and is a safe and effective carrier for M. catarrhalis detoxified lipooligosaccharide (LOS)-based conjugates (18). Serum immunoglobulin G (IgG) antibodies specific to CD are present in infants with otitis media (25) and in children with otitis media with effusion (11). Analysis of salivary immunoglobulin A (IgA) in children with acute respiratory tract infection indicates that CD may be one of the outer membrane antigens eliciting a mucosal immune response (27). IgA antibodies against CD as well as several other surface components of M. catarrhalis are also detected in the saliva of healthy adults (28). Furthe...

We have cloned an 82-base-pair region spanning the site of normal 3' end formation of Saccharomyces cerevisiae CYCI mRNA into an integrative vector carrying the 5' end of the actin gene (including its intron) fused in frame to HIS4ABC sequences. This vector can confer the ability to grow on histidinol if HIS4C (encoding histidinol dehydrogenase) is sufficiently expressed. With the CYCI fragment cloned in its wild-type (forward) orientation within the actin intron, transformants cannot grow on histidinol, whereas cells transformed with the vector carrying the reverse orientation ofthis fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate <40% of the control level of full-length mRNA and reveal the presence ofa short, stable (=300 nucleotides) poly(A) RNA that represents 60-70% ofthe transcripts originating from the same promoter. The reverse orientation of the insert allows nearnormal levels offull-length mRNA. Mapping ofthe 3' end ofthe truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYCI transcript. Dominant and recessive suppressor mutations permit growth on histidinol despite the inserted fragment. Genetic analyses indicate that most of the dominant mutants are cis-acting and that the recessive mutants define a minimum of three complementation groups, indicating that defects in several different genes can restore higher levels of HIS4C expression.Sherman (3) when they found that the cycl-512 mutation [a 38-base-pair (bp) deletion spanning the wild-type CYCI mRNA 3' end point] resulted in a 90%o reduction in the overall accumulation oftranscripts encoding the CYCI product; many of the mRNAs were longer, and all were polyadenylylated. Several other yeast genes share limited homology to a subset of the sequences deleted by the cycl-512 mutation. Local sequence changes in these regions can restore partial function (4-6); however, no definitive sequence has yet been proved to specify 3' end formation. Analysis of cycl-512 revertants demonstrated that mutations in trans-acting factors can also act to restore the efficiency of 3' end formation and accumulation of stable mRNAs (4, 7).In this report we describe a combined genetic and biochemical approach to understanding 3' end formation and polyadenylylation of yeast mRNA. We have used an in-frame fusion of the actin and HIS4 genes (8) (8); genetic markers include ura3-52 for selection of URA3 transformants, his4-401 [a 2-kilobase (kb) deletion encompassing the promoter and most of the coding sequences], and the dominant HOLI mutation to permit uptake of the histidine precursor, histidinol. In addition, either ade2 or ade5 markers were introduced into the strains to permit selection of diploids used in complementation tests. Yeast were transformed after spheroplasting (9), and other standard genetic techniques and growth media were as described by Sherman et al. (10). In SC + Hol medium, 0.064% histidinol was included in s...

In the present study we investigated the role of factor XIIIa reactive Gln and Lys sites of staphylococcal FnbA receptor in cross-linking reaction with alpha chains of fibrin. For this purpose we produced two recombinant FnbA mutants in which either a single Gln103 site (1Q FnbA) or all identified reactive Gln103, 105, 783, 830 and Lys157, 503, 620, 762 sites (4Q4K FnbA) were substituted with Ala residues. The results of FXIIIa-catalyzed incorporation of dansylcadaverine and dansylated peptide patterned on the NH2-terminal segment of fibronectin revealed that the reactivity of Gln substrate sites was drastically reduced in 1Q FnbA and 4Q4K FnbA mutants, while the reactivity of Lys substrate sites was only moderately decreased in 4Q4K FnbA. When it was tested in the FXIIIa-mediated fibrin cross-linking reaction, the 1Q FnbA mutant exhibited about 70-85% reduction in reactivity compared to that of the wild-type FnbA. These results demonstrate that FnbA participates in cross-linking to alpha chains of fibrin predominantly via its Gln103 reactive site. Several minor sites, including residues replaced in 4Q4K FnbA mutant, contributed to an additional 15-30% of the total fibrin cross-linking reactivity of FnbA. Comparison of amino acid sequences that follow the major reactive Gln site in FnbA and several known substrate proteins revealed that FXIIIa displays a preference for the glutamine residue in an xQAxBxPx sequence, where Q represents reactive glutamine, x is any amino acid residue, A is a polar residue, B is either valine or leucine, and P is proline.