Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under-the control of a strong promoter (PL, from bacteriophage A) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.Bacteroides nodosus is the essential causative agent of ovine footrot (4, 11). This anaerobe contains numerous surface filaments, about 6 nm in diameter and ranging up to several micrometers in length (14, 46, 50), termed fimbriae (or common pili), which play a central role in both pathogenesis and immunity (for a recent review, see reference 29).Fimbriae have adherent functions and appear to be a mechanism for the colonization of epithelial tissues in eucaryotic hosts. The properties of B. nodosus fimbriae (14) suggest that they belong in the category of type 4, as proposed by Ottow (38), citing Pseudomonas aeruginosa (6) as a prototype. Fimbriae of this type have a polar location on the cell and appear to be involved in surface translocation by a phenomenon known as twitching motility (21). The same characteristics are also observed in the fimbriae found in a broad range of gram-negative species classified within the genera Acinetobacter, Alteromonas, Bacteroides, Eikenella, Moraxella, Neisseria, and Pseudomonas, among others (6,17,20,21).This grouping is supported by recent protein and DNA sequence analyses of the structural subunits of the fimbriae of B. nodosus (12, 31), Moraxella nonliquefaciens (16), Moraxella bovis (28), Neisseria gonorrhoeae (22,33,44), Neisseria meningitidis (22, 36), and P. aeruginosa (42). These subunits, which range in size from about 145 to 160 amino acids among different species and serotypes, all share the distinctive feature of an unusual modified amino acid, methylphenylalanine (MeF), as the first residue in the mature protein, as well as a striking degree of sequence conservation throughout the amino-terminal region. This region is highly hydrophobic and exhibits at least 90% homology with the following 32-amino-acid consensus sequence:MeF T L I E L M I V

No abstract

The acid-labile subunit (ALS) is a glycosylated 85-kDa member of the leucine-rich repeat (LRR) protein superfamily and circulates in ternary complexes with the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). These complexes are thought to regulate the serum IGFs by restricting IGF movement out of the circulation. However, little is known about how ALS binds to IGFBP-3 or -5, which link the IGFs to ALS. To investigate potential sites of interaction, the ALS structure has been modeled with the crystal structure of the LRR protein porcine ribonuclease inhibitor as a template. ALS is predicted to be a donut-shaped molecule with an internal diameter of 1.7 nm, an external diameter of 7.2 nm, and a thickness of 3.6 nm. These dimensions are supported by rotary shadowing electron microscopy of ALS. The internal face is lined with a substantial region of electronegative surface potential that could interact with the positively charged region on IGFBP-3 known to be involved in ALS binding. The model also predicts that three potential N-linked oligosaccharide sites within the LRR domain are clustered together, which may be important in light of recent studies showing ALS glycan involvement in complex formation with IGFBP-3.The majority of serum insulin-like growth factors (IGFs) 1 circulate within 130 -150-kDa ternary complexes containing either IGF-I or -II, IGF-binding protein (IGFBP)-3, and the acid-labile subunit (ALS), an 85-kDa glycoprotein. It is thought that the size of these complexes prevents IGF access to target cells, while free IGFs and IGFs in binary complexes with the IGFBPs can easily cross the capillary endothelial barrier. Furthermore, the ALS-containing complex significantly increases the serum half-lives of both the IGFs and IGFBP-3 and in this way maintains a circulating store of these molecules (1, 2). Therefore, the association of ALS to the IGF complex is an important event in serum IGF regulation. ALS binding is the limiting step in complex formation, since the affinity of ALS for the IGF⅐IGFBP-3 complex is up to 2000-fold less than the affinity of IGFBP-3 for the IGFs in physiological salt concentrations, pH and temperature (3). Recently, Twigg and Baxter (4) showed that ALS and the IGFs can also form a ternary complex in vitro with IGFBP-5 (which has high homology to IGFBP-3 in the ALS binding domain) and that this IGFBP-5 complex is found in low concentrations in serum.Although the structures of the IGFs have been solved (5, 6), the structures of the proteins which interact with and regulate the serum IGFs have not been elucidated. Therefore, it is not yet clear how ALS interacts physically with IGFBP-3 to form the ternary complexes. However, one major structural feature of ALS is that around 75% of its residues are ordered into 18 tandem repeats of 24 amino acids plus two partial repeats, all of which contain the consensus motif for the leucine-rich repeat (LRR) superfamily of proteins. (7). All the members of this superfamily are involved in protein-protein interactions...

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Type 4 fimbriae (pili) are found in a wide variety of gram-negative bacteria and are composed of small structural subunits which share significant sequence homology among different species, especially at their amino-terminal ends. Previous studies demonstrating morphogenetic expression of Bacteroides nodosus fimbriae from cloned subunit genes in Pseudomonas aeruginosa suggested that there is a common mechanism for type 4 fimbriae assembly and that the structural subunits are interchangeable (J. S. Mattick et al., J. Bacteriol. 169:33-41, 1987). Here we have examined the expression of Moraxella bovis fimbrial subunits in P. aeruginosa. M. bovis subunits were assembled into extraceilular fimbriae in this host, in some cases as a homopolymer but in others as a mosaic with the indigenous subunit, indicating structural equivalence. This result contrasts with other studies in which recombinant P. aeruginosa expressing different subunits produced fimbriae composed almost exclusively of one subunit or the other (T. C. Elleman and J. E. Peterson, Mol. Microbiol. 1:377-380, 1987). Both observations can be explained by reversibility of subunit-subunit interactions at the site of assembly, with the forward equilibrium favoring chain extension between compatible subunits.Fimbriae, classified as type 4 by Ottow (20), are found in a wide range of bacterial pathogens, including Moraxella bovis, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, and Pseudomonas aeruginosa (7,14,18,19,22). The characteristics of type 4 fimbriae include a predominantly polar location on the cell, association with a phenomenon known as "twitching motility," and certain conserved features of the structural subunit which composes the fimbrial strand (7, 18). The structural subunits vary from about 145 to 160 amino acids long (approximately 16,000 molecular weight) between different genera, species, and serotypes, but all contain a highly homologous and hydrophobic amino-terminal region. In addition, most contain an unusual modified amino acid, N-methylphenylalanine, as the first residue in the mature protein, which is produced from a preprotein by cleavage of a short (six-to seven-amino-acid) positively charged leader sequence. Intergenera, interspecies, and interserotype variations occur primarily in the carboxy-terminal two-thirds of the protein (7, 18).The conserved amino-terminal region of the subunit is thought to contain important signals for the structure and assembly of the fimbrial strand. The high degree of sequence conservation would also suggest that these various type 4 fimbriate organisms use a common mechanism for fimbrial biosynthesis (18). This was supported by the demonstration of high-level morphogenetic expression of B. nodosus fimbriae from a cloned subunit gene (under appropriate promoter control) in P. aeruginosa (18) and the subsequent use of this material to vaccinate sheep against footrot (8).In this study we used a similar approach to demonstrate morphogenetic expression of M. bovis fimbriae from a cloned ...