Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa

Abstract

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

Footrot in Clawed and Hoofed Animals: Symptoms, Causes and Treatments

“…Transcription of fimA increases when cells are grown on solid media (56). This explains the observations of Mattick et al (68), e.g. an increase in the number of fimbriae when cells are cultured on a solid surface.…”

“…The fimbriae are composed of small, 145 to 160 аmino аcid (14 kDa to 20 kDa) polypeptide subunits named PilA, or pilin (2). The highly conserved amino-terminal part of the subunit is hydrophobic, whilst the variable central and carboxy-terminal region is hydrophilic (69,70). The polypeptides isolated from fimbriae range from 77 kDa to 88 kDa in size (2,68).…”

“…Stewart et al (94) Mattick et al (69) showed that fimbriae proteins of D. nodosus, obtained by gene cloning and expression by P. aeruginosa, could be used with the same success for vaccine preparation as the natural D. nodosus fimbriae. This may lead to higher yields of fimbriae proteins and the developing of a multivalent vaccine active against different serotypes or even species.…”

“…In similar studies, less effective vaccination has been reported when non-fimbriated cells were used (90,97,106). This led to the development of a series of vaccines based on pilin (68,69,112). The effectivity of the vaccines depended on the level of purification (92).…”

The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

“…Transformation ofP. aeruginosa strains was performed as described (13). Antibiotics were used at the following concentrations: E. coli, ampicillin (50 ,g/ml)/ tetracycline (40 ,g/ml); P. aeruginosa, carbenicillin (500 ,tg/ ml)/tetracycline (200 ,ug/ml).…”