Gram-negative bacteria are known to produce two types of surface organelles: flagella, which are required for motility and chemotaxis, and pili (fimbriae), which play a part in the interaction of bacteria with other bacteria and with eukaryotic host cells. Here we report a third class of E. coli surface organelles for which we propose the name curli. Curli are coiled surface structures composed of a single type of subunit, the curlin, which differs from all known pilin proteins and is synthesized in the absence of a cleavable signal peptide. Although the gene encoding this structural subunit, crl, is present and transcribed in most natural isolates of E. coli, only certain strains are able to assemble the subunit protein into curli. This assembly process occurs preferentially at growth temperatures below 37 degrees C. The ability of curli to mediate binding to fibronectin may be a virulence-associated property for wound colonization and for the colonization of fibronectin-coated surfaces.

Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli.

A gene encoding a fibronectin binding protein (FnBP) has recently been isolated and sequenced from Staphylococcus aureus strain 8325–4. In the same bacterial strain, 682 bp downstream to the stop codon of this gene (fnbA), a second gene termed fnbB has now been discovered, encoding another FnBP (FnBPB). The two genes show in large parts striking sequence homologies. The complete amino acid sequence encoded by fnbB has been deduced and compared to that deduced from fnbA. In FnBPB a stretch of 66 amino acids downstream to the signal peptide has 75% identity with the corresponding region in FnBPA. At the C-terminal site another 394 amino acid stretch is almost identical in both gene products. This stretch contains the 38 amino acid long D repeats, the wall spanning Wr repeats and the hydrophobic membrane spanning domain. In FnBPA each of the three D repeats has been identified as a fibronectin binding structure. These structures are highly conserved in FnBPB and most likely represent the major Fn-binding domain of this protein. However, a subclone of gene fnbB lacking the coding region for the D repeats also clearly expresses fibronectin binding activity. This additional binding site is so far unique for FnBPB and interacts like the D domains with the N-terminal 24–31-kDa fragment of fibronectin. The purified recombinant FnBP fragment (not containing the D repeats) completely inhibits the binding of fibronectin to whole cells of S. aureus.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1991.tb16468.x

Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene.

Campylobacter jejuni, a Gram-negative bacterium, is a common cause of gastrointestinal disease. By analogy with other enteric pathogens such as Salmonella and Shigella, the ability of C. jejuni to bind to host cells is thought to be essential in the pathogenesis of enteritis. Scanning electron microscopy of infected INT407 cells suggested that C. jejuni bound to a component of the extracellular matrix. Binding assays using immobilized extracellular matrix proteins and soluble fibronectin showed specific and saturable binding of fibronectin to C. jejuni. Ligand immunoblot assays using 125I-labelled fibronectin revealed specific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel-purified protein, reacted with a 37 kDa protein in all C. jejuni isolates (n = 15) as tested by immunoblot analysis. Antibodies present in convalescent serum from C. jejuni-infected individuals also recognized a 37 kDa protein. The gene encoding the immunoreactive 37kDa protein was cloned and sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a calculated molecular mass of 36872Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhesin protein from Pseudomonas fluorescens. Isogenic C. jejuni mutants which lack the 37 kDa outer membrane protein, which we have termed CadF, displayed significantly reduced binding to fibronectin. Biotinylated fibronectin bound to a protein with an apparent molecular mass of 37 kDa in the outer membrane protein extracts from wild-type C. jejuni as judged by ligand-binding blots. These results indicate that the binding of C. jejuni to fibronectin is mediated by the 37 kDa outer membrane protein which is conserved among C. jejuni isolates.

Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni.

The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitro-constructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M. O'Reilly, J.C.S. de Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathogen. 1:125-138, 1986). A double Spa- Hly- mutant was constructed by transduction. The virulence of Spa- and Hly- mutants was tested by experimental infection of mice. When subcutaneous injections were given, Hly- mutants formed a flat, darkened lesion, whereas Hly+ strains caused a raised, cream lesion. Alpha-toxin was shown to be a major factor in forming subcutaneous lesions and in causing the death of mice injected intraperitoneally. Spa- mutants were slightly less virulent than their Spa+ counterparts, which suggests that protein A is also a virulence factor of S. aureus.

https://iai.asm.org/content/iai/55/12/3103.full.pdf

Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.

Fibronectin, a large and essential multidomain glycoprotein, with multiple adhesive properties, functioning as a key link between cells and their extracellular matrices, is now recognized to be the target for a large number of bacterial proteins, which are generally considered to function as bacterial adhesins. In the last decade, an avalanche of bacterial fibronectin-binding proteins (FnBPs) has been identified, and the bioinformatics, structural biology, biological function and role in the virulence of a growing number of both Gram-positive and Gram-negative proteins have begun to emerge. The evidence suggests that fibronectin has a wider biological remit than was previously thought and that bacterial FnBPs have actions over and above that of simple adhesion. This article provides an update on our current understanding of FnBPs from both Gram-negative and Gram-positive bacteria and their proposed roles in bacterial colonization, bacterial virulence and bacteria–host interactions.

Fibronectin: a multidomain host adhesin targeted by bacterial fibronectin-binding proteins.

Binding of Escherichia coli to fibronectin. A mechanism of tissue adherence.

Fibronectin (fn) is a major protein of plasma and connective tissue which binds to Staphylococcus aureus and strains of other staphylococcal species often isolated from pyogenic infections. Strains of pyogenic streptococci (group A, C, and G strains) also bind fn in a similar time-dependent, saturable, and functionally irreversible way. Fibronectin-binding components released from staphylococcal and streptococcal cells after treatment with bacteriolytic or proteolytic enzymes are sensitive to protease degradation. Cross-absorption experiments with fn peptides show that the major binding for both organisms occurs to the NH2 terminal (29 kdalton) fragment while enterotoxigenic E. coli strains of the 06 serogroup show binding to one or two fn fragments outside the NH2 terminal domain. S. aureus cells bind to fn immobilized on plastic surfaces, suggesting that binding to biomaterials is an important portal of entry for pyogenic cocci to invade subepithelial tissues. Recent observations that other pathogens such as Treponema pallidum, tissue invasive parasites, and Candida albicans also bind fn indicate that many microbial pathogens may invade tissues by first binding to fn, or other connective tissue matrix proteins such as collagens or laminin. We recently defined uropathogenic E. coli and S. pyogenes strains binding to laminin or collagen. Finally, one protein A mutant of S. aureus (SA113-spA26) which is low in fn binding has a low virulence in experimental infections, indicating that fn binding is an important step in colonization of tissues and development of localized tissue infections.

A. Faris

Papers

Binding of Escherichia coli to fibronectin. A mechanism of tissue adherence.

Binding of Microbial Pathogens to Connective Tissue Fibronectin: An Early Step in Localized and Invasive Infections