Chaperone-usher fimbriae in a diverse selection of Gallibacterium genomes

Background

Fimbriae are bacterial cell surface organelles involved in the pathogenesis of many bacterial species, including Gallibacterium anatis, in which a F17-like fimbriae of the chaperone-usher (CU) family was recently shown to be an important virulence factor and vaccine candidate. To reveal the distribution and variability of CU fimbriae 22 genomes of the avian host-restricted bacteria Gallibacterium spp. were investigated. Fimbrial clusters were classified using phylogeny-based and conserved domain (CD) distribution-based approaches. To characterize the fimbriae in depth evolutionary analysis and in vitro expression of the most prevalent fimbrial clusters was performed.

Results

Overall 48 CU fimbriae were identified in the genomes of the examined Gallibacterium isolates. All fimbriae were assigned to γ4 clade of the CU fimbriae of Gram-negative bacteria and were organized in four-gene clusters encoding a putative major fimbrial subunit, a chaperone, an usher and a fimbrial adhesin. Five fimbrial clusters (Flf-Flf4) and eight conserved domain groups were defined to accommodate the identified fimbriae. Although, the number of different fimbrial clusters in individual Gallibacterium genomes was low, there was substantial amino acid sequence variability in the major fimbrial subunit and the adhesin proteins. The distribution of CDs among fimbrial clusters, analysis of their flanking regions, and evolutionary comparison of the strains revealed that Gallibacterium fimbrial clusters likely underwent evolutionary divergence resulting in highly host adapted and antigenically variable fimbriae. In vitro, only the fimbrial subunit FlfA was expressed in most Gallibacterium strains encoding this protein. The absence or scarce expression of the two other common fimbrial subunits (Flf1A and Flf3A) indicates that their expression may require other in vitro or in vivo conditions.

Conclusions

This is the first approach establishing a systematic fimbria classification system within Gallibacterium spp., which indicates a species-wide distribution of γ₄ CU fimbriae among a diverse collection of Gallibacterium isolates. The expression of only one out of up to three fimbriae present in the individual genomes in vitro suggests that fimbriae expression in Gallibacterium is highly regulated. This information is important for future attempts to understand the role of Gallibacterium fimbriae in pathogenesis, and may prove useful for improved control of Gallibacterium infections in chickens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1093) contains supplementary material, which is available to authorized users.     

Chaperone-Usher Fimbriae of Escherichia coli

Chaperone-usher (CU) fimbriae are adhesive surface organelles common to many Gram-negative bacteria. Escherichia coli genomes contain a large variety of characterised and putative CU fimbrial operons, however, the classification and annotation of individual loci remains problematic. Here we describe a classification model based on usher phylogeny and genomic locus position to categorise the CU fimbrial types of E. coli. Using the BLASTp algorithm, an iterative usher protein search was performed to identify CU fimbrial operons from 35 E. coli (and one Escherichia fergusonnii) genomes representing different pathogenic and phylogenic lineages, as well as 132 Escherichia spp. plasmids. A total of 458 CU fimbrial operons were identified, which represent 38 distinct fimbrial types based on genomic locus position and usher phylogeny. The majority of fimbrial operon types occupied a specific locus position on the E. coli chromosome; exceptions were associated with mobile genetic elements. A group of core-associated E. coli CU fimbriae were defined and include the Type 1, Yad, Yeh, Yfc, Mat, F9 and Ybg fimbriae. These genes were present as intact or disrupted operons at the same genetic locus in almost all genomes examined. Evaluation of the distribution and prevalence of CU fimbrial types among different pathogenic and phylogenic groups provides an overview of group specific fimbrial profiles and insight into the ancestry and evolution of CU fimbriae in E. coli.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli

Abstract: Fimbriae are long filamentous polymeric protein structures located at the surface of bacterial cells. They enable the bacteria to bind to specific receptor structures and thereby to colonise specific surfaces. Fimbriae consist of so-called major and minor subunits, which form, in a specific order, the fimbrial structure. In this review emphasis is put on the genetic organisation, regulation and especially on the biosynthesis of fimbriae of enterotoxigenic Escherichia coli strains, and more in particular on K88 and related fimbriae, with ample reference to the well-studied P and type 1 fimbriae. The biosynthesis of these fimbriae requires two specific and unique proteins, a periplasmic chaperone and an outer membrane located molecular usher ('doorkeeper'). Molecular and structural aspects of the secretion of fimbrial subunits across the cytoplasmic membrane, the interaction of these subunits with the periplasmic molecular chaperone, their translocation to the inner site of the outer membrane and their interaction with the usher protein, as well as the (ordered) translocation of the subunits across the outer membrane and their assembly into a grwoing fimbrial structure will be described. A model for K88 fimbriae is presented.     

Environmental signals implicated in Dr fimbriae release by pathogenic Escherichia coli

Afa/Dr diffusely adhering Escherichia coli have been shown to cause urinary tract infections and enteric infections. Virulence of Dr-positive IH11128 bacteria is associated with the presence of Dr fimbriae. In this report, we show for the first time that the Dr fimbriae are released in the extracellular medium in response to multiple environmental signals. Production and secretion of Dr fimbriae are clearly thermoregulated. A comparison of the amounts of secreted fimbriae showed that the secretion is drastically increased during anaerobic growth in minimal medium. The effect of anaerobiosis on secretion seemed to depend on both the growth phase and the culture medium. The secretion was maximal during the logarithmic-phase growth and corresponded to 27 and 57% of total Dr fimbriae produced by bacteria grown in mineral medium+glucose and LB broth, respectively. Thus, the anaerobic environment of the colon would favour the secretion of Dr fimbriae during bacterial multiplication. The controlled release of the Dr fimbriae, which is carried out in the absence of cellular lysis, appears independent of the action of proteases or a process of maturation. The mechanism employed in the liberation of Dr fimbriae thus seems different from that described for the adhesins FHA and Hap of Bordetella pertussis and Haemophilus influenzae.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

A possible association between CFA (I) fimbriae and K99 fimbriae on Escherichia coli and bacterial adherence to human lymphocytes

We have explored a possible association between Escherichia coli binding to human lymphocytes and plasmid coded fimbriae on the bacterial surface. E. coli with or without the plasmid coded membrane CFA(I), K99 and K88 were mixed with freshly-drawn human peripheral blood lymphocytes. When the lymphocytes were mixed with E. coli possessing the CFA(I) fimbriae, 59% of the lymphocytes bound bacteria onto the surface, whereas only 22% of the lymphocytes bound the CFA(I)- derivative. The lymphocytes bound 53% and 56% of two K9+ strains, whereas 22% and 8% of the lymphocytes adhered the same strains without the K99 fimbriae. Twelve per cent and 7% of lymphocytes bound bacteria when the strain was K88+ or K88-, respectively. Likewise a low (8%) adherence to lymphocytes was found when the E. coli did not possess fimbriae or flagella.     

Surface organelles assembled by secretion systems of Gram-negative bacteria: diversity in structure and function

Gram-negative bacteria express a wide variety of organelles on their cell surface. These surface structures may be the end products of secretion systems, such as the hair-like fibers assembled by the chaperone/usher (CU) and type IV pilus pathways, which generally function in adhesion to surfaces and bacterial-bacterial and bacterial-host interactions. Alternatively, the surface organelles may be integral components of the secretion machinery itself, such as the needle complex and pilus extensions formed by the type III and type IV secretion systems, which function in the delivery of bacterial effectors inside host cells. Bacterial surface structures perform functions critical for pathogenesis and have evolved to withstand forces exerted by the external environment and cope with defenses mounted by the host immune system. Given their essential roles in pathogenesis and exposed nature, bacterial surface structures also make attractive targets for therapeutic intervention. This review will describe the structure and function of surface organelles assembled by four different Gram-negative bacterial secretion systems: the CU pathway, the type IV pilus pathway, and the type III and type IV secretion systems.     

Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways

Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram‐negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone‐usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P‐fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P‐fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.     

Fimbriation ofEscherichia coli in urinary tract infections. Comparisons between bacteria in the urine and subcultured bacterial isolates

Midstream urine samples from 37 patients with urinary tract infections were studied by electron microscopy, hemagglutination, and the salt aggregation test (SAT) to measure the hydrophobicity of the bacterial surface.Escherichia coli subcultured from these urine samples were tested in the same way. Fimbriae were visualized onE. coli in the urine of 31 specimens, and all these urines containedE. coli that expressed pronounced surface hydrophobicity and aggregated in ammonium sulfate of 0.1–1.6 M final concentration. Hemagglutination of human and/or guinea pig erythrocytes was expressed by 21E. coli in the urine. TheE. coli strains subcultured from these 31 urine samples were also fimbriated, but the number of fimbriae per bacterium as well as the percentage of fimbriated bacteria varied compared with the directly collected strains. The surface hydrophobicity and hemagglutination were similar to the results with the directly collected bacteria. However, after serial transfer in CFA-broth under static conditions, all non-hemagglutinating strains expressed mannose-sensitive hemagglutination of guinea pig erythrocytes, and three strains also expressed weak mannose-resistant hemagglutination of human erythrocytes. Following serial transfer, fimbriae were also visualized on the sixE. coli strains that appeared non-fimbriate in the urine. It is thus concluded thatE. coli causing urinary tract infection are often fimbriated and express surface hydrophobicity in the urine. Based on these findings, a rapid method to isolate hydrophobic, possibly fimbriated bacteria was tried in which the urine was mixed with a hydrophobic gel. Hydrophobic bacteria bound to the gel and could be eluted from the sedimented gel.     

Adhesins of escherichia coli

E. coli has got increasing importance as a causative agent of intestinal and extra-intestinal diseases. In both these infections adhesion of the bacteria to mucous surface cells are initial events for coionization and development of infection. Adhesins are bacterial recognition proteins which specifically interact with carbohydrate moieties of glycoproteins or glycolipids on mammalian cells. The adhesiveness of bacteria is associated with filamentous surface appendages, designated as fimbriae or pili, as well as with non-fimbrial components. Some recent data on the nomenclature, classification, disease association, receptor specificity, and topographic arrangement are presented. The correlation between E. coli O : K : H serovar and fimbrial antigens is demonstrated on the basis of E. coli isolated from patients with urinary tract infections. Hitherto unknown non-fimbrial adhesins are briefly described.     

Chaperone-usher pathways: diversity and pilus assembly mechanism

Up to eight different types of secretion systems, and several more subtypes, have been described in Gram-negative bacteria. Here, we focus on the diversity and assembly mechanism of one of the best-studied secretion systems, the widespread chaperone-usher pathway known to assemble and secrete adhesive surface structures, called pili or fimbriae, which play essential roles in targeting bacterial pathogens to the host.     

Chaperone–usher pathways: diversity and pilus assembly mechanism

Up to eight different types of secretion systems, and several more subtypes, have been described in Gram-negative bacteria. Here, we focus on the diversity and assembly mechanism of one of the best-studied secretion systems, the widespread chaperone–usher pathway known to assemble and secrete adhesive surface structures, called pili or fimbriae, which play essential roles in targeting bacterial pathogens to the host.     

A two-plasmid Escherichia coli system for expression of Dr adhesins

This paper presents a very efficient expression system for production of Dr adhesins. The system consists of two plasmids. One is the pACYCpBAD-DraC-C-His, which contains the draC gene under the control of the arabinose promoter (pBAD), encoding the DraC usher. The second is the pET30b-syg-DraBE, which contains the draB and draE genes under the control of the T7lac promoter, encoding the DraB chaperone and the DraE adhesin, respectively. Those plasmids have different origin of replication and can therefore coexist in one cell. Since different promoters are present, the protein expression can be controlled. The Dr adhesion expression system constructed opens up a lot of possibilities, and could be very useful in experiments focusing on understanding the biogenesis of Gram-negative bacteria adhesins. For this purpose we showed that the AfaE-III adhesin (98.1% identity between the DraE and the AfaE-III adhesins, with three divergent amino acids within the sequences) was able to pass through the DraC channel in the Escherichia coli BL21(DE3) strain. Immunoblotting analysis and immunofluorescence microscopy showed the presence of AfaE-III on the bacterial cell surface. In addition, the system described can be useful for displaying the immune-relevant sectors of foreign proteins on the bacterial cell. The heterologous epitope sequence of the HSV1 glycoprotein D was inserted into the draE gene in place of the N-terminal region of surface exposed domain 2. Chimeric proteins were exposed on the bacterial surface as evidenced by immunoblotting and immunofluorescence microscopy. The effective display of peptide segments on Dr fimbriae expressed at the bacterial cell surface, can be used for the development of a fimbrial vaccine.     

Fimbriae and adherence of Stenotrophomonas maltophilia to epithelial cells and to abiotic surfaces

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen associated with several infectious diseases and opportunistic infections, especially in immunocompromised patients. These bacteria adhere avidly to medical implants and catheters forming a biofilm that confers natural protection against host immune defences and different antimicrobial agents. The nature of the bacterial surface factors involved in biofilm formation on inert surfaces and in adherence of S. maltophilia to epithelial cells is largely unknown. In this study, we identified and characterized fimbrial structures produced by S. maltophilia grown at 37 degrees C. The S. maltophilia fimbriae 1 (SMF-1) are composed of a 17 kDa fimbrin subunit which shares significant similarities with the N-terminal amino acid sequences of several fimbrial adhesins (G, F17, K99 and 20K) found in Escherichia coli pathogenic strains and the CupA fimbriae of Pseudomonas aeruginosa. All of the clinical S. maltophilia isolates tested produced the 17 kDa fimbrin. Antibodies raised against SMF-1 fimbriae inhibited the agglutination of animal erythrocytes, adherence to HEp-2 cells and biofilm formation by S. maltophilia. High resolution electron microscopy provided evidence of the presence of fimbriae acting as bridges between bacteria adhering to inert surfaces or to cultured epithelial cells. This is the first characterization of fimbriae in this genus. We provide compelling data suggesting that the SMF-1 fimbriae are involved in haemagglutination, biofilm formation and adherence to cultured mammalian cells.     

Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences

A strategy has been designed for the construction of recombinant bacterial strains which eventually may become useful as live vaccines and which may also be relevant for the preparation of conventional vaccines. The approach used is the fusion of small antigenic peptide sequences into specific segments of a protein whose location on the bacterial surface ensures that the recombinant organism is able to present the inserted antigen to the host (animal or human) infected by the bacterium. The chosen surface protein is a naturally occurring polymer of Escherichia coli, viz., type 1 fimbriae. The results obtained show that fusion of such foreign sequences into selected points of the structural protein of the fimbriae results in the production of functionally normal type 1 fimbriae. Furthermore, hybrid fimbriae carrying such small epitope sequences can be recognized by antibodies directed against the foreign parent protein. This observation is an important prerequisite for the eventual design of useful vaccines. The analysis of the fimbrial protein and its potential as a carrier of foreign peptides from hepatitis B surface antigen, foot-and-mouth disease virus and poliovirus indicated that there may be several positions in the protein which may turn out to be relevant for this purpose and be important fusion sites.     

Pili in gram-positive pathogens

Most bacterial pathogens have long filamentous structures known as pili or fimbriae extending from their surface. These structures are often involved in the initial adhesion of the bacteria to host tissues during colonization. In gram-negative bacteria, pili are typically formed by non-covalent interactions between pilin subunits. By contrast, the recently discovered pili in gram-positive pathogens are formed by covalent polymerization of adhesive pilin subunits. Evidence from studies of pili in the three principal streptococcal pathogens of humans indicates that the genes that encode the pilin subunits and the enzymes that are required for the assembly of these subunits into pili have been acquired en bloc by the horizontal transfer of a pathogenicity island.     

Role of cranberry juice on molecular-scale surface characteristics and adhesion behavior of Escherichia coli

Cranberry juice has long been believed to benefit the prevention and treatment of urinary tract infections (UTIs). As the first step in the development of infection, bacterial adhesion is of great research interest, yet few studies have addressed molecular level adhesion in this context. P-fimbriated Escherichia coli play a major role in the development of a serious type of UTI, acute pyelonephritis. Experiments were conducted to investigate the molecular-scale effects of cranberry juice on two E. coli strains: HB101, which has no fimbriae, and the mutant HB101pDC1 which expresses P-fimbriae. Atomic force microscopy (AFM) was used to investigate both bacterial surface characteristics and adhesion forces between a probe surface (silicon nitride) and the bacteria, providing a direct evaluation of bacterial adhesion and interaction forces. Cranberry juice affected bacterial surface polymer and adhesion behavior after a short exposure period (<3 h). Cranberry juice affected the P-fimbriated bacteria by decreasing the adhesion forces between the bacterium and tip and by altering the conformation of the surface macromolecules on E. coli HB101pDC1. The equilibrium length of polymer (P-fimbriae) on this bacterium decreased from approximately 148 to approximately 48 nm upon being exposed to cranberry juice. Highly acidic conditions were not necessary for the prevention of bacterial adhesion, since neutralization of cranberry juice solutions to pH = 7.0 allowed us to observe differences in adhesion between the E. coli strains. Our results demonstrate molecular-level changes in the surfaces of P-fimbriated E. coli upon exposure to neutralized cranberry juice.