Kinetics of phase variation between S and type-1 fimbriae of Escherichia coli

Abstract The rate of fimbrial phase variation in Escherichia coli strain 3040 was determined. The strain has type-1 and S fimbriae. The bacterial culture was fractionated into homogeneous subpopulations expressing either one of the fimbrial types only; the subpopulations were inoculated into broth and the fimbriation of individual cells was assayed by immunofluorescence as a function of time. The rate of the shift from S- or type-1-fimbriate cells to non-fimbriate ones was of the order of 10⁻² per cell generation and more rapid than a direct shift from one fimbrial phase to another, although both types of phase variations were observed.     

Immunofluorescence study of fimbrial phase variation in Escherichia coli KS71

An immunofluorescence assay was developed to study fimbrial phase variation in a pyelonephritogenic Escherichia coli strain, KS71. By using fluorochrome-labeled antibodies specific for either P, type-1C, or type-1 fimbriae of strain KS71, it was shown that in a broth culture of strain KS71 the fimbrial types mostly occurred on different cells. Only 9% of the cells carried more than one fimbrial type. The KS71 cell population was fractionated into subpopulations expressing only one of the fimbrial types or lacking fimbriae. Immunofluorescence assay of the subpopulations revealed a rapid phase variation in fimbrial synthesis. Kinetic analyses of a nonfimbriated cell population suggested that a change from one fimbrial phase to another was not totally random.     

Occurrence of type-1C fimbriae on Escherichia coli strains isolated from human extraintestinal infections

Two monoclonal antibodies specific for type-1C fimbriae of Escherichia coli were produced. In enzyme-linked immunosorbent assay and immunoblotting the antibodies, which were of the IgG1 isotype, reacted with type-1C, but not with P or type-1 fimbriae of E. coli strain KS71. Immunoblotting and immunoprecipitation of crude fimbrial extracts from 25 strains invariably gave an apparent molecular weight of 17 000 for the type-1C fimbrillin. A total of 313 E. coli strains, isolated from patients with extraintestinal infection or from faeces of healthy children, were screened for the presence of type-1C fimbriae using both the monoclonal and polyclonal antibodies. Of these, 45 (14%) strains had type-1C fimbriae, with the highest frequency (27%) on strains isolated from patients with pyelonephritis. No faecal strain had type-1C fimbriae, and the frequency on the other diagnostic groups ranged from 11 to 15%. Thus, no direct correlation between type-1C fimbriae and bacterial virulence in human extraintestinal infections was found. Type-1C fimbriae were detected on only a few E. coli serotypes, notably on all O6:K2:H1 and O22:K13:H1 strains tested.     

Organization of fimbriate cells in colonies of Escherichia coli strain 3040

Immunofluorescence staining with fimbria-specific antibodies was used to study the organization of fimbriate cells in colonies of Escherichia coli strain 3040. The strain has both type-1 and S fimbriae and shows fast phase variation between the fimbrial types. Colonies stained in sectors whose length and number per colony were dependent on the fimbrial phase of progeny cells. It is proposed that such sectors result from fimbrial phase variation.     

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue-binding specificity of the type-3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild-type Klebsiella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type-3 fimbriae, as well as the purified type-3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type-3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type-3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type-3 fimbrial gene cluster in association with the pap-encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type-3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type-3 fimbriate strains of Yersinia and Salmonella, showing that the ability to use type V collagen as tissue target is widespread among enteric bacteria.     

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin

Adherence of type-1-fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type-1-fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non-fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside-inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae-coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type-1 -fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type-1-fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type-1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type-1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.     

Molecular basis for the enterocyte tropism exhibited by Salmonella typhimurium type 1 fimbriae

Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae. The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces. The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression. Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested. To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE. The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E. coli fimbriae. Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S. typhimurium type 1 fimbriae.     

Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit.

Each of five monoclonal antibodies (mAbs) prepared against the type 1 fimbriae of Actinomyces viscosus T14V reacted with a 54 kDa cloned protein previously identified as a fimbrial subunit. This purified protein completely inhibited the reaction of a specific anti-type-1-fimbria rabbit antibody with A. viscosus whole cells. Maximum values for the number of antibody molecules bound per bacterial cell ranged from 7 x 10(3) to 1.2 x 10(4) for the different 125I-labelled mAbs and was approximately 7 x 10(4) for 125I-labelled rabbit IgG or Fab against either type 1 fimbriae or the 54 kDa cloned protein. Although the different mAbs, either individually or as a mixture, failed to inhibit the type-1-fimbria-mediated adherence of A. viscosus T14V to saliva-treated hydroxyapatite, each rabbit antibody gave 50% inhibition of adherence when approximately 5 x 10(4) molecules of IgG were bound per cell. However, binding of each corresponding rabbit Fab had no significant effect on bacterial attachment unless much higher concentrations were used. These findings suggest that antibodies directed solely against the 54 kDa fimbrial subunit do not react with the putative receptor binding sites of A. viscosus T14V type 1 fimbriae. Instead, inhibition of attachment by the polyclonal antibodies may depend on an indirect effect of antibody binding that prevents the fimbria-receptor interaction.     

complex interplay between type 1 fimbrial expression and flagellum-mediated motility of uropathogenic Escherichia coli

Type 1 fimbriae and flagella have been previously shown to contribute to the virulence of uropathogenic Escherichia coli (UPEC) within the urinary tract. In this study, the relationship between motility and type 1 fimbrial expression was tested for UPEC strain CFT073 by examining the phenotypic effect of fimbrial expression on motility and the effect that induction of motility has on type 1 fimbrial expression. While constitutive expression of type 1 fimbriae resulted in a significant decrease in motility and flagellin expression (P < 0.0001), a loss of type 1 fimbrial expression did not result in increased motility. Additionally, hypermotility and flagellar gene over- and underexpression were not observed to affect the expression of type 1 fimbriae. Hence, it appeared that the relationship between type 1 fimbrial expression and motility is unidirectional, where the overexpression of type 1 fimbriae dramatically affects motility and flagellum expression but not vice versa. Moreover, the constitutive expression of type 1 fimbriae in UPEC cystitis isolate F11 and the laboratory strain E. coli K-12 MG1655 also resulted in decreased motility, suggesting that this phenomenon is not specific to CFT073 or UPEC in general. Lastly, by analyzing the repression of motility caused by constitutive type 1 fimbrial expression, it was concluded that the synthesis and presence of type 1 fimbriae at the bacterial surface is only partially responsible for the repression of motility, as evidenced by the partial restoration of motility in the CFT073 fim L-ON DeltafimAICDFGH mutant. Altogether, these data provide further insight into the complex interplay between type 1 fimbrial expression and flagellum-mediated motility.     

Type II secretory pathway for surface secretion of DraD invasin from the uropathogenic Escherichia coli Dr+ strain

The virulence of the uropathogenic Escherichia coli Dr(+) IH11128 strain is associated with the presence of Dr fimbrial structures and a DraD invasin which can act as a fimbrial capping domain at the bacterial cell surface. However, a recent study suggests that the DraD protein is surface exposed in two forms: fimbria associated and fimbria nonassociated (prone to interaction with the N-terminal extension of the DraE protein located on the fimbrial tip). The actual mechanism of DraD surface secretion is presently unknown. We identified a previously unrecognized type II secretory pathway (secreton) in the uropathogenic E. coli Dr(+) strain which is well conserved among gram-negative bacteria and used mainly for secretion of virulence determinants. An active secreton is composed of 12 to 15 different proteins, among which GspD functions as an outer-membrane channel to permit extrusion of proteins in a folded state. Therefore, we inactivated the pathway by inserting the group II intron into a gspD gene of the type II secretion machinery by site-specific recombination. DraD secretion by the E. coli Dr(+) and gspD mutant strains was determined by immunofluorescence microscopy (with antibodies raised against DraD) and an assay of cell binding between bacteria and HeLa cells. The specificity of DraD-mediated bacterial binding for the integrin receptor was confirmed by examination of the adhesion of DraD-coated beads to HeLa cells in the presence and absence of alpha(5)beta(1) monoclonal antibodies. The investigations that we performed showed that type II secretion in E. coli Dr(+) strains leads to DraD translocation at the bacterial cell surfaces.     

Immunological and genetical relatedness of type-1 and type-2 fimbriae in salmonellas of serotypes Gallinarum, Pullorum and Typhimurium

The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial antiserum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.     

Immunological and genetical relatedness of type-1 and type-2 fimbriae in salmonellas of serotypes Gallinarum, Pullorum and Typhimurium

The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial anti-serum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.     

K88大肠杆菌菌毛蛋白发酵工艺条件优化研究

分别采用LB培养基、牛肉膏蛋白胨培养基、强化营养培养基、玉米浆培养基对大肠杆菌K88进行发酵培养,选出最适于大肠杆菌K88生长的玉米浆培养基;采用正交实验对玉米浆培养基的C／N、K2HPO4／KH2PO4、Mg^2＋的配比进行优化,筛选最适于大肠杆菌K88生长的营养配比;研究生长曲线、接种量以及菌体和菌毛生产量的相关性,根据实验结果优化发酵培养条件,确定菌种的最佳发酵工艺,以收获最多的K88菌毛蛋白。研究表明,K88大肠杆菌在玉米浆培养基C／N、K2HP04／KH2PO4的配比分别为5／11、1／1,Mg^2＋为0．1g/mL,pH值为7．2,转速为200r／min,接种量为4．5％的条件下发酵26个小时,菌体和菌毛生产量均达到高峰,同时得出菌毛蛋白产生量和菌体量成正相关。     

The distinct binding specificities exhibited by enterobacterial type 1 fimbriae are determined by their fimbrial shafts

Type 1 fimbriae of enterobacteria are heteropolymeric organelles of adhesion composed of FimH, a mannose-binding lectin, and a shaft composed primarily of FimA. We compared the binding activities of recombinant clones expressing type 1 fimbriae from Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium for gut and uroepithelial cells and for various soluble mannosylated proteins. Each fimbria was characterized by its capacity to bind particular epithelial cells and to aggregate mannoproteins. However, when each respective FimH subunit was cloned and expressed in the absence of its shaft as a fusion protein with MalE, each FimH bound a wide range of mannose-containing compounds. In addition, we found that expression of FimH on a heterologous fimbrial shaft, e.g. K. pneumoniae FimH on the E. coli fimbrial shaft or vice versa, altered the binding specificity of FimH such that it closely resembled that of the native heterologous type 1 fimbriae. Furthermore, attachment to and invasion of bladder epithelial cells, which were mediated much better by native E. coli type 1 fimbriae compared with native K. pneumoniae type 1 fimbriae, were found to be dependent on the background of the fimbrial shaft (E. coli versus K. pneumoniae) rather than the background of the FimH expressed. Thus, the distinct binding specificities of different enterobacterial type 1 fimbriae cannot be ascribed solely to the primary structure of their respective FimH subunits, but are also modulated by the fimbrial shaft on which each FimH subunit is presented, possibly through conformational constraints imposed on FimH by the fimbrial shaft. The capacity of type 1 fimbrial shafts to modulate the tissue tropism of different enterobacterial species represents a novel function for these highly organized structures.     

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.     

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.     

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.     

Penetration of fimbriate enteric bacteria through basement membranes: A hypothesis

A mechanism for penetration of basement membranes by Escherichia coli is presented. The mechanism is based on the ability of the S fimbriae of meningitis-associated E. coli to bind to vascular endothelium and choroid plexuses in brain and to basement membranes. On the other hand, the S and the type 1 fimbriae of E. coli immobilize plasminogen and tissue-type plasminogen activator; this process generates proteolytic plasmin activity on the surface of fimbriate cells. Our hypothesis is that bacterium-bound plasma activity, directed to basement membranes through fimbrial binding, promotes bacterial penetration through basement membranes.     

Bacterial lectins, cell-cell recognition and infectious disease

Numerous bacterial strains produce surface lectins, commonly in the form of fimbriae that are filamentous assemblies of protein subunits. Among the best characterized of these are the type 1 (mannose specific) fimbrial lectins of Escherichia coli that consist almost exclusively of one class of subunit with a molecular mass of 17 kDa. They possess an extended combining site corresponding to a trisaccharide and preferentially bind carbohydrate units of oligomannose or hybrid type. Type 1 fimbriae also possess a hydrophobic region close to the carbohydrate-binding site, since aromatic alpha-mannosides inhibit strongly (up to 1000-times more than methyl alpha-mannoside) the agglutination of yeasts by the bacteria and the adherence of the latter to pig ileal epithelial cells. The combining sites of type 1 fimbriae of the salmonellae and of other enteric bacteria are different from those of E. coli in that they are smaller and do not possess a hydrophobic region. The various bacterial surface lectins appear to function primarily in the initiation of infection by mediating bacterial adherence to epithelial cells, e.g. in the urinary and gastrointestinal tracts. The mannose specific lectins also act as recognition molecules in lectinophagocytosis (i.e. phagocytosis of the bacteria in the absence of opsonins) by mouse, rat and human peritoneal macrophages, and human polymorphonuclear leukocytes. Affinity chromatography of membrane lysates from human polymorphonuclear leukocytes on immobilized type 1 fimbrial lectin, using methyl alpha-mannoside as eluent, showed that glycoproteins with apparent molecular masses of 70-80, 100 and 150 kDa act as receptors for the bacteria. Inhibition experiments with monoclonal antibodies suggest that the glycoprotein bands of 100 and 150 kDa may be identical with the alpha and beta subunits of leukocyte complement receptors and adhesion glycoproteins involved in complement-mediated opsonophagocytosis. The systems described serve as a fine illustration for the biological role of lectin-carbohydrate interactions. Further studies of these systems will lead to a deeper understanding of the molecular basis of infectious diseases, and perhaps also to new approaches for their prevention.     

Role of fimbrial adhesins in the pathogenesis of Escherichia coli infections.

Escherichia coli strains are able to cause intestinal (enteritis, diarrhoeal diseases) and extraintestinal (urinary tract infections, sepsis, meningitis) infections. Most pathogenic E. coli strains produce specific fimbrial adhesins, which represent essential colonization factors: intestinal E. coli strains very often carry transferable plasmids with gene clusters specific for fimbrial adhesins, like K88 and K99, or colonization factor antigens (CFA) I and II. In contrast, the fimbrial gene clusters of extraintestinal E. coli strains, such as P, S, or F1C fimbriae, are located on the chromosomes. The fimbrial adhesin complexes consist of major and minor subunit proteins. Their binding specificity can generally be assayed in hemagglutination tests. In the case of fimbrial adhesins of intestinal E. coli strains, the major subunit proteins preferentially represent the hemagglutinating adhesins, whereas minor subunit proteins are the hemagglutinins of extraintestinal E. coli strains. Recently "alternative" adhesin proteins were identified, which have the capacity to bind to eukaryotic structures different from the receptors of the erythrocytes. Fimbrial adhesins are not constitutively expressed but are stringently regulated on the molecular level. Extraintestinal E. coli wild-type strains normally carry three or more fimbrial adhesin determinants, which have the capacity to influence the expression of one another (cross talk). Furthermore the fimbrial gene clusters undergo phase variation, which seems to be important for their contribution to pathogenesis of E. coli.