Type 3 fimbriae of Klebsiella sp.: molecular characterization and role in bacterial adhesion to plant roots.

Type 3 fimbriae of Klebsiella were purified and characterized. The fimbriae were 4 to 5 nm in diameter and 0.5 to 2 microns long. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the fimbrillin had an apparent molecular weight of 23,500, and it differed from enterobacterial type 1 fimbrillins in its amino acid composition. Hydrophobic amino acids comprised 33.6% of all amino acids in the fimbrillin, which lacked cystine, phenylalanine, and arginine. Serologically, the type 3 fimbriae were also distinct from the type 1 fimbriae. Purified type 3 fimbriae agglutinated tannin-treated human blood group O erythrocytes; this confirms the role of type 3 fimbriae as hemagglutinins. Purified 125I-labeled type 3 fimbriae bound to the roots of Poa pratensis, and this binding could be inhibited by Fab fragments to the purified fimbriae. Anti-type 3 fimbriae Fab fragments also inhibited bacterial adhesion to plant roots. These results demonstrate that type 3 fimbriae mediate adhesion of klebsiellas to plant roots. Eight nitrogen-fixing strains of Klebsiella also produced type 3 fimbriae when grown under anaerobic nitrogen fixation conditions. It is proposed that type 3 fimbriae are involved in the establishment of the plant-bacterium association concerning nitrogen-fixing Klebsiella strains.     

Do type 1 fimbriae promote inflammation in the human urinary tract?

Type 1 fimbriae have been implicated as virulence factors in animal models of urinary tract infection (UTI), but the function in human disease remains unclear. This study used a human challenge model to examine if type 1 fimbriae trigger inflammation in the urinary tract. The asymptomatic bacteriuria strain Escherichia coli 83972, which fails to express type 1 fimbriae, due to a 4.25 kb fimB-fimD deletion, was reconstituted with a functional fim gene cluster and fimbrial expression was monitored through a gfp reporter. Each patient was inoculated with the fim+ or fim- variants on separate occasions, and the host response to type 1 fimbriae was quantified by intraindividual comparisons of the responses to the fim+ or fim- isogens, using cytokines and neutrophils as end-points. Type 1 fimbriae did not promote inflammation and adherence was poor, as examined on exfoliated cells in urine. This was unexpected, as type 1 fimbriae enhanced the inflammatory response to the same strain in the murine urinary tract and as P fimbrial expression by E. coli 83972 enhances adherence and inflammation in challenged patients. We conclude that type 1 fimbriae do not contribute to the mucosal inflammatory response in the human urinary tract.     

Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes

The adhesion of Escherichia coli to eukaryotic cells is mediated by proteinaceous surface appendages called fimbriae and complementary receptors on host cells. Although type 1 fimbriae, which contain a D-mannose-reactive lectin, have been well studied little is known about the binding mechanism of isolated fimbriae to individual cell receptors. This report describes the isolation and purification of a guinea pig erythrocyte receptor for type 1 fimbriae. Erythrocyte membranes were dissolved in 0.5% Triton X-100 and the receptor isolated and purified by affinity chromatography using type 1 fimbriae immobilized on Sepharose. The 65-kDa receptor, which inhibits the agglutination of guinea pig erythrocytes by type 1 fimbriated E. coli, has a pI of 8.5-8.7, and binds concanavalin A and type 1 fimbriae in a dose-dependent and saturable manner. The fimbrial binding activity of the receptor was reduced when treated with sodium metaperiodate, endoglycosidase H, trypsin, and V8 protease, suggesting the isolated receptor is a glycoprotein with N-linked carbohydrate units. Isolated type 1 fimbriae inhibited the binding of fimbriated E. coli to purified receptor in a dose- and time-related fashion. The calculated binding affinity was 6 X 10(6) M-1, a value consistent with the low binding affinity expected from previous studies of the agglutination of guinea pig erythrocytes by isolated type 1 fimbriae.     

Structural and genetic characterization of the Shigella boydii type 13 O antigen

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coli constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coli and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected 1H, 13C and 1H, 31P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.     

Structural and genetic characterization of the Shigella boydii type 10 and type 6 O antigens

Comparison of the O antigens of Shigella boydii types 10 and 6 by chemical analysis and nuclear magnetic resonance spectroscopy showed that their structures are similar, with the only difference being the presence or absence of d-ribofuranose, which is the immunodominant sugar in S. boydii type 10. In S. boydii type 6, a residue previously reported as alpha-d-GlcpA, was shown to be beta-d-GlcpA as in S. boydii type 10. S. boydii types 10 and 6 are reported not to cross-react serologically, and the role of d-ribofuranose in the specificity of S. boydii was confirmed by making a mutant of type 10 that lacked d-ribofuranose. However, S. boydii type 11, which has a d-ribofuranose but with different linkage does show cross-reaction with type 10. The O-antigen gene loci of S. boydii types 10 and 6 were shown to be virtually identical except that orf8 (wbaM), which was confirmed as the ribofuranosyltransferase gene, is interrupted by IS629 in type 6. Therefore, it is proposed that the O-antigen gene cluster of S. boydii type 6 was derived from type 10 by an IS element insertion.     

Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.

Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.     

Virulence of Porphyromonas gingivalis is altered by substitution of fimbria gene with different genotype

Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and DeltafimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor alpha5beta1-integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL-1beta and IL-6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.     

Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose-sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline-sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim-lip conditional mutant synthesized type 1 fimbriae when grown in Mueller-Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independently isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim-lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA- fimbriae. The HA+ phenotype of both conditional and non-fimbriated mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.     

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.     

The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit.

The role of type 1 fimbriae in the mannose-sensitive attachment of Escherichia coli to eucaryotic cells was investigated by deletion mutation analysis of a recombinant plasmid, pSH2, carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. A mutant, pUT2002, containing a deletion remote from the structural gene encoding the 17-kilodalton subunit protein of type 1 fimbriae failed to agglutinate guinea pig erythrocytes even though the bacteria expressed fimbriae morphologically and antigenically indistinguishable from those produced by the intact recombinant plasmid. Fimbriae isolated from pUT2002 failed to agglutinate guinea pig erythrocytes, but reacted with a monoclonal antibody specific for quaternary structural determinants of type 1 fimbriae. Moreover, the dissociated fimbrial subunits from this mutant were indistinguishable from normal fimbriae by their migration during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, by their reactivity with a monoclonal antibody directed against a subunit-specific epitope, and in enzyme-linked immunosorbent assays with monospecific antisera. These results indicate that the adhesive functions in type 1 fimbriae are dependent on a factor(s) encoded by a gene other than those required for synthesis, assembly, and expression of the structural 17-kilodalton subunit.     

Adherence and pathogenesis of Salmonella enteritidis in mice

Adherence of many pathogenic organisms to the host cells has been associated with the presence of fimbriae. The exact role of these organelles in the adherence and pathogenesis of Salmonella enteritidis is not well established. Utilizing hemagglutination tests, S. enteritidis was shown to possess type 1 and type 3 fimbriae. Polyacrylamide gel electrophoresis of the isolated fimbriae showed that type 1 and 3 fimbriae of S. enteritidis had subunit M.r of 17 and 22 kDa, respectively. In vitro adherence assays suggested that S. enteritidis utilized type 1 fimbriae to adhere to human buccal and mouse small intestine epithelial cells. In addition, antibody produced against type 1 and type 3 fimbriae protected the mice from infection with a lethal dose of S. enteritidis. These results suggest that type 1 and possibly type 3 fimbriae are involved in the adherence and pathogenesis of S. enteritidis. The data further suggest that they may have a role in the adherence and pathogenesis of the other enteric organisms.     

Structural and genetic characterization of Shigella boydii type 17 O antigen and confirmation of two new genes involved in the synthesis of glucolactilic acid

Shigella strains are human pathogens and normally identified based on their O antigens. The chemical structure and gene cluster of Shigella boydii type 17 O antigen were studied. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen of S. boydii type 17 has a linear trisaccharide O unit, which consists of two residues of N-acetylgalactosamine (GalNAc) and a 4-O-[(R)-1-carboxyethyl]-d-glucose (glucolactilic acid). The O antigen gene cluster of S. boydii type 17 was sequenced and genes encoding UDP-N-acetylglucosamine C4 epimerase for GalNAc synthesis, O unit flippase, O antigen polymerase, and glycosyltransferases were putatively identified based on sequence similarities and the presence of conserved motifs. Two genes, whose functions could not be clearly indicated by homology search, were confirmed to be involved in the synthesis of glucolactilic acid by mutation and structural verification of the O antigens from the mutants. To our knowledge, this is the first time that genes involved in the synthesis of glucolactilic acid have been reported. Two genes specific to S. boydii type 17 were also identified.     

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.     

Reciprocal exchange of minor components of type 1 and F1C fimbriae results in hybrid organelles with changed receptor specificities.

Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found on strains associated with urinary tract infections. The specific receptor binding of the fimbriae is due to the presence of receptor recognition proteins present in the organelles as minor structural elements. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structures of the organelles, are very similar, although the actual sequence homology of the structural elements is not remarkable; notably, the sequence identity between the minor components of the type 1 and F1C fimbriae is only 34 to 41%. Type 1 fimbriae mediate agglutination of guinea pig erythrocytes, whereas F1C fimbriae do not confer agglutination of any types of erythrocytes tested. However, F1C fimbriae mediate specific adhesion to epithelial cells in the collecting ducts of the human kidney as well as to cells of various cell lines. This report addresses the question of fimbrial promiscuity. Our data indicate that minor fimbrial structural elements can be exchanged between the two fimbrial systems, resulting in hybrid organelles with changed receptor specificity. This is the first study on reciprocal exchange of structural components from two different fimbrial systems.     

Assembly of a chemically synthesized peptide of Escherichia coli type 1 fimbriae into fimbria-like antigenic structures.

Escherichia coli type 1 fimbriae are composed of subunits, each of which comprises 158 amino acids. We synthesized a copy of a 13-residue peptide, located near the NH2 terminus of the fimbrial subunit, that assumed some of the properties of type 1 fimbriae. At pH 5.5 the synthetic peptide autoassembled into fibrillar structures that resembled type 1 fimbriae except that they appeared less rigid and rodlike. A quaternary structure-specific monoclonal antibody against type 1 fimbriae recognized the synthetic peptide in the assembled but not the unassembled state. Furthermore, when the synthetic peptide was injected in its fimbrial conformation into rabbits, it evoked antibodies that reacted with type 1 fimbriae isolated from E. coli.     

Fragmentation of Escherichia coli type 1 fimbriae exposes cryptic D-mannose-binding sites.

Cells of the gram-negative bacterium Escherichia coli are able to attach to various host cells by means of a mannose-specific adhesin associated with type 1 fimbriae. Here we show that fragmentation of type 1 fimbriae by freezing and thawing results in increased mannose-binding activity as demonstrated by increased hemagglutination, increased stimulation of human lymphocyte proliferation, and increased binding of the mannose-containing enzyme horseradish peroxidase. Increased activity in all three assays was mannose sensitive and was not exhibited by FimH- mutant type 1 fimbriae lacking the adhesin. Scatchard analysis of the data from peroxidase binding assays showed that unfrozen and frozen fimbriae contain binding sites displaying two classes of affinity. Frozen and thawed fimbriae expressed an increase in the number of high-affinity binding sites. These results show that fragmentation of the fimbrial structure exposes cryptic mannose-binding activity associated with type 1 fimbriae, presumably that of internally located adhesin molecules. Our data support earlier observations that adhesin moieties of type 1 fimbriae are located both at the tips and at intervals along the length of the fimbriae. In addition, our data suggest that only the adhesin moieties that are located at the fimbrial tips are functional in binding mannose. Adhesins located along the length of the fimbriae have their mannose-binding activity buried within the fimbrial structure and hence are not functional. We propose an updated model for the structure of type 1 fimbriae that is in agreement with the above observations.     

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.     

Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization

Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications.     

Type 1 fimbria-mediated adhesion of enteric bacteria to grass roots

Type 1 fimbriae of Klebsiella pneumoniae and Enterobacter agglomerans mediated bacterial adhesion to the roots of bluegrass, Poa pratensis. Purified, radiolabeled fimbriae bound to grass roots in vitro; binding was inhibited by alpha-methyl-d-mannoside or Fab fragments to the fimbriae. Anti-type 1 fimbriae Fab fragments and alpha-methyl-d-mannoside also inhibited adhesion of type 1-fimbriated bacteria to P. pratensis roots. It is proposed that associative nitrogen fixation by Klebsiella and Enterobacter strains also involves type 1 fimbriae, in addition to the type 3 fimbriae of Klebsiella spp. (T. K. Korhonen, E. Tarkka, H. Ranta, and K. Haahtela, J. Bacteriol. 155:860-865, 1983).     

Mitogenic stimulation of human B lymphocytes by the mannose-specific adhesin on Escherichia coli type 1 fimbriae

Escherichia coli type 1 fimbriae contain in association with the major structural protein a lectin-like adhesin moiety that mediates attachment of E. coli to mannose-containing receptors on the surface of host cells. We have investigated the lymphocyte mitogenic activity of this mannose-specific adhesin by comparing the ability of purified wild type type 1 fimbriae containing the adhesin and mutant type 1 fimbriae lacking the adhesin to stimulate proliferation in human lymphocytes. Both fimbriae stimulated a peak of proliferation at 8 days whereas only the wild type fimbriae stimulated an additional peak of proliferation occurring at 3 days. Proliferation at 3 days but not at 8 days could be blocked by the addition of alpha-methyl-D-mannoside. Neonatal lymphocytes from umbilical cord blood responded to both wild type and mutant fimbriae in a fashion similar to adult cells. Stimulation of separated T and non-T cell populations indicated that the proliferation seen at 3 days was solely due to non-T cells whereas the 8-day response was due to T cell proliferation. The addition of gamma-irradiated T cells did not appear to enhance the 3-day response of the non-T cells. However, the 8-day response by T cells was dependent on the presence of gamma-irradiated non-T cells. In cultures of unseparated cells, wild type fimbriae stimulated more than 75% of the B cells to enter the S and G2 phase at 3 days whereas at 8 days cycling T cells were present in both wild type and mutant fimbriae-stimulated cultures. Taken together, our observations suggest that the adhesin molecule stimulates a polyclonal mitogenic response in B cells that peaks at 3 days, and other structural components of the fimbriae are responsible for evoking an 8-day (probably immune) response in T cells.