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.     

Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits. We made a construct for the expression of recombinant one-chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active. The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers. Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar.     

Characterization of type 1 and mannose-resistant fimbriae of Erwinia spp.

Type 1 fimbriae from Erwinia carotovora subsp. carotovora and mannose-resistant fimbriae from Erwinia rhapontici were purified and characterized. The type 1 fimbrillin had an apparent molecular weight of 16,500; that of the mannose-resistant fimbrillin was 18,000. The amino-terminal amino acid sequences of the two fimbrillins were related, but tryptic peptide maps showed significant differences between the proteins. No serological cross-reaction was found between the two fimbrial filaments, nor did they cross-react with type 1 or type 3 fimbriae purified from other enterobacterial species. Immunofluorescent staining of bacterial populations revealed that they were heterogeneous with respect to fimbriation.     

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.     

The pma1 and pma2 H(+)-ATPases from Schizosaccharomyces pombe are functionally interchangeable.

The pma2 gene of Schizosaccharomyces pombe codes for a polypeptide having a predicted Mr of 110,126 and which is 79% identical to the plasma membrane H(+)-ATPase encoded by the pma1 gene. The pma2 gene, unlike pma1, is weakly expressed and not essential to mitotic growth. By constructing yeast strains in which the chromosomal pma2 gene is under control of the adh promoter, it has been possible to identify the overproduced ATPase in plasma membrane via formation of a phosphoenzyme. In a pma1-1 mutant strain whose ATPase activity is insensitive to vanadate, the overexpressed pma2 gene restores vanadate sensitivity. It also rescues a pma1 null mutant from lethality. These results demonstrate that the two H(+)-ATPases are functionally interchangeable in vivo but differently expressed.     

Immunoelectron microscopic analysis of elongation of type 1 fimbriae in Escherichia coli.

Using 10- and 20-nm-diameter gold particles conjugated to an antifimbrial monoclonal antibody, we analyzed the location of assembly of newly formed subunits on growing type 1 fimbriae of Escherichia coli. Fimbriae were removed from an E. coli K-12-derived strain, CSH50, by blending. Blended cells were allowed to regenerate their fimbriae in growth medium for approximately 25 min, after which they were labeled with a 20-nm-gold-monoclonal antibody probe. Continued outgrowth of these labeled fimbriae was allowed for additional time intervals, after which they were labeled with a 10-nm-gold-monoclonal antibody probe. The resulting fimbriae, double labeled with 10- and 20-nm-diameter gold particles, were examined in an electron microscope. The pattern of labeling on individual fimbrial organelles indicated morphologically that newly synthesized subunits are added to a growing organelle at its base.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

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.     

Role of type 1 and type 3 fimbriae on the adherence and pathogenesis of Salmonella enteritidis in mice.

Through hemagglutination tests two isogenic strains of Salmonella enteritidis were shown to possess type 1 fimbriae (strain V) and type 1 and type 3 fimbriae (strain A). The two strains bound to human buccal and mouse small intestine epithelial cells. Strain A attached to the epithelial cells more readily and in larger numbers in comparison to strain V. Adherence of both strains were sensitive to the presence of D-mannose and pretreatment of the epithelial cells with tannic acid did not promote D-mannose resistant type binding of strain A S. enteritidis to human buccal and mouse small intestine epithelial cells. Furthermore, results from LD50 study indicated that, when the tests were carried out through oral inoculation of the mice the highly fimbriated stain A appeared to be more virulent. However, when the tests were carried out through intraperitoneal inoculation strain V was more virulent. These results indicate that adherence is a major contributing factor to the virulence of S. enteritidis and both type 1 and type 3 fimbriae contribute to this phenomenon.     

Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.     

The PARP and rRNA promoters of Trypanosoma brucei are composed of dissimilar sequence elements that are functionally interchangeable.

The African trypanosomes express two major surface proteins, the variant surface glycoprotein (VSG) and the procyclic acidic repetitive protein (PARP). The RNA polymerase that transcribes the VSG and PARP genes shares many characteristics with RNA polymerase I. We show that although there is very little similarity in nucleotide sequence, the functional structure of a trypanosome rRNA promoter is almost identical to that of the PARP promoter. Further, domains from the PARP promoter can functionally substitute for the corresponding parts of the rRNA promoter, and vice versa.     

Graft-vs-host reactions in F1 mice induced by parental lymphoid cells: nature of recruited F1 cells.

Graft versus host (GVH) reactivity of parental lymph node (LN) cells was assayed by measurements of 3H-thymidine incorporation in vivo. Mitomycin (Mit.) treatment of parental cells abolished their proliferative activity but the combination of such Mit.-treated parental cells with F1 LN cells resulted in much higher proliferation than either one population alone. This recruitment into proliferation of F1 cells was prominent on days 3 and 4 after cell injection and amounted to 35 to 51% of the total activity seen after injection of untreated parental cells alone. The F1 cell sensitive to recruitment was resistant to anti-Thy 1.2 treatment, was not removed by carbonyl iron-magnet separation; and was not present in thymus. The parental cell inducing recruitment was, however, sensitive to anti-Thy 1.2. When spleen cells from hapten immune F1 donors were injected together with Mit.-treated parental LN cells and boosted with hapten on another carrier, a typical "allogeneic effect" was observed in the anti-hapten immune response. It was concluded that Mit.-treated parental T cells exerted a mitogenic effect on F1 B cells resulting in extensive recruitment similar to that seen in murine mixed lymphocyte reactions.     

Location of binding sites on common type 1 fimbriae from Escherichia coli.

Common type 1 fimbriae were isolated from Escherichia coli and their length distribution profile was determined before and after treatment with ultrasound. As fimbriae were shortened, so their haemagglutinating capacity decreased, but their ability to bind to erythrocytes did not decrease to the same extent. Isolated fimbriae did not agglutinate inside-out vesicles prepared from horse erythrocytes or liposomes, suggesting that the binding mechanism was not based on non-specific hydrophobic interactions. The results support a lateral rather than a terminal location for the fimbrial binding site responsible for haemagglutination.     

Identification of major and minor chaperone proteins involved in the export of 987P fimbriae.

The 987P fimbriae of Escherichia coli consist mainly of the major subunit, FasA, and two minor subunits, FasF and FasG. In addition to the previously characterized outer membrane or usher protein FasD, the FasB, FasC, and FasE proteins are required for fimbriation. To better understand the roles of these minor proteins, their genes were sequenced and the predicted polypeptides were shown to be most similar to periplasmic chaperone proteins of fimbrial systems. Western blot (immunoblot) analysis and immunoprecipitation of various fas mutants with specific antibody probes identified both the subcellular localizations and associations of these minor components. FasB was shown to be a periplasmic chaperone for the major fimbrial subunit, FasA. A novel periplasmic chaperone, FasC, which stabilizes and specifically interacts with the adhesin, FasG, was identified. FasE, a chaperone-like protein, is also located in the periplasm and is required for optimal export of FasG and possibly other subunits. The use of different chaperone proteins for various 987P subunits is a novel observation for fimbrial biogenesis in bacteria. Whether other fimbrial systems use a similar tactic remains to be discovered.     

Kinetic analysis of the synthesis and assembly of type 1 fimbriae of Escherichia coli.

The adhesive organelles (type 1 fimbriae) of K-12 and other isolates of Escherichia coli are composed of identical 17,000-dalton subunits. We examined the assembly of these subunits into fimbrial organelles. After synthesis, the nascent subunits were first processed and then assembled into the organelles; the assembly step took almost 3 min in log-phase cultures at 37 degrees C. Even during blockage of protein synthesis, the free subunits continued to assemble until the pool was depleted. This pool was small in comparison with the amount of total fimbrial protein already assembled into surface organelles and was not sufficient to regenerate new detectable organelles after the removal of preexistent ones by blending. Assembly appeared to slow when the metabolic rate of the bacterial cells slowed, since subunits took longer to appear in the organelles at lower than optimal temperatures or as a culture entered the stationary phase. The synthetic rate of subunits slowed sooner than that of total cellular proteins as a culture approached the stationary phase and ceased completely as the culture entered the stationary phase. The amount of fimbrial antigen expressed on the surface of the cells remained relatively constant during growth of a culture.     

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.