Biomedical subjects

J Hacker

Publications and source records attributed to J Hacker.

At least 37 records · Page 2Linked to original sources

PubMed1994

Adhesin regulatory genes within large, unstable DNA regions of pathogenic Escherichia coli: cross-talk between different adhesin gene clusters.

The uropathogenic Escherichia coli strain 536 possesses two large, unstable DNA regions on its chromosome, which were termed pathogenicity islands (pais). Deletions of pais, which occur with relatively high frequency in vitro and in vivo, lead to avirulent mutants. The genetic determinants for production of haemolysin (Hly) and P-related fimbriae (Prf) are located in one of these islands. Deletion of this pathogenicity island (paill) not only removes the hly- and prf-specific genes, but also represses S fimbriae (Sfa), although the sfa genes of this virulence factor are not located on paill. We have identified two regulatory genes, prfB and prfl, of the prf gene cluster that are homologous to the sfa regulatory genes sfaB and sfaC, respectively. Mutations in sfaB and sfaC that inhibit transcription of the major fimbrial subunit gene sfaA were complemented by the homologous prf genes, suggesting communication between the two fimbrial gene clusters in the wild-type strain. Chromosomal mutagenesis of the two prf regulators in strain 536 repressed transcription of sfaA, detected by Northern hybridization and a chromosomal sfaA-lacZ fusion. In addition, haemagglutination assays measured a lower level of S fimbriae in these mutants. Expression of the cloned prf regulators in trans reversed the effect of the mutations; furthermore, constitutive expression of prfB or prfl could also over-come the repression of S fimbriae in a strain that had lost the pathogenicity islands. Virulence assays in mice established that the prf mutants were less virulent than the wild-type strain. The results demonstrate that cross-regulation of two unlinked virulence gene clusters together with the co-ordinate loss of large DNA regions significantly influences the virulence of an extraintestinal E. coli wild-type isolate.

Amino Acid Sequence↗

PubMed1994

Emergence of fluoroquinolone-resistant Escherichia coli at a cancer center.

Prophylactic treatment with fluoroquinolones of patients with profound neutropenia has been found to be useful for preventing gram-negative bacteremia and has become a standard preventive-therapy strategy in many cancer centers, but the development of bacterial resistance is a cause of concern. During the past few years, we have observed an increasing number of patients with leukemia from whom fluoroquinolone-resistant strains of Escherichia coli were isolated. The increase was significant in this patient population, and among patients with other underlying diseases, the rates of isolation of such strains per number of discharges were significantly lower and did not increase. Most of the leukemia case patients (16 of 19) had been pretreated with an oral quinolone (ofloxacin), with cumulative doses until the first isolation of a resistant E. coli strain ranging from 0 to 97.8 g (median, 14.4 g). Repeated isolation of such strains was seen in 8 of 17 patients during a follow-up period of > or = 4 weeks and in 1 of 6 patients during a follow-up period of > or = 16 weeks. Ten patients developed bacteremia (mortality, 1 of 10). On the basis of the number of patients with leukemia admitted to the hematology-oncology service, the incidence of bacteremia caused by fluoroquinolone-resistant E. coli increased from < 0.5% in 1988-1989 and 0.8% in 1990-1991 to 4.5% in 1992-1993 (P < 0.01). MICs for nine isolates obtained from cultures of blood from different patients ranged between 8 and 16 microgram/ml (ciprofloxacin and PD 131628), 8 and 32 microgram/ml (ofloxacin and BAY Y 3118), and 16 and 32 microgram/ml (sparfloxacin) and indicated resistance to trimethoprim-sulfamethoxazole, ampicillin, doxycycline, and chloramphenicol. Of nine isolates obtained from cultures of blood from different patients and that were subjected to genomic DNA typing by pulsed-field gel electrophoresis of XbaI digests, seven were typeable. Among these, four different genotypes were identified, suggesting both the independent development and the horizontal spread of resistant clones of E. coli.

Anti-Infective Agents↗

PubMed1994

Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen.

Uropathogenic Escherichia coli 536 (O6:K15:H31) carries two unstable DNA regions, which were shown to be responsible for virulence. These regions, on which the genes for hemolysin production (hly) and P-related fimbriae (prf) are located, are termed pathogenicity islands (PAI) I and II, and were mapped to positions 82 and 97, respectively, on the E. coli K-12 linkage map. Sequence analysis of the PAI region junction sites revealed sequences of the leuX and selC loci specific for leucine and selenocysteine tRNAs. The tRNA loci function as the targets for excision events. Northern (RNA) blot analysis revealed that the sites of excision are transcriptionally active in the wild-type strain and that no tRNA-specific transcripts were found in the deletion mutant. The analysis of deletion mutants revealed that the excision of these regions is specific and involves direct repeats of 16 and 18 nucleotides, respectively, on both sides of the deletions. By using DNA long-range mapping techniques, the size of PAI I, located at position 82, was calculated to be 70 kb, while PAI II, mapped at position 97, comprises 190 kb. The excision events described here reflect the dynamics of the E. coli chromosome.

Base Sequence↗

PubMed1994

Sequence determination and mutational analysis of the lly locus of Legionella pneumophila.

The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus.

Amino Acid Sequence↗

PubMed1993

Adhesion of S-fimbriated Escherichia coli to brain glycolipids mediated by sfaA gene-encoded protein of S-fimbriae.

In an attempt to further assess the role of S-fimbriae in the pathogenesis of Escherichia coli meningitis, the adherence of E. coli strains with or without S-fimbriae were examined for this study to purified glycolipids using thin layer chromatography overlay assays. Only S-fimbriated E. coli strains bound to sulfatide, seminolipid, galactosyl ceramide, and lactosyl ceramide but not to gangliosides including sialyl neolacto-series and other neutral glycolipids. The binding of S-fimbriated E. coli to sulfatide was temperature dependent (i.e. maximal at 37 degrees C) and inhibited by S-fimbriae, anti-S-fimbriae, and anti-S-adhesin antibodies as well as by sulfatide, galactosyl ceramide, and lactosyl ceramide. E. coli transformants which lack the sfaA gene from the Sfa gene cluster showed no binding to the glycolipids, while other transformants lacking the adhesin gene sfaS or sfaG or H and mutants obtained by site-directed mutagenesis in the sfaS gene exhibited a similar binding to the glycolipids compared to the parent S-fimbriated strain. A large amount of sulfated glycolipids was demonstrated on brain endothelial cells and the binding of S-fimbriated E. coli to brain endothelial cells was inhibited by these glycolipids. These findings suggest that the binding of S-fimbriated E. coli to brain endothelial cells occurs in part via glycolipids containing terminal Gal(3SO4)beta-1 residues and in part by S-fimbriae protein SfaA. S-adhesin was not involved in the binding of S-fimbriae to these glycolipids.

Adhesins, Escherichia coli↗

PubMed1993

Clonal differentiation of uropathogenic Escherichia coli isolates of serotype O6:K5 by fimbrial antigen typing and DNA long-range mapping techniques.

Escherichia coli isolates of serotype O6:K5 are the most common causative agents of cystitis and pyelonephritis in adults. To answer the question, as to whether strains of this particular serotype represent one special clonal group, out of a collection of 34 serotype O6:K5 isolates [Zingler et al. (1990) Zentralbl. Bakteriol Mikrobiol Hyg [A] 274:372-381] 15 strains were selected and analyzed in detail. The flagellar (H) antigen and the outer membrane protein (OMP) pattern were determined. Further serum resistance properties and the genetic presence and expression of other virulence factors, including hemolysin, aerobactin, P fimbriae, S/F1C fimbriae and type 1 fimbriae was evaluated. In addition the XbaI-macrorestriction pattern of ten representative isolates was elaborated and the fimbrial (F) antigen type of the P fimbriae was determined, to obtain the complete O:K:H:F pattern. These analyses could clearly show that the O6:K5 isolates do not represent one clonal group. The XbaI-macrorestriction profiles were heterogeneous and marked differences in the hybridization patterns, using virulence-associated gene probes in Southern hybridization of long-range-separated genomic DNA, were observed among the strains. However, some of strains showed similarities in the genomic profiles, arguing for clonal groupings among the O6:K5 isolates. Interestingly the strains grouped together exhibited the same fimbrial F type that many indicate a coincidence of this phenotypic trait with clonality.

Adhesins, Escherichia coli↗

PubMed1993

Transcriptional analysis and regulation of the sfa determinant coding for S fimbriae of pathogenic Escherichia coli strains.

The sfa determinant codes for S fimbrial adhesins which constitute adherence factors of pathogenic Escherichia coli strains. We have recently shown that the sfa determinant is transcribed from three promoters, pA, pB, and pC. In comparison with the promoters pB and pC, promoter pA, which is located in front of the structural gene sfaA, showed very weak activity. Here we have determined the exact positions of the mRNA start points by primer extension studies. We have also shown that mRNAs of 500, 700 and 1400 bases can be detected using oligonucleotide probes specific for the genes sfaB, sfaC and sfaA. SfaB and SfaC are positive regulators influencing fimbriation and the production of the S-specific adhesin which is encoded by the gene sfaS located in the distal half of the determinant. In addition, it is demonstrated that SfaB and SfaC interfere with the regulatory effect of the histone-like protein H-NS, encoded by a locus termed drdX or osmZ. In a drdX+ strain the regulators are necessary for transcription of the sfa determinant. In contrast, sfa expression is activator-independent in a drdX- strain. In this latter genetic background, a substantial fraction of the sfa transcripts is initiated from promoter pA. On the basis of these data we discuss a model for the regulation of this adhesin-specific determinant.

Adhesins, Escherichia coli↗

PubMed1993

Regulation and binding properties of S fimbriae cloned from E. coli strains causing urinary tract infection and meningitis.

S fimbriae are able to recognize receptor molecules containing sialic acid and are produced by pathogenic E. coli strains causing urinary tract infection and menigitis. In order to characterize the corresponding genetic determinant, termed S fimbrial adhesin (sfa) gene cluster, we have cloned the S-specific genes from a urinary pathogen and from a meningitis isolate. Nine genes are involved in the production of S fimbriae, two of these, sfaB and sfaC code for regulatory proteins being necessary for the expression of S fimbriae. Two promoters, PB and PC, are located in front of these genes. Transcription of the sfa determinant is influenced by activation of the promoters via SfaB and SfaC, the action of the H-NS protein and an RNaseE-specific mRNA processing. In addition, a third promoter, PA, located in front of the major subunit gene sfaA, can be activated under special circumstances. Four genes of the sfa determinant code for the subunit-specific proteins, SfaA (16 kda), SfaG (17 kda), SfaS (14 kda) and SfaH (29 kda). It was demonstrated that the protein SfaA is the major subunit protein while SfaS is identical to the sialic-acid-specific adhesin of S fimbriae. The introduction of specific mutations into sfaS revealed that a region of six amino acids of the adhesin which includes two lysine and one arginine residues is involved in the receptor specific interaction of S fimbriae. Additionally, it has been shown that SfaS is necessary for the induction of fimbriation while SfaH plays a role in the stringency of binding of S fimbriae to erythrocytes.

Adhesins, Escherichia coli↗

PubMed1993

Adhesion and internalization of E. coli strains expressing various pathogenicity determinants.

The adhesion of Escherichia coli to host epithelial cells is the very first step of urinary tract infections followed by the internalization of some bacteria into these cells. These steps are influenced by several surface antigens or products of the pathogen, e.g. fimbriae or adhesins, K antigen, and hemolysin. The bacterial adherence and the internalization of several mutants of an E. coli O18:K5 strain differing in the expression of K5 antigen, hemolysin, and fimbriae were measured using a permanent line of porcine tubuloepithelial cells (LLC-PK1). Strains with K5 antigen were reduced in adherence and internalization in comparison to the K-negative strains. The expression of hemolysin by these strains lead to an increase of adherence and internalization. The internalization of bacteria is influenced mainly by their adherence to the epithelial cells. Thus, the internalization of attached bacteria is rather a kind of endocytosis than an invasion of the bacteria. To confirm this thesis, we investigated the influence of cytoskeletal inhibitors (cytochalasine B, cytochalasine D, colchicine, and chloroquine) on bacterial adherence and internalization. The cytoskeletal inhibitors lead to a significant inhibition of internalization of the bacteria tested. The receptor-mediated endocytosis of bacteria by tubuloepithelial cells may be of importance in the pathogenesis of recurrent urinary tract infection.

Animals↗

PubMed1993

Analysis of virulence factors of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease is a facultative intracellular bacterium, which in the course of human infection multiplies in lung macrophages predominantly manifesting as pneumonia. The natural habitat of Legionella is found in sweet water reservoirs and man-made water systems. Virulent L. pneumophila spontaneously convert to an avirulent status at a high frequency. Genetic approaches have led to the identification of various L. pneumophila genes. The mip (macrophage infectivity potentiator) determinant remains at present the sole established virulence factor. The Mip protein exhibits activity of a peptidyl prolyl cis trans isomerase (PPIase), an enzyme which is able to bind the immunosuppressant FK506 and is involved in protein folding. The recently cloned major outer membrane protein (MOMP) could play a role in the uptake of legionellae by macrophages. Cellular models are useful in studying the intracellular replication of legionellae in eukaryotic cells. Human cell lines and protozoan models are appropriate for this purpose. By using U 937 macrophage-like cells and Acanthamoeba castellanii as hosts, we could discriminate virulent and avirulent L. pneumophila variants since only the virulent strain was capable of intracellular growth at 37 degrees C. By using these systems we further demonstrated that a hemolytic factor cloned and characterized in our laboratory, legiolysin (lly), had no influence on the intracellular growth of L. pneumophila.

Bacterial Outer Membrane Proteins↗

PubMed1993

Dissection of pathogenetic determinants and their genomic positions for the evaluation of epidemic strains and infection routes.

The epidemiology of infectious diseases intends to disclose the infection routes and sources of the respective diseases in order to provide preventive measures. The success of such a task is directly dependent on the knowledge about the special biological quality and the environmental distribution of the causative agents. This will imply extensive laboratory analysis rather than statistical surveys. The laboratory analysis for epidemiological purposes comprises a set of sophisticated methods for the subdifferentiation of the isolates (e.g. serofermentative and electrotyping, genomic fingerprinting). These methods provide circumstantial evidence of genetic and thus, epidemiological relatedness. Also the dissection of the pathogenic personality of the respective infectious agents was shown to have epidemiologic implications. However, the virulence pattern and the pattern of the genomic position of the respective determinants were found to be well conserved. For this reason, the dissection of virulence factors in comparison to other clonal properties must be regarded only as an additional marker for epidemiological discrimination. Moreover, any form of epidemic virulence remains to be detected by future analysis.

Bacteria↗

PubMed1993

Isolation of a nicotinamide-requiring clone of Escherichia coli O18:K1:H7 from women with acute cystitis: resemblance to strains found in neonatal meningitis.

During a study of the nutritional requirements of clinical isolates of Escherichia coli, we found that 21 (7.0%) of 301 strains required nicotinamide to grow in minimal medium. The nicotinamide-requiring strains were present in 16 (15.8%) of 101 cultures of urine from young women with acute cystitis, in 5 (5.0%) of 100 stool specimens from healthy adults, and in none of 100 blood samples from adult patients with bacteremia. Most of the strains belonged to serogroup O18:K1:H7, were hemolytic, possessed type 1 fimbriae, and exhibited similar patterns of antibiotic susceptibility. Two of the urinary isolates expressed S fimbriae, and all 16 urinary isolates contained the sfaS homologue gene on their chromosomes. One of the stool isolates contained the sfaS gene. The urinary isolates closely resembled a large clone of E. coli that is reportedly associated with neonatal meningitis and sepsis. It may be possible to detect this and related clones by their requirement for nicotinamide and to screen strains for S fimbriae by relatively inexpensive hemagglutination methods, including the use of avian P1 antigens to detect mannose-resistant, non-P-fimbriated E. coli; the agglutination of bovine erythrocytes; and the use of bovine mucin to detect sialyl galactosides in S fimbriae.

Acute Disease↗

PubMed1993

Immunophilins: structure-function relationship and possible role in microbial pathogenicity.

Immunophilins are housekeeping proteins present in a wide variety of organisms. Members of two protein superfamilies, cyclophilins (Cyps) and FK506-binding proteins (FKBPs) belong to this class of immunophilins. Despite the fact that the amino acid sequences of Cyp and FKBPs do not exhibit noticeable homology to each other, proteins of both classes are able to ligate immunosuppressive peptide derivatives. Cyps form complexes with the cyclic undercapeptide cyclosporin A and FKBPs are able to bind FK506 as well as rapamycin, both of which have a pipecolyl bond within their structure. In a ligand-bound form, immunophilins interfere with signal transduction in T cells. In addition, immunophilins have peptidyl prolyl cis-trans isomerase (PPlase) activity and are able to accelerate the rate of conformational events in proline-containing polypeptides. Microorganisms produce proteins that exhibit extensive sequence homologies to cyclophilins and FKBPs of higher organisms and which have considerable PPlase catalytic activity. While cyclophilins seem to be present in most if not all microbial species investigated, FKBPs are produced by yeasts as well as by a number of pathogenic bacteria, such as Legionella pneumophila, Chlamydia trachomatis and Neisseria meningitidis. The Mip protein of L. pneumophila is a virulence factor that plays an essential role in the ability of the bacteria to survive and multiply in phagocytic cells. Some results are summarized on the structure and putative functions of immunophilins and place special emphasis on the contribution of these polypeptides to the virulence of pathogenic microorganisms.

Amino Acid Isomerases↗

PubMed1993

Inhibition of adhesion of S-fimbriated E. coli to buccal epithelial cells by human skim milk is predominantly mediated by mucins and depends on the period of lactation.

Expression of S-fimbriae is frequent in Escherichia coli strains causing sepsis and meningitis in the newborn period. We analysed the ability of human skim milk to inhibit adhesion of S-fimbriated E. coli to human buccal epithelia. Adhesion was inhibited by up to 90% using colostrum (5%) and up to 50% with mature milk (5%), indicating that this anti-infective mechanism depends on the period of lactation. Elimination of up to 99% of immunoglobulins and 91% of lactoferrin by affinity chromatography had no effect on the inhibition of adhesion. After separation of high- (> 10 kD) and low-molecular-weight fractions of skim milk, only the fraction > 10 kD was found to be able to inhibit bacterial adhesion. In order to further characterize receptor molecules for bacteria, we investigated binding of isolated S-fimbriae to glycoprotein bands on Western blot strips. Fimbriae mainly bound to a high-molecular-weight band (> 200 kD). According to molecular weight and staining behaviour, this band most likely represents mucins. We conclude that carbohydrate residues on secreted mucins of human skim milk are able to inhibit bacterial adhesion to mucosal surfaces. This could provide protection against neonatal sepsis and meningitis caused by E. coli.

Age Factors↗

PubMed1993

Sialyloligosaccharide chains of laminin as an extracellular matrix target for S fimbriae of Escherichia coli.

S fimbriae purified from recombinant Escherichia coli HB101(pANN801-13) bound strongly to extracellular matrices of cultured endothelial and epithelial cells; only poor binding was seen with the fimbriae purified from the sfaS mutant strain HB101(pANN801-1321). E. coli HB101(pANN801-13) adhered strongly to laminin immobilized on glass; no adhesion was seen to type I, III, IV, or V collagen. Strain HB101(pANN801-1321) failed to adhere to any of the target proteins. Adhesion to laminin of strain HB101(pANN801-13) was inhibited by sialyl-alpha-2,3-lactose as well as by periodate oxidation and neuraminidase treatment of laminin. In Western blotting, the purified S fimbriae recognized more strongly the A chain than the B chains of laminin.

Bacterial Adhesion↗

PubMed1993

Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate.

S fimbrial adhesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newborn meningitis. We recently described the cloning and molecular characterization of a determinant, termed sfaI, from the chromosome of an E. coli urinary tract infection strain. Here we present data concerning a S fimbria-specific gene cluster, designated sfaII, of an E. coli newborn meningitis strain. Like the SfaI complex, SfaII consists of the major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding the subunit proteins of SfaII were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with those of the SfaI complex subunits. Although the sequences of the two major SfaA subunits differed markedly, the sequences of the minor subunits showed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of the meninigitis isolate. These data were confirmed by the isolation and characterization of the SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of SfaI and SfaII revealed that differences between the two Sfa complexes with respect to their capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other than SfaS.

Adhesins, Escherichia coli↗

PubMed1993

Specificity of S fimbriae on recombinant Escherichia coli: preferential binding to gangliosides expressing NeuGc alpha (2-3)Gal and NeuAc alpha (2-8)NeuAc.

The adhesins of Escherichia coli strains HB101(pANN801-13) and HB101(pAZZ50), which express S fimbriae encoded by a recombinant plasmid containing the sfaI and sfaII gene clusters, respectively, were characterized with regard to the detailed structural requirements of their binding to sialyloligosaccharides on (neo)glycoproteins and (neo)glycolipids. From binding and binding inhibition studies in solid-phase enzyme immunoassays with isolated S fimbriae, several major conclusions can be drawn. S fimbriae bind specifically to sialic acid on gangliosides. The most active structural variant of sialic acid on GM3 ganglioside is N-glycolylneuraminic acid (NeuGc). In contrast to previous reports, high binding activities were measured also for b-series gangliosides expressing NeuAc alpha (2-8)NeuAc. In agreement with earlier studies, the site of sialic acid substitution to subterminal sugars strongly influences the binding to sialyloligosaccharides, i.e., alpha-6-linked sialic acid is only poorly recognized by the adhesin compared with alpha-3-linked sialic acid. C-8 and C-9 hydroxyl groups form essential structural elements of sialic acid in the binding event.

Animals↗

PubMed1993

Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.

Adhesins, Escherichia coli↗