Frits K. de Graaf

Characterization of the antigenic and adhesive properties of FaeG, the major subunit of K88 fimbriae

The two K88 serotypes, K88ab and K88ac, differ in terms of antigenic and adhesive properties. The structural determinants of the serotype‐specific epitopes and the identity of the amino acid residues involved in fimbriae‐receptor interaction were studied by the construction and analysis of K88 hybrid proteins in which various parts of the K88ab and K88ac fimbrial subunit FaeG were exchanged, and by in vitro mutagenesis of non‐conserved amino acid residues. Using a set of monoclonal antibodies, several regions or amino acid residues involved in the formation of serotype‐specific antigenic determinants were located. The haemagglutinating activity of the hybrid and mutant proteins revealed several amino acid residues involved in the formation of the receptor binding site. A clear correlation was found between the receptor binding site and the serotype‐specific antigenic determinants.

Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99.

Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin‐layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non‐acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor‐active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGcα‐3Galß4GlcßCer (NeuGc‐GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc‐GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N‐glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein‐linked sequences as well and thus explain the resistance of adult pigs to infection with E. coli K99.

Leucine‐responsive regulatory protein, IS 1 insertions, and the negative regulator FaeA control the expression of the fae (K88) operon in Escherichia coli

Nucleotide sequence analysis of the fae operon encoding the biosynthesis of K88 fimbriae revealed the presence of two divergently transcribed regulatory genes, faeA and faeB, separated by two inverted iS 1 insertions. The amino acid sequences of the regulatory proteins FaeA and FaeB show similarity to the primary structure of corresponding regulatory proteins involved in the biosynthesis of Pap and S fimbriae. Expression of faeA is positively controlled by the FaeA protein, whereas K88 fimbriae production is negatively controlled by the co‐operative activity of FaeA and the leucine‐responsive regulatory protein (Lrp). Exchange of FaeA for Papl, a positive regulator of Pap fimbriae expression, also represses K88 production indicating that the combination Papl/Lrp has opposite effects on fae and pap expression. Mutations in faeB had no effect on the biosynthesis of K88 fimbriae. The presence of the two iS 1 insertions is hypothesized to neutralize part of the repression of K88 biosynthesis by FaeA/Lrp. Like pap, the fae operon does not respond to exogenous leucine.

Detection and identification of FaeC as a minor component of K88 fibriilae of Escherichia coli

A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit‐like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab‐encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.

Negative control of fae (K88) expression by the‘global’regulator Lrp is modulated by the‘local’regulator FaeA and affected by DNA methylation

Expression of the K88 (fae) operon is negatively controlled by the co‐operative binding of Lrp and FaeA to the fae regulatory region and is dependent on the methylation status of three GATC sites present in this region. In this paper, we describe the binding of Lrp to a T‐rich DNA helix between GATC site I and site II. FaeA stabilized and modified the Lrp binding, thereby extending the Lrp footprint over GATC site I and site III. Methylation of GATC site I prevented the binding of Lrp/FaeA at this site and appeared to be essential for the cells, since mutation of this site into GTTC resulted in a lethal overproduction of K88 fimbriae. Methylation of GATC site II and site III reduced the stability of Lrp/FaeA binding. Moreover, methylation of GATC site III stimulated faeB promoter activity. The plasmid population in cells harbouring multiple copies of a K88 plasmid consisted of two differentially methylated forms. Form A plasmids with a methylated GATC site I and site III and a non‐methylated site II (+,‐,+) represented 20% of the population and were responsible for high‐level expression. Form B plasmids with a methylated GATC site I and a non‐methylated site II and site III (+,‐,‐) represented 80% of the population and were responsible for low‐level expression. Apparently, K88 fimbriae expression in vivo is balanced at its maximal possible level by modulation of the methylation status of GATC site III. The ratio (1:4) between these populations is stabilized by a constitutive synthesis of FaeA resulting from the presence of an IS1 insertion upstream of faeA. This IS1 insertion separates the faeA promoter from the FaeB‐binding sites, thereby neutralizing the control by FaeB activity on expression of FaeA. Instead, faeA transcription is stimulated by binding of FaeA to the faeA promoter region.

Characterization of FapR, a positive regulator of expression of the 987P operon in enterotoxigenic Escherichia Coli

Expression of the 987P gene cluster is activated by the adjacent IS1 element of an STpa transposon. Nucleotide sequence analysis of the 987P‐DNA region contiguous with this IS1 element revealed the presence of an open reading frame designated fapR, encoding a basic protein of 260 amino acid residues with a molecular mass of 30349 Daltons. The gene product, FapR, possesses similarity to a number of positive regulators of gene expression: VirF, Rns, AppY and EnvY. Moreover, a 43‐amino‐acid residue sequence in the C‐terminal part of FapR is similar to the C‐terminal domain of AraC, RhaR, and RhaS. Expression of fapR is dependent on the adjacent IS1 element. The FapR protein appears to be required for activation of the silent promoter of the fimbrial subunit gene, fapC

Subcellular localization and topology of the K88 usher FaeD in Escherichia coli.

The subcellular localization of the K88 usher FaeD was studied in Escherichia coli whole ceils by using iso‐pycnic sucrose density gradient centrifugation of isolated membranes, the detergents Triton X‐100 and sodium lauryl sarcosinate and immunoblotting with a specific FaeD antiserum. Cells containing the complete K88 operon, as well as cells containing the sub‐cloned faeD gene in various expression vectors, were used. Most of the FaeD was present in the outer membranes in a detergent‐resistant form. Agglutination experiments with E coli cells expressing FaeD confirmed an outer membrane localization and indicated the presence of FaeD at the cell surface. Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amino acid residues. Twelve different FaeD–PhoA fusion proteins were prepared and characterized by nucleotide sequencing and immuno‐blotting. Most of these fusion sites were located in the amino‐terminal and carboxyl‐terminal regions of FaeD. Six amino‐terminal fusion proteins were soluble proteins in the peripiasm, whereas the other fusion proteins were associated with the outer membrane. The protease accessibility of FaeD and of the six outer membrane‐bound FaeD–PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, and isolated membranes. Collagenase H, kallikrein, trypsin and proteinase K were used. Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane‐spanning segments and two relatively large periplasmic regions, at the amino‐terminal and carboxyl‐terminal end of the protein, respectively.

Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae

Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved In the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site‐specific frame‐shift mutation in lanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.

Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coii

The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG.

Transcriptional organization of the DNA region controlling expression of the K99 gene cluster

SummaryThe transcriptional organization of the K99 gene cluster was investigated in two ways. First, the DNA region, containing the transcriptional signals was analyzed using a transcription vector system with Escherichia coli galactokinase (GalK) as assayable marker and second, an in vitro transcription system was employed. A detailed analysis of the transcription signals revealed that a strong promoter PA and a moderate promoter PB are located upstream of fanA and fanB, respectively. No promoter activity was detected in the intercistronic region between fanB and fanC. Factor-dependent terminators of transcription were detected and are probably located in the intercistronic region between fanA and fanB (T1), and between fanB and fanC (T2). A third terminator (T3) was observed between fanC and fanD and has an efficiency of 90%. Analysis of the regulatory region in an in vitro transcription system confirmed the location of the respective transcription signals. A model for the transcriptional organization of the K99 cluster is presented. Indications were obtained that the trans-acting regulatory polypeptides FanA and FanB both function as anti-terminators. A model for the regulation of expression of the K99 gene cluster is postulated.