Adherence of F18 fimbrial

Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein. In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E. coli adhesion to porcine enterocytes. Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein.The operons of many fimbrial adhesins of Escherichia coli are well characterized (4). They contain genes coding for the major subunit protein, molecular chaperone and usher proteins, minor subunits, adhesin, and proteins of unknown function (4,11,12). The genes involved in the biosynthesis of F18 fimbria have been only partially described (5, 6). The major protein of the F18 fimbria, FedA, is not sufficient for recognizing the F18 receptor (5). Two additional genes from the fed gene cluster, fedE and fedF, have been described as essential for fimbrial adhesion and fimbrial length (6). However, so far it has not been possible to assess F18 adhesion function with regard to either of the two gene products.In this study, we sequenced the unknown region of the E. coli fed gene cluster and produced and purified FedF and FedE as fusion proteins with maltose binding protein (MBP) for raising antisera for adhesion studies. Furthermore, using indirect immunofluorescence microscopy and adhesion inhibition tests, we have characterized the FedF proteins as the adhesin of F18 fimbriae.Sequencing of the plasmid pIH120. The entire gene cluster encoding E. coli F18 fimbria was sequenced from the plasmid pIH120 (6) with an ABI 310 sequencer according to the manual of the manufacturer (PE Applied Biosystems). pIH120 was transferred into an E. coli HB101 host, resulting in strain ERF2055. Sequence analyses revealed that the fed gene cluster is composed of five genes. The gene coding for the major protein of F18 fimbria (fedA) and the genes encoding two minor proteins (fedE and fedF) were described earlier (5, 6). Two additional open reading frames were found between fedA and fedE and were designated fedB and fedC. FedB showed the highest similarity (83% identity) to the AfrB protein (AAC28316) from E. coli RDEC-1 (Fig. 1) and significant homology to other usher proteins involved in the biosynthesis of microbial pili (3). The second open reading frame (fedC) overlapped the 3Ј end of fedB, and its product had high identity (82%) with the periplasmic chaperone AfrC (AAC228317) from E. coli RDEC-1. Both FedB and FedC possess a predicted signal peptide for transmembrane secretion with a putative cleavage site for a signal peptidase between amino acids 23 and 24. The calculated molecular masses of the mature FedB and FedC are 86,001 and 23,418 Da, respectively. The fedF gene was also PCR cloned and sequenced from a Finnish E. coli O141 isolate (data not shown) and found to have 99.6% identity with the fedF derived from pIH120. In addition to the previously reported transcription terminator, located downstream of fedA (5), an inverted repeat (⌬G of Ϫ17.3 kcal mol Ϫ1 ) for th...

F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF.

A gene (htrA) coding for a stress-inducible HtrA-like protein from Lactobacillus helveticus CNRZ32 was cloned, sequenced, and characterized. The deduced amino acid sequence of the gene exhibited 30% identity with the HtrA protein fromEscherichia coli; the putative catalytic triad and a PDZ domain that characterize the HtrA family of known bacterial serine proteases were also found in the sequence. Expression of the L. helveticus htrA gene in a variety of stress conditions was analyzed at the transcriptional level. The strongest induction, resulting in over an eightfold increase in the htrAtranscription level, was found in growing CNRZ32 cells exposed to 4% (wt/vol) NaCl. Enhanced htrA mRNA expression was also seen in CNRZ32 cells after exposure to puromycin, ethanol, or heat. The reporter gene gusA was integrated in theLactobacillus chromosome downstream of the htrApromoter by a double-crossover event which also interrupted the wild-type gene. The expression of gusA in the stress conditions tested was similar to that of htrA itself. In addition, the presence of an intact htrA gene facilitated growth under heat stress but not under salt stress.

Three Lactobacillus helveticus 53/7 genes essential for the biosynthesis of isopentenyl diphosphate and the gene coding for a putative carotenoid biosynthesis protein were for the first time identified from lactic acid bacteria. The deduced amino acid sequences of the mevalonate pathway gene products share significant identity with corresponding proteins of a few gram-positive cocci and Streptomyces species.