Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit gene.

Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.     

Expression of cloned plasmid regions encoding colonization factor antigen I (CFA/I) in Escherichia coli

Molecular cloning from a plasmid encoding colonization factor antigen I (CFA/I) and heat-stable enterotoxin isolated two regions, 1 and 2, that are required for the production of CFA/I fimbriae. The level of CFA/I synthesis measured by ELISA was similar in an Escherichia coli K12 strain carrying regions 1 and 2 cloned separately on compatible plasmid vectors to that in the same strain containing the parental plasmid. The structural gene for the CFA/I fimbrial subunit was within region 1. This region directed production in E. coli minicells of at least six independent polypeptides, of which the fimbrial subunit and at least three others appeared to be synthesized as precursor molecules that underwent processing. Cloned DNA containing CFA/I region 2 specified three polypeptides in minicells. Attempts to reduce the size of the cloned region 1 resulted in a derivative plasmid that carried the CFA/I structural gene but did not complement a region-2 recombinant plasmid to restore production of CFA/I fimbriae.     

Characterization of two genes encoding antigenically distinct type-1 fimbriae of Klebsiella pneumoniae

A uropathogenic isolate of Klebsiella pneumoniae was shown to exhibit a mannose-sensitive hemagglutinating phenotype and to produce type-1 fimbriae consisting of subunits with a different electrophoretic mobility than those previously investigated. The gene cluster encoding expression of fimbriae was cloned and the genetic organization of the encoded polypeptides was determined. The gene encoding the major fimbrial subunit was localized and further examined by nucleotide sequence analysis. Comparison of two K. pneumoniae fimbrial genes revealed a nucleotide sequence agreement of 73%, and amino acid sequence agreement of 84% for the mature fimbrial subunits. Predictions of putative antigenic sites were correlated with regions demonstrating amino acid variability. In agreement with these predictions, no serological cross-reactivity between both fimbrial proteins could be demonstrated using an enzyme-linked immunosorbent assay (ELISA).     

Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells.

A fimbrial adhesin, designated F1845, was found to be responsible for the diffuse HEp-2 cell adherence of a diarrheal Escherichia coli isolate. The genetic determinant of F1845 was cloned, and the order of the genes necessary for production of F1845 was determined by maxicell analysis. Five polypeptides with apparent sizes of 10, 95, 27, 15.5, and 14.3 kilodaltons (kDa) were found to be encoded in that order by the F1845 determinant. The nucleotide sequence of the 14.3-kDa subunit gene was determined and found to share extensive homology in its signal sequence with the gene encoding the structural subunit of the AFA-1 hemagglutinin of a uropathogenic E. coli strain (A. Labigne-Roussel, M.A. Schmidt, W. Walz, and S. Falkow, J. Bacteriol. 162:1285-1292, 1985) but not in the region encoding the mature protein. Southern blot hybridizations indicated that the F1845 determinants are of chromosomal origin. Hybridization studies using a probe from the region encoding the 95-kDa polypeptide indicated that related sequences may be plasmid associated in some strains and chromosomal in others. Additional hybridization studies of E. coli isolates possessing sequence homology to the F1845 determinant suggest that the sequences in the 5' region of the F1845 structural subunit gene are more highly conserved than sequences in the 3' region.     

Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139: H28

An oligonucleotide, derived from the N-terminal amino acid sequence of the CS1 fimbrial subunit protein was used to identify the subunit gene on recombinant plasmid pDEP23 containing the structural genes of the CS1 fimbrial operon. The nucleotide sequence of the subunit gene (csoA), encoding a protein of 171 amino acids, was determined. Flanking it upstream, a gene (csoB) encoding a protein of 238 amino acids was found. The CsoB and CsoA proteins are homologous to the CfaA and CfaB proteins in the CFA/I fimbrial operon. For all the CS1 producing strains investigated the structural genes are located on plasmids. Like CFA/I fimbriae, CS1 fimbriae are only expressed in the presence of a positive regulator, CfaD for CFA/I and Rns for CS1, respectively. The promoter region upstream of the csoB gene was cloned in front of the promoterless alkaline phosphatase (phoA) gene of the promoter-probe vector pCB267. PhoA activity was enhanced approximately two-fold by the introduction of compatible plasmids containing either rns or cfaD.     

Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of Escherichia coli

Four genes encoding for polypeptides with apparent molecular weights of 17,000, 26,000 (the fimbrial subunit), 27,000, and 81,000 have been implicated in the biosynthesis of the K88ab fimbria (Mooi et al., J. Bacteriol. 150:512-521, 1982). Escherichia coli mutants with defects in these genes were examined for the presence of fimbrial precursors. An analysis of these mutants revealed that fimbrial subunits accumulated transiently in the periplasmic space before being translocated across the outer membrane. The 81,000-dalton (d) polypeptide is probably involved in translocating fimbrial subunits across the outer membrane, because in the absence of this polypeptide the fimbrial subunits remained in the periplasmic space, where they were found to be associated with the 17,000- and 27,000-d polypeptides. In mutants with a deletion in the gene for the 27,000-d polypeptide, fimbrial precursors were not detected, because the fimbrial subunits were degraded. The 27,000-d polypeptide might be involved in stabilizing a conformation of the fimbrial subunit required to translocate it across the outer membrane. In the absence of the 17,000-d polypeptide, most fimbrial subunits were found in the periplasmic space associated with the 27,000-d polypeptide. However, small amounts of subunits were also translocated across the outer membrane. These extracellular subunits did not adhere to brushborders, suggesting that fimbrial subunits must be modified by the 17,000-d polypeptide to be assembled into functional fimbriae. A model for the biosynthesis of the K88ab fimbria is proposed.     

Heterogeneous post-translational modification of Actinobacillus actinomycetemcomitans fimbrillin

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle-forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography-electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310-a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post-translationally modified. Modification of the fimbrial peptides was also suggested by an N-terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C-terminal region. A periodate oxidation/biotin-hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.     

Identification of the polypeptides of the major light-harvesting complex of photosystem II (LHCII) with their genes in tomato.

Using an improved SDS-PAGE system, the polypeptides of the major chlorophyll a/b light-harvesting complex of PSII (LHCII) from tomato leaves were resolved into five polypeptide bands. All the polypeptides were matched with the genes encoding them by comparing amino acid sequences of tryptic peptides with gene sequences. The two major LHCII bands (usually comigrating as a '27 kDa' polypeptide) were encoded by cab1 and cab3 (Type I LHCII) genes. A third strong band of about 25 kDa was encoded by cab4 (Type II) genes. Polypeptides from two minor bands of 23-24 kDa were not N-terminally blocked; their N-terminal sequences showed they were Type III LHCII proteins. One complete cDNA clone and several incomplete clones for Type III polypeptides were sequenced. Combined with the peptide sequences, the results indicate that there are at least four different Type III genes in tomato, encoding four almost identical polypeptides. Thus, all the LHCII CAB polypeptides have been identified, and each type of LHCII polypeptide is encoded by distinct gene or genes in tomato.     

Characterization and molecular cloning of the PCF8775 CS5 antigen from an enterotoxigenic Escherichia coli 0115:H40 isolated in Central Australia

The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purity with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14 kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Summary Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.     

Mucosal vaccination of mice with recombinant Proteus mirabilis structural fimbrial proteins

Proteus mirabilis, a common cause of urinary tract infection (UTI), expresses several types of fimbria including mannose-resistant/Proteus-like fimbriae (MRP), uroepithelial cell adhesin (UCA), renamed non-agglutinating fimbriae (NAF) by some authors, and P. mirabilis fimbriae (PMF), which are potentially involved in adhesion to the uroepithelium. In this study, we immunised different groups of mice with recombinant structural subunits of these fimbriae (MrpA, UcaA and PmfA) using two mucosal routes (nasal and transurethral) and we transurethrally challenged the animals with a P. mirabilis uropathogenic isolate. Induction of specific serum and urine IgG and IgA was measured to assess the potential role of the humoral immune response in protection against experimental ascending P. mirabilis UTI. Intranasally MrpA- and UcaA-immunised mice were protected against P. mirabilis ascending UTI, since recovery of bacteria from kidneys and bladders was significantly lower than in PBS-treated mice, and both fimbrial subunits significantly induced specific serum and urine antibodies. Only MrpA and PmfA transurethrally immunised animals were protected only at the kidney level, and in this case only MrpA-immunised mice exhibited significant serum IgG induction. Correlation analysis did not show a significant relationship between serum and urine specific antibody response and protection observed against infection. Our results suggest that an immunisation strategy based on structural fimbrial proteins may be useful to prevent P. mirabilis UTI. Further studies are being carried out to characterise the immune and inflammatory response induced by P. mirabilis recombinant fimbrial subunits.     

The beta subunit of tomato fruit polygalacturonase isoenzyme 1: isolation, characterization, and identification of unique structural features.

We have purified and isolated cDNAs encoding the beta subunit of tomato fruit polygalacturonase isoenzyme 1 (PG1), a cell wall protein that associates with, and apparently regulates, the catalytic PG2 polypeptides. Expression of the beta subunit is fruit specific and temporally separated from the expression of PG2 during fruit development. The 37- to 39-kD beta subunit is encoded as a 69-kD precursor protein containing a signal sequence and two propeptide domains. The mature protein is composed almost entirely of the novel 14-amino acid motif FTNYGxxGNGGxxx in which many of the phenylalanine residues are post-translationally modified. The unique structural features of the motif suggest an important role in the function of the protein and hence in the activity of PG1. The beta subunit may represent a class of bifunctional plant proteins that interact both with structural components of the cell wall and catalytic proteins to localize and/or regulate metabolic activities within the cell wall.     

Cloning, characterization, and sequence analysis of the clcE gene encoding the maleylacetate reductase of Pseudomonas sp. strain B13.

A 3,167-bp PstI fragment of genomic DNA from Pseudomonas sp. strain B13 was cloned and sequenced. The gene clcE consists of 1,059 nucleotides encoding a protein of 352 amino acids with a calculated mass of 37,769 Da which showed maleylacetate reductase activity. The protein had significant sequence similarities with the polypeptides encoded by tcbF of pP51 (59.4% identical positions), tfdF of pJP4 (55.1%), and tftE of Burkholderia cepacia AC1100 (53.1%). The function of TcbF as maleylacetate reductase was established by an enzyme assay.     

Cloning and genetic organization of the gene cluster encoding F71 fimbriae of a uropathogenic Escherichia coli and comparison with the F72 gene cluster

Abstract The gene cluster coding for expression of F7₁ fimbriae of the uropathogenic Escherichia coli strain AD110 has been cloned by a cosmid-cloning procedure. A positive clone was further subcloned to a plasmid of 17.5 kilobases (kb), pPIL110-75. Analysis of pPIL110-75 showed that at least six genes are present encoding proteins with apparent M _rs of 75 000, 36 000, 23 000, 20 000, 17 000 and 14 000. The 20-kDa protein, encoding the F7₁ fimbrial subunit is dispensable for expression of the MRHA phenotype. Complementation experiments of mutants in the F7₂ gene cluster by gene products of the F7₁ gene cluster show that the two gene clusters are related.