Birgit Ludwig

Confirmation of the existence of a third family among peptidyl‐prolyl cis/trans isomerases Amino acid sequence and recombinant production of parvulin

In addition to the major cyclophilin‐like peptidyl‐prolyl cis/trans isomerases (PPIases) of Escherichia coli an enzyme of very low relative molecular mass (10.1 kDa) was discovered in this organism which gave first indication of the existence of a novel family in this enzyme class [1994, FEBS Lett. 343, 65–69]. In the present report we describe the chemically determined amino acid sequence of four peptides derived from the 10.1 kDa protein by the treatment with either cyanogen bromide or endoproteinase Lys‐C. Together with a continuous run of 75 amino acids starting N‐terminally, the sequence of the mature enzyme, 92 residues in length, was elucidated. Cloning and determination of the primary structure of a DNA fragment encoding this enzyme were also performed. Overexpression of the enzyme by using multicopies of plasmid pSEP38 in E. coli and detecting an enhanced PPIase activity attributed to the 10.1 kDa enzyme provided additional proof that the 92 amino acid protein was a PPIase. The enzyme was called parvulin (lat.: parvulus, very small). Homology analyses indicated that several parvulin‐like proteins could be found in the database screened. To further elucidate the functional role of PPIases it might be of some importance that homologous proteins like the PrtM protein of Lactococcus lactis and the PrsA lipoprotein of Bacillus subtilis are known to be involved in the protein export and maturation machinery of the bacteria.

Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity.

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macro‐phage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506‐binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl‐prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K‐12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP‐type PPlases of eukaryotes. However, the N‐terminal extension of Mip and/or the substitutions of the variable amino acids in the C‐terminal FKBP core lead to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc‐Ala‐Xaa‐Pro‐Phe‐4‐nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein‐folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.

Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesin determinant: nucleotide sequence of the genes sfa B, C, D, E, F

The S fimbrial adhesin (sfa) determinant of E. coli comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfa C situated proximal to the main structural gene sfaA is described. Sfa-LacZ fusions show that the two genes are transcribed in opposite directions. The isolation of mutants in the proximal region of the sfa gene cluster, the construction of sfa-phoA gene fusions and subsequent transcomplementation studies indicated that the genes sfa B and sfa C play a role in regulation of the sfa determinant. In addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfa A and sfa G responsible for S subunit proteins, were determined. It is suggested that these genes are involved in transport and assembly of fimbrial subunits. Thus the entire genetic organization of the sfa determinant is presented and compared with the gene clusters coding for P fimbriae (pap), F1C fimbriae (foc) and type I fimbriae (fim). The evolutionary relationship of fimbrial adhesion determinants is discussed.

Characterization of legiolysin (lly), responsible for haemolytic activity, colour production and fluorescence of Legionella pneumophila.

A genomic library of Legionella pneumophila, the causative agent of Legionnaires’disease in humans was constructed in Escherichia coli K12 and the recombinant clones were tested for haemolysis and other phenotypic properties. Seven clones were identified which were able to confer haemolysis of human, sheep, and canine erythrocytes but which were unable to mediate proteolytic activities or cyto‐toxic effects on CHO‐ or Vero cells. Clones that exhibited this haemolytic property were also able to produce a brown colour and a yellow‐green fluorescence activity detected on M9 plates containing tyrosine. The genetic determinant encoding these properties, termed legiolysin (lly) was mapped by Tn 1000 mutagenesis and by subcloning experiments. Southern hybridization with an lly‐specific gene probe showed that this determinant is part of the genome of L. pneumophila but is not identical to a protease gene of L. pneumophila which also mediates haemolysis. Minicell analysis of lly‐specific plasmids exhibited a protein of 39kDa. Polyclonal antibodies generated against a LacZ‐Lly hybrid protein also recognized a 39kDa protein produced either by the recombinant legiolysin‐positive E. coli K12 clones or by L. pneumophila wild‐type strains.

A homodimer represents an active species of the peptidyl‐prolyl cis/trans isomerase FKBP25mem from Legionella pneumophila

The molecular mass of the native FK506‐binding peptidyl‐prolyl cis/trans isomerase (PPIase) FKBP25mem from Legionella pneumophila (Mip (macrophage infectivity potentiator) protein) was determined by two methods. By gel‐permeation chromatography we found no indication of the presence of the monomeric enzyme. However, an oligomeric state with a molecular mass of about 62 kDa was detected. By cross‐linking with dimethyl pimelimidate and subsequent SDS‐PAGE of either the surface proteins of intact L. pneumophila cells or the purified recombinant FKBP25mem in solution, we observed an immunoreactive band indicative of a mass in the dimer range. In contrast to human recombinant FKBP12, the enzymatic activity of Legionella FKBP25mem was strongly dependent on the protein concentration, pointing to a dimer as the most active species. However, the inhibition by FK506 yielded a nearly constant value of K i of about 250 nM when measured in the same range of FKBP25mem concentration. These results may be explained by the fact that monomeric FKBP25mem has little, if any, influence on enzymatic activity when compared with the homodimer.

A cyclophilin‐like peptidyl‐prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506‐binding protein, FKBP25mem), belongs to the enzyme family of peptidyl‐prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N‐terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164‐amino‐acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19‐amino‐acid polypeptide extension including a 6‐histi‐dine tag and an enterokinase cleavage site exhibits

Legiolysin, a New Hemolysin from L. pneumophila

Legionella pneumophila generates exotoxins, cytolysins, proteases or hemolysins that damage host cells like erythrocytes or tissue culture cells. The gene for a new L. pneumophila hemolysin without a proteolytic activity was identified, cloned in E. coli and sequenced. The gene product was analysed by SDS-Polyacrylamide-gel-electrophoresis.