Schmoll, T. (Institut für Genetik und Mikrobiologie, University of Würzburg, Röntgenring 11, 0';'8700, Würzburg, Germany), J. Morschhäuser, M. Ott, B. Ludwig, I. van Die and J. Hacker. 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. Microbial Pathogenesis 1990; 9: 331-343. The S fimbrial adhesin (sfa) determinant of E. co/i comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfaC 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 sturlies indicated that the genes sfaB and sfaC play a role in regulation of the sfa determinant. ln addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfaA and sfaG responsible for S subunit proteins, were determined. lt 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), F1 C fimbriae (foc) and type I fimbriae ( fim). The evolutionary relationship of fimbrial adhesin determinants is discussed.

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

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocytes, macrophages, and protozoan organisms. The Mip (macrophage infectivity potentiator) protein represents a factor of L. pneumophila necessary for optimal intracellular survival. Interestingly, Mip belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. In order to identify amino acids most likely to be involved in the enzymatic activity of Mip, site-directed mutagenized Mip proteins were constructed and characterized. It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitution resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% of the activity of the wild-type Mip, respectively). Genes coding for the wild-type and for site-directed-mutagenized Mip proteins were used to complement three different Mip-negative mutants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. While Mip protein expression could be restored in the corresponding complementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investigate the influence of the PPIase activity of Mip on intracellular survival of L. pneumophila, invasion assays were performed using the macrophage-like cell line U937, human blood monocytes, and Acanthamoeba castellanii. The Mip-negative mutants were approximately 50- to 100-fold less infective for A. castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains. The wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains. In addition, no differences in intracellular survival were observed between the wild-type isolates and the Legionella strains exhibiting strongly reduced PPIase activity. These data indicated that the enzymatic activity of Mip does not contribute to intracellular survival of L. 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.

The Mip (‘macrophage infectivity potentiator’) protein of Legionella pneumophila has been shown to be an essential virulence factor, exhibiting peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. The cloning and sequencing of mip genes from three different L. pneumophila strains revealed a single amino acid substitution which did not affect the isomerase property of the enzyme. Mip proteins isolated from two wild-type L. pneumophila strains and from two corresponding Escherichia coli K-12 recombinant clones derived from these strains exhibited identical enzymatic properties and the precursor proteins are processed at identical cleavage sites. The mature Mip proteins exist in an oligomeric form. Site-directed mutagenesis demonstrated that a substitution of an Asp residue at position 142 by a Leu residue affects PPIase activity of Mip.

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.

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.

Summary 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.

A genomic library of Legionella pneumophila, the causative agent of Legionnaires disease in humans, was constructed in Escherichia coli K-12, and the recombinant clones were screened by immuno-colony blots with an antiserum raised against heat-killed L. pneumophila. Twenty-three clones coding for a Legionella-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found to be associated with the peptidoglycan layer both in L. pneumophila and in the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions with a monospecific polyclonal 19-kDa protein-specific antiserum. The protein was termed peptidoglycan-associated protein of L. pneumophila (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb ClaI fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18.9 and 16.8 kDa, respectively, were located on the ClaI fragment. Exonuclease III digestion studies confirmed that pplA is the gene coding for the peptidoglycan-associated 19-kDa protein of L. pneumophila. The amino acid sequence of PplA exhibits a high degree of homology to the sequences of the Pal lipoproteins of E. coli K-12 and Haemophilus influenzae.

Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as well as within protozoan organisms.Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human embryo lung fibroblastic cells, it is demonstrated thatLegionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival ofLegionella a genomic library of strainLegionella pneumophila Philadelphia I was established inEscherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further studies will be necessary to determine the exact role of the cloned proteins in the pathogenesis ofLegionella.

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.